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Articles by Kathleen Kulka in JoVE

 JoVE Immunology and Infection

Growth of Mycobacterium tuberculosis Biofilms

1Department of Infectious Diseases and Microbiology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh


JoVE 3820

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Other articles by Kathleen Kulka on PubMed

Non-nuclear Staining of Thick Tissue Sections

[Does Clonidine Modify the Hypnotic Effect of Propofol?]

The administration of alpha 2-adrenoceptor agonists before the induction of anaesthesia leads to a significant reduction in the amount of anaesthetic medication required, probably due to an attenuation of haemodynamic stress responses in centrally mediated sympathicolysis. However, it is not yet known whether alpha 2-adrenoceptor agonists influence the hypnotic action of anaesthetics. Therefore, this study was performed to evaluate the influence of the alpha 2-adrenoceptor agonist clonidine on the potency and the duration of the hypnotic action of anaesthetic agents. METHOD. The study was approved by the local ethical committee. To study the effect of clonidine on the potency of propofol we determined the ED50 of propofol with and without clonidine pretreatment. To this end, 100 unpremedicated patients (ASA I or II) were randomly assigned to receive 4 micrograms/kg body weight clonidine or placebo, each of which was dissolved in 100 ml NaCl and infused over a period of 15 min starting 30 min before the induction of anaesthesia. According to the results of a pilot study, patients who had been treated with clonidine received either 0.25, 0.5, 0.75, 1 or 1.25 mg/kg propofol for anaesthesia induction. Patients in the placebo group received 0.5, 1, 1.5, 2 or 2.5 mg/kg propofol. The success of anaesthesia induction was evaluated clinically (eye opening on command, eyelid reflex). On the basis of these data the ED50 of propofol with and without clonidine pretreatment was calculated using the modified probit analysis according to Spearman and Kärber. The effect of clonidine on the duration of anaesthesia was compared in six groups of 10 patients each, who received 1, 1.5 or 2 mg/kg propofol for anaesthesia induction with and without prior clonidine treatment. RESULTS. In the placebo group a dose of 0.5 mg propofol per kg did not produce a hypnotic effect in any patient, while 2.5 mg propofol per kilogram of body weight was effective in all patients. In the clonidine group 0.25 mg propofol per kilogram of body weight had no hypnotic effect, while 1.25 mg propofol per kilogram of body weight was effective in all patients. Increasing the dose of propofol resulted in an increasing number of successful anaesthesia inductions in the placebo as well as in the clonidine group. According to these data, the ED50 of propofol with clonidine was calculated at 0.675 +/- 0.23 mg/kg with clonidine and 1.5 +/- 0.58 mg/kg without clonidine pretreatment. Increasing the dose of propofol did not result in a significant increase in the duration of anaesthesia (1 mg/kg: 260 +/- 114 s; 1.5 mg/kg: 270 +/- 103 s; 2 mg/kg: 295 +/- 152 s). However, premedication with clonidine almost doubled the duration of the hypnotic action of propofol (1 mg/kg: 457 +/- 239 s; 1.5 mg/kg: 501 +/- 249 s; 2 mg/kg: 582 +/- 254 s) (P < 0.01). CONCLUSION. According to these findings the administration of clonidine prior to anaesthesia induction significantly increases the potency and the duration of the hypnotic action of propofol. From our data we conclude that the influence of clonidine on the hypnotic action of anaesthetics is an important factor in the reduction of anaesthetic requirements observed after clonidine pretreatment.

Blue Paraffin Wax Blocks: a Solution to the Problem

Chromosome Aberrations in Peripheral Lymphocytes from Occupants of Houses with Elevated Indoor Radon Concentrations

Chromosome analyses were performed in blood lymphocytes of 25 subjects continuously living in houses with indoor radon (222Rn) concentrations exceeding 4-60-fold the German average of 50 Bqm-3. The mean frequency of cells containing dicentrics + ring chromosomes (1.3 +/- 0.3/1000 cells) and the incidence of dicentrics + ring chromosomes per cell (1.5 +/- 0.4 x 10(-3)) were significantly increased compared to the control levels (0.54 +/- 0.11 x 10(-3) for both endpoints). Taking into account the individual radiation history over the last 10 years prior to blood sampling and the life time of peripheral lymphocytes, weighted cumulative radon exposures at the time of blood sampling between 700 and 6300 Bqm-3a were derived. Although individual exposures could not be inferred from the aberration rates, a tendency for an exposure-effect relationship became apparent for two groups of subjects with a mean weighted cumulative radon exposure above and below 1800 Bqm-3a.

Polymicrobial and Monomicrobial Bacteraemic Urinary Tract Infection

Polymicrobial blood or urine cultures in bacteraemic urinary tract infection (UTI) are relatively common. There is, however, very little information available on the clinical and bacteriological features that distinguishes between monomicrobial and polymicrobial urosepsis. During 1980-84, 68 of 198 episodes (34%) of urosepsis with at least one identical organism in blood and urine, had multiple growth in either one or the other. Comparison between monomicrobial and polymicrobial infectious episodes showed that the latter were more often hospital-acquired and more frequently associated with urinary catheters. Pseudomonas aeruginosa was more often associated with polymicrobial than with monomicrobial infections, whereas Escherichia coli was more common in monomicrobial infections. Mortality was higher in polymicrobial infections, and was further increased if multiple organisms grew from blood rather than from urine. Thus, there are clinical, microbiological and prognostic characteristics that distinguish polymicrobial from monomicrobial bacteraemic UTIs.

Regulated Degradation of the Transcription Factor Gcn4

We report that Gcn4, a yeast transcriptional activator of the bZIP family involved in the regulation of the biosynthesis of amino acids and purines, is rapidly turned over. This degradation is inhibited under conditions of starvation for amino acids. Degradation is also inhibited by single amino acid alterations in a region adjacent to the Gcn4 activation domain. Furthermore, we show that degradation of Gcn4 proceeds through the ubiquitin pathway, a major proteolytic system for cytoplasmic proteins, and is dependent on two specific ubiquitin conjugating enzymes, Cdc34 (Ubc3) and Rad6 (Ubc2). As a first step towards reconstituting the Gcn4 degradation pathway in vitro, we show that purified Cdc34 and Rad6 proteins are able to direct the specific ubiquitination of Gcn4.

Cutaneous Ultraviolet Radiation Inhibits Herpes Simplex Virus-induced Lymphoproliferation in Latently Infected Subjects

Exposure of herpes simplex virus (HSV) latently infected subjects to ultraviolet irradiation (UVR) (1 minimum erythema dose, 90% body surface) caused a significant inhibition of HSV and phytohemagglutinin-induced lymphoproliferation. The inhibition was observed on Day 3 post-UVR and lasted at least 9 days. UVR-induced downregulation of HSV-specific lymphoproliferation was associated with increased levels of activated transforming growth factor beta. However, the relationship between UVR-induced immune downregulation and the development of recurrent HSV lesions was incomplete.

Synergistic Antiviral Activities of Oligonucleoside Methylphosphonates Complementary to Herpes Simplex Virus Type 1 Immediate-early MRNAs 4, 5, and 1

An oligonucleoside methylphosphonate (ONMP) complementary to the splice acceptor site of immediate-early (IE) pre-mRNAs 4 and 5 (IE4,5SA) inhibits herpes simplex virus type 1 (HSV-1) growth in vitro and in infected animals. The antiviral effect appears to be due to inhibition of IE pre-mRNA 4 and 5 splicing and/or IE4 gene expression (M. Kulka, M. Wachsman, S. Miura, R. Fishelevich, P. S. Miller, P. O. P. Ts'o, and L. Aurelian, Antiviral Res. 20:115-130, 1993). We describe the potentiation of antiviral activity when we targeted two IE genes with different ONMPs. A psoralen derivative of an ONMP complementary to the IE mRNA 1 (IE1) translation initiation site (IE1TI) covalently bound a 2.8-kb transcript that hybridized with a 20-base oligonucleotide complementary to the 5' leader sequence of IE1 but not a 20-base oligonucleotide complementary to the first intron of IE1. IE1TI inhibited IE1 gene expression and virus replication in cells infected with HSV-1 in vitro. Inhibition was specific because it was not observed with oligomers mutated in two (IE1TImu1) or four (IE1TImu2) central residues or in cells infected with an IE1 deletion mutant (HSV-1 dl1403). IE1TI potentiated the antiviral activity of IE4,5SA (synergistic effect), while potentiation was not observed when IE4,5SA was mixed with IE1TImu1. A similar synergistic effect was seen when IE1TI was mixed with an ONMP complementary to the translation initiation site of IE mRNA 4 but not with an ONMP complementary to the translation initiation site of IE mRNA 5. These findings suggest that synergistic antiviral activity is mediated by targeting at least two IE genes (IE1 and IE4).

Role of the C-terminus of Saccharomyces Cerevisiae Ubiquitin-conjugating Enzyme (Rad6) in Substrate and Ubiquitin-protein-ligase (E3-R) Interactions

The product of the RAD6 (UBC2) gene of Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme (Rad6) which is implicated in DNA repair, induced mutagenesis, retrotransposition, sporulation and the degradation of proteins with destabilizing N-terminal amino acid residues. Deletion of the 23-residue acidic C-terminus of Rad6 impairs sporulation and N-end rule protein degradation in vivo but does not affect other functions such as DNA repair and induced mutagenesis. We have investigated the role of the C-terminus of Rad6 in in vitro interactions with various substrates and with a putative ubiquitin-protein ligase, E3-R. The removal of the Rad6 C-terminus had significant different effects on enzyme activity for individual substrates. Although the 23-residue truncated Rad6-149 protein had markedly impaired activity for histone H2B and micrococcal nuclease, the activity for cytochrome c was the same as that of the intact Rad6 protein. Similarly, truncation of Rad6 had no effect on its activity for several poor substrates, namely, beta-casein, beta-lactoglobulin and oxidized RNase. E3-R stimulated the activities of both Rad6 and Rad6-149 for the latter three substrates to similar degrees. E3-R appears to act by enhancing the low intrinsic affinity of Rad6 and Rad6-149 for these substrates. Thus Rad6 can act in three different modes in vitro depending on the substrate, namely unassisted C-terminus-dependent, unassisted C-terminus-independent and E3-R-assisted C-terminus-independent modes. We also examined the results of removing the C-terminal acidic region of Cdc34 (Ubc3), a ubiquitin-conjugating enzyme closely related to Rad6. Truncation of Cdc34 like that of Rad6 had no effect on activity for beta-casein, beta-lactoglobulin or oxidized RNase in the presence or absence of E3-R.

Excision Adequacy and Characteristics of Impalpable Masses Assessed by Specimen Mammography in a Tetrahedron

Hamartomas of the Breast: Six Novel Diagnostic Features in Three-dimensional Thick Sections

Fifteen hamartomas of the breast have been studied by dissecting microscopy of thick sections at a depth of 1.0 to 2.5 mm, and by conventional 5 microns deep sections. This combined approach has revealed six distinctive features, three in the parenchymal structure and three in the connective tissue components. Ducts (invariably of penetrating or arcuate configuration) and discrete lobules, neither of which are seen in lesions accepted as fibroadenomas, were invariably found. In one-third of hamartomas, up to 10% of the surface area was occupied by Herãti-style nodules composed of concentric rings of epithelium. In the hyaline interlobular connective tissue characteristic drifts of caraway seed-like fibrocytes, encasement of adipocytes by hyaline collagen, or spider-naevus vascular abnormalities were found in approximately half of the mammary hamartomas when examined in thick sections. Hitherto the positive diagnosis of hamartomas of the breast has relied on a combined clinical, radiological and pathological assessment. We suggest that the additional features described here, taken in conjunction with those already known, may facilitate the recognition of mammary hamartomas by histopathological examination alone.

Peritraumatic Dissociation and Posttraumatic Stress in Male Vietnam Theater Veterans

The aim of this study was to determine the reliability and validity of a proposed measure of peritraumatic dissociation and, as part of that effort, to determine the relationship between dissociative experiences during disturbing combat trauma and the subsequent development of posttraumatic stress disorder (PTSD).

Automated Metaphase Finding: an Assessment of the Efficiency of the METAFER2 System in a Routine Mutagenicity Assay

The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparations from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure. METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-stained second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.

Inhibition of Herpes Simplex Virus Replication by Antisense Oligo-2'-O-methylribonucleoside Methylphosphonates

Antisense oligonucleoside methylphosphonates complementary to the 12 nucleotides found at the intron/exon junction of the splice acceptor site of herpes simplex virus type 1 (HSV-1) immediate early mRNAs 4 and 5 were synthesized. The methylphosphonate oligomers contained either 2'-deoxyribose nucleosides, d-OMPs, or 2'O-methylribose nucleosides, mr-OMPs. At 37 degrees C, the affinity of the mr-OMP for a complementary 12-mer RNA target was approximately four times higher than that of the corresponding d-OMP as measured by a constant activity gel electrophoresis mobility shift assay. An mr-OMP whose sequence contained two mismatched bases did not bind to the RNA target under these conditions. The mr-OMP also showed improved ability to inhibit HSV-1 replication in HSV-1 infected Vero cells in culture. Thus the IC50 of the mr-OMP was five times less than that of the d-OMP. No inhibition was observed by the mismatched mr-OMP, and no inhibition of herpes simplex virus type 2 (HSV-2) replication was observed with any of the oligomers. These results demonstrate a direct correlation between oligomer binding affinity and antisense activity in cell culture and suggest that oligo-2'-O-methylribonucleoside methylphosphonates are promising candidates for development of effective antisense reagents.

Intracellular Internalization and Signaling Pathways Triggered by the Large Subunit of HSV-2 Ribonucleotide Reductase (ICP10)

The large subunit of the HSV-2 ribonucleotide reductase (RR) (ICP10) is a chimera consisting of a serine threonine (Ser/Thr) protein kinase domain at the amino terminus and the RR domain at the carboxy terminus. Transformed human cells that constitutively express ICP10 (JHLa1) were stained with anti-LA-1 antibody (recognizes ICP10 amino acids 13-26) and immunogold-conjugated goat anti-rabbit IgG and were examined by electron microscopy. ICP10-associated gold particles were observed on the cell surface and in structures with ultrastructural characteristics of endocytic vesicles, multivesicular bodies, and lysosomes, consistent with endocytic internalization. ICP10 was also associated with the cytoskeleton fraction of JHLa1 cells and, at least in part, it colocalized with actin filaments. This was evidenced by immunoprecipitation of [35S]methionine-labeled cell fractions and immunofluorescent staining of Triton-treated cells with anti-LA-1 antibody and phalloidin. Endocytic localization of gold particles was not seen in cells that constitutively express the ICP10 transmembrane (TM)-deleted mutant p139TM (JHL15). p139TM did not associate with the cytoskeleton and was almost entirely localized within the cytoplasm. raf and Erk evidenced decreased mobility consistent with an activated state in JHLa1, but not JHL15, cells, and chloramphenicol acetyl transferase (CAT) expression from a c-fos/cat hybrid construct was significantly increased in JHLa1 but not JHL15 cells. The data indicate that effector molecules downstream of ras are activated in JHLa1 cells and the ICP10 TM segment plays a critical role in ICP10 intracellular localization and its ability to activate signaling pathways. This behavior is analogous to that of an activated growth factor receptor kinase.

Combined FISH Painting and Harlequin Staining for Cell Cycle-controlled Chromosome Analysis in Human Lymphocytes

We present a simple method that allows scoring of FISH-painted chromosomes exclusively in the first division human lymphocytes. It consists of a combination of FISH with chromosome-specific libraries and a differential sister chromatid staining after BrdU treatment of lymphocyte cultures. The method allows a precise quantification of induced chromosome damage for human biodosimetry.

One of the Two Common Mutations Causing Factor XI Deficiency in Ashkenazi Jews (type II) is Also Prevalent in Iraqi Jews, Who Represent the Ancient Gene Pool of Jews

In recent years four mutations causing factor XI deficiency have been identified in Jews of Ashkenazi (European) origin. Two of them, type II (a nonsense mutation) and type III (a missense mutation), were found to prevail among 125 unrelated Ashkenazi Jews with severe factor XI deficiency. A finding of type II mutation in four unrelated Iraqi-Jewish families raised the possibility that this mutation is also common in Iraqi Jews, who represent the ancient gene pool of the Jews. A molecular-based analysis performed in 1,040 consecutively hospitalized patients disclosed the following results: Among 531 Ashkenazi-Jewish patients, the type II allele frequency was 0.0217 and among 509 Iraqi-Jewish patients, 0.0167 (P = .50). The type III allele frequency in the Ashkenazi-Jewish patients was 0.0254, whereas none of 502 Iraqi-Jewish patients examined had this mutation. These data suggest that the type II mutation was present in Jews already 2.5 millenia ago. The data also indicate that the estimated risk for severe factor XI deficiency in Ashkenazi Jews (due to either genotype) is 0.22% and in Iraqi Jews, 0.03%, and that the estimated risk of heterozygosity in Ashkenazi Jews is 9.0% and in Iraqi Jews, 3.3%. As patients with severe factor XI deficiency are prone to bleeding after injury and patients with partial deficiency may have similar bleeding complications when an additional hemostatic derangement is present, the observed high frequencies should be borne in mind when surgery is planned for individuals belonging to these populations.

Dose-response Effects of Intravenous Clonidine on Stress Response During Induction of Anesthesia in Coronary Artery Bypass Graft Patients

This study was designed to evaluate the dose-response effects of different doses of clonidine on the stress response to laryngoscopy and endotracheal intubation. In a randomized, double-blind study, 48 coronary artery bypass grafting (CABG) patients received 0, 2, 4, or 6 micrograms/kg clonidine as an intravenous (IV) infusion during a 15-min period 30 min prior to induction of anesthesia with etomidate (0.3 mg/kg), fentanyl (5-7 micrograms/kg), and pancuronium (0.1 mg/kg). Sedation was assessed prior to induction of anesthesia. Cardiovascular variables and catecholamine plasma levels were measured at predefined intervals. Additional bolus doses of etomidate and fentanyl for suppression of stress-induced reactions were administered if predefined limits of heart rate and blood pressure were exceeded. Clonidine 4 and 6 micrograms/kg significantly attenuated hemodynamic and adrenergic reactions to stress, reduced pharmacologic interventions, and increased sedation. However, clonidine 6 micrograms/kg was not more effective than 4 micrograms/kg, and clonidine 2 micrograms/kg was equally effective as placebo. We conclude that clonidine 4 micrograms/kg IV is the appropriate dose to attenuate the stress response to laryngoscopy in CABG patients. Side effects limiting the use of IV clonidine were not observed.

[Lymphoepithelial Carcinoma of the Nasolacrimal Duct--a Case Report]

A 59-year-old patient with an extremely rare lymphoepithelioma of the nasolacrimal duct was treated with radiation therapy and chemotherapy.

Antiviral Activity of an Oligo(nucleoside Methylphosphonate) That Targets HSV-1 Immediate-early Pre-mRNA 4,5 is Augmented by Cotreatment with Replication-defective Adenovirus

Replication-defective adenovirus p259A caused a 400-fold increase in the sequence-specific antiherpetic activity of oligo(nucleoside methylphosphonate) (ONMP) IE4,5SA. Herpes simplex virus type 1 (HSV-1) growth was not inhibited in cells exposed to p259A in the absence of IE4,5SA or in cells cotreated with IE4,5SA and heated (10 minutes, 90 degrees C) p259A virus. Fluorescent microscopy of Vero cells treated with BODIPY-conjugated IE4,5SA revealed intracellular localization within endocytic-like vesicles with minimal cytoplasmic and intranuclear distribution. Diffuse staining over the entire cell was observed in cell cotreated with the BODIPY-conjugated IE4,5SA and p259A virus. This effect was not observed in cells cotreated with the BODIPY-conjugated ONMP and heated p259A virus. We interpret these findings to indicate that p259A augments IE4,5SA antiherpetic activity presumably via its ability to increase ONMP uptake and release from endocytic-like vesicles.

Fine Lines

Karinthy's Journey Round My Skull

Detection of Centromeres in Vinblastine- and Radiation-induced Micronuclei of Human Lymphocytes Using FISH with an Alpha Satellite Pancentromeric DNA Probe

Fluorescence in situ hybridisation (FISH) with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in micronuclei of human lymphocytes induced by gamma irradiation and by Vinblastine sulfate. In a cytokinesis-block micro-nucleus assay a dose-dependent increase of micronuclei was detected for both agents. 72-89% of vinblastine-induced micronuclei, but only 7-48% of radiation-induced micronuclei showed centromere-positive fluorescence signals. Vinblastine treatment frequencies of centromere-negative micronuclei did not increase compared to control values, nor did frequencies of centromere-positive micronuclei in irradiated lymphocytes. Since FISH with an alpha satellite DNA probe allows the direct detection of centromeric DNA sequences the spindle damaging or clastogenic effectiveness of a compound can be easily and reliably examined in a cytokinesis-block micronucleus assay in human lymphocytes.

ATP and SH3 Binding Sites in the Protein Kinase of the Large Subunit of Herpes Simplex Virus Type 2 of Ribonucleotide Reductase (ICP10)

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein. It consists of a ribonucleotide reductase and a serine/threonine protein kinase (PK) domain, which has three proline-rich motifs consistent with SH3-binding sites at positions 140, 149, and 396. We used site-directed mutagenesis to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys176 or Lys259 reduced PK activity (5-8-fold) and binding of the 14C-labeled ATP analog rho-fluorosulfonylbenzoyl 5'-adenosine (FSBA) but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu209 (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg2+ or Mn2+ ions, suggesting that Glu209 functions in ion-dependent PK activity. ICP10 bound the adaptor protein Grb2 in vitro. Mutation of the ICP10 proline-rich motifs at positions 396 and 149 reduced Grb2 binding 20- and 2-fold, respectively. Binding was abrogated by mutation of both motifs. Grb2 binding to wild type ICP10 was competed by a peptide for the Grb2 C-terminal SH3 motif, indicating that it involves the Grb2 C-terminal SH3.

Preoperative Alpha2-adrenergic Receptor Agonists Prevent the Deterioration of Renal Function After Cardiac Surgery: Results of a Randomized, Controlled Trial

To evaluate the influence of the alpha2-adrenergic receptor agonist clonidine on creatinine clearance as a measure of renal function.

Herpes Simplex Virus-mediated Activation of Human Immunodeficiency Virus is Inhibited by Oligonucleoside Methylphosphonates That Target Immediate-early MRNAs 1 and 3

IE1 and IE3 mRNAs and their protein products (IE110 and IE175, respectively) were detected in HSV-1-infected U937 cells at 4-15 hours postinfection. In transient expression assays with infectious HIV or an HIV-LTR-directed chloramphenicol acetyltransferase construction (HIV-LTRcat), HSV-1 caused HIV activation (86.7% +/- 6.4% conversion). Electrophoretic mobility shift assays with DNA sequences that encompass the LBP-1 binding site revealed increased levels of DNA-protein complex formation with nuclear extracts from HSV-1 infected as compared with uninfected U937 cells. Novel bands were not seen. HSV-1 mutants respectively deleted in IE110 (dl1403) or IE175 (d120) activated HIV as well as wild-type virus. However, HSV-1-mediated activation was inhibited (26% conversion) by simultaneous treatment with oligonucleoside methylphosphonates (ONMP) that specifically inhibit expression of IE110 (IE1TI) or IE175 (IE3TI). ONMP did not inhibit activation when used individually (83.8% and 67.8% conversion with IETI1 and IE3TI, respectively). Combinations of mutant ONMP that do not inhibit IE110 or IE175 expression did not reduce the levels of HSV-1-mediated activation. These findings suggest that HSV genes IE1 and IE3 can independently activate HIV in monocytic cells and ONMP that target HSV IE genes can be used to inhibit HIV activation.

Genotoxicity of 4-chloro-o-toluidine in Salmonella Typhimurium, Human Lymphocytes and V79 Cells

In the absence of a metabolizing system (S9 mix) 4-chloro-o-toluidine (4-COT) was found to be ineffective in a combination of assays for gene mutations in Salmonella typhimurium, for chromosome aberrations and sister chromatide exchanges in human lymphocytes, and for the induction of spindle disturbances in V79 Chinese hamster cells. In the presence of S9, 4-COT was also ineffective in producing structural or numerical changes in mammalian cells, but the yields of 4-COT induced revertants in S. typhimurium strains TA 100 and TA 98 were about 2-fold higher than those in controls.

Traumatic Arterial Damage After Fine-needle Aspirational Cytology in Mammary Complex Sclerosing Lesions

We retrospectively reviewed 107 consecutive cases of radial scar and complex sclerosing lesions in a 54 month period seeking vascular lesions using archival H & E stained 5 micron thick sections. Eight showed vascular abnormalities, five being false aneurysms which were limited to 62 lesions > 10 mm diameter; no false aneurysm was found in 45 radial scars < or = 10 mm. All cases displayed arterial lesions, but two also showed associated venous fibrosis. Traumatic false aneurysms with loss or severe thinning of the media and intimal, medial or extravascular spindle cell proliferation were found in the arteries of five cases with histological evidence of local reaction to previous fine-needle aspirational biopsy and clinical documentation of aspiration 18 to 121 days before. The diameters of the false aneurysms correlated with the original widths of the arteries. The other lesions with mature intimal fibrosis in arteries or veins lacked such local post-aspiration expansile reactive changes, or a history of fine needle aspiration. We conclude that traumatic false aneurysms found in complex sclerosing lesions seem to be iatrogenic, resulting from fine needle aspiration. Mature fibrosis in veins or arteries are probably unrelated processes.

Role of the Conserved Carboxy-terminal Alpha-helix of Rad6p in Ubiquitination and DNA Repair

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal alpha-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal alpha-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal alpha-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal alpha-helix mutants to different degrees. This indicates that the C-terminal alpha-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.

Chromosome Translocations in Thyroid Tissues from Belarussian Children Exposed to Radioiodine from the Chernobyl Accident, Measured by FISH-painting

Chromosome painting of chromosomes 1, 4 and 12 was performed on metaphase preparations of cultured thyroid cells to analyse the frequency of radiation-induced stable chromosome translocations in papillary thyroid carcinomas from 40 Belarussian children exposed to radioiodine from the Chernobyl accident, and from 31 reference case. As expected, we found the highest translocation frequencies in secondary thyroid tumours after radiotherapy, but there were also high frequencies in tumour tissues as well as in non-tumourous tissues from childhood papillary carcinoma samples from Belarus. Among the Belarussian tumours the cases from the Gomel region exhibited the highest frequency of translocations and five cases lie within the range of frequencies observed in secondary thyroid tumours after radiotherapy. The findings support the assumption that radiation was the principal cause of the tumours in Belarus, but they indicate also that only a minority of the Belarus cases, which have developed papillary carcinomas, were exposed to very high doses of radioiodine.

Quantification of FISH-painted Chromosome Aberrations After Domestic Radon Exposure

Chromosome painting (target chromosomes 1, 4, 12) was performed in peripheral lymphocytes from 25 occupants of nine houses with indoor radon concentrations of 210-3000 Bqm-3. Compared to a control group, the mean frequency of symmetrical translocations of the radon group was slightly but not significantly (p < 0.10) increased. A similar tendency became apparent for a comparison of two groups of subjects with cumulative radon exposures above and below 2800 Bqm-3 y. It is concluded that FISH-based measurements of stable symmetrical translocations should reflect the cumulative radon exposure to haematopoietic compartments such as the red bone marrow rather than to mature blood lymphocytes. Since, however, radon-derived bone marrow doses are low and control frequencies of translocations are very high (about 10-fold higher than the value for conventionally scored dicentrics), the observed relative increase (1.5-fold) of the translocation frequency in blood lymphocytes is too small to discriminate chronic radon exposure from background.

Benign and Malignant Stellate Breast Lesions: Structural Differences in 5 Microns Sections and Thick Tissue Slices

Complex sclerosing lesions have presented a diagnostic problem since they were identified amongst benign breast lesions. Their differentiation from stellate carcinomas may cause serious difficulties for both radiologists and pathologists. In the present study the conventional and thick-slice appearances of 15 complex sclerosing lesions and 15 well differentiated and tubular carcinomas were compared, with the main emphasis on the stellate zone of the lesions. There was a marked morphological difference between the stellate extensions: the majority was formed by epithelial structures in complex sclerosing lesions as opposed to the composition of the extensions of the stellate carcinomas, where fibrovascular tissue dominates. Well differentiated carcinomas originating in complex sclerosing lesions showed an intermediate ratio of epithelial and fibrous stellate extensions. The structural differences may explain the radiomorphological differences of benign and malignant stellate breast lesions. We suggest these structural characteristics ought to be included in the pathomorphologic differential diagnostic features.

New Avenues in 3-D Computerised (stereopathological) Imaging of Breast Cancer (review)

Multimodal methods of three-dimensional (3-D) imaging of breast cancer are described. These involve scanning confocal microscopy, using 50 MHz acoustic or near-infrared images, four-view (tetrahedral) radiography and x-ray projection microscopy. Computerised volume data from these techniques can be used to produce three-dimensional images of tissue ranging from 500 microns to approximately 4 mm in thickness. Preliminary findings indicate that stereoscopic images or 3-D computerised reconstructions are capable of advancing the understanding of the structure of ductal carcinoma in situ, lesions simulating microinvasive breast carcinoma, surgical clearance of high-grade calcifying ductal carcinoma in situ, and the 3-D growth patterns of invasive forms of breast carcinoma. In the future computerised image fusion techniques seem likely to be able to take advantage of multimodal imaging of breast cancers, thus correcting primary imaging artefacts, improving robustness, and combining complementary information. In addition, the use of computerised tetrahedral radiography may change the intraoperative assessment of breast cancers, which mostly depend at present upon subsequent laboratory procedures that take days to perform.

[Alpha 2-adrenoceptor Agonists for the Treatment of Chronic Pain]

The antinociceptive effect of alpha(2)-adrenoceptor agonists is mediated by activation of descending inhibiting noradrenergic systems, which modulates 'wide-dynamic-range' neurones. Furthermore, they inhibit the liberation of substance P and endorphines and activate serotoninergic neurones. Despite this variety of antinociceptive actions, there is still little experience with alpha(2)-adrenoceptor agonists as therapeutic agents for use in chronic pain syndromes. Studies in animals and patients have shown that the transdermal, epidural and intravenous administration of the alpha(2)-adrenoceptor agonist clonidine reduces pain intensity in neuropathic pain syndromes for periods varying from some hours up to 1 month. Patients suffering from lancinating or sharp pain respond best to this therapy. Topically applied clonidine (200-300 microg) relieves hyperalgesia in sympathetically maintained pain. Epidural administration of 300 microg clonidine dissolved in 5 ml NaCl 0.9 % has also been shown to be effective. In patients suffering from cancer pain tolerant to opioids, pain control has proved possible again with combinations of opioids and clonidine. In isolated cases clonidine has been administered epidurally at a dose of 1500 microg/day for almost 5 months without evidence for any histotoxic property of clonidine. Side effects often observed during administration of alpha(2)-adrenoceptor agonists are dry mouth, sedation, hypotension and bradycardia. Therapeutic interventions are usually not required.

[Effectiveness of Autologous Blood Donation in Coronary Surgery--a Retrospective Analysis]

This study was performed to evaluate whether autologous blood donation prior to coronary artery bypass graft surgery (cabg) reduces homologous blood requirements and the risk of homologous blood transfusion.

Biological Monitoring of Hospital Pharmacy Personnel Occupationally Exposed to Cytostatic Drugs: Urinary Excretion and Cytogenetics Studies

For evaluation of the risk borne by hospital pharmacy personnel exposed to antineoplastic agents, the incorporation of cyclophosphamide, ifosfamide, and platinum-containing drugs was quantified by the determination of urinary concentrations. In addition, the induction of micronuclei (MN) and sister-chromatid-exchange (SCE) rates in peripheral blood lymphocytes were studied for correlation with the urinary excretion of cytostatic drugs. Cyclophosphamide and ifosfamide were determined in 24-h urine samples using gas chromatography with electron capture (detection limit 2.5 micrograms/l). Voltammetric analysis enabled the determination of platinum concentrations of 4 ng/l. Heparinized blood (20 ml) was drawn and lymphocytes were cultured for MN and SCE studies. In all, 13 hospital pharmacists and pharmacy technicians regularly involved in the preparation of cytostatic drugs participated in this investigation (7 persons represent a follow-up group). All subjects applied standard safety precautions, including the use of a vertical laminar air-flow hood, protective gowns, and latex gloves. On the day of urine sampling an average of 4,870 mg cyclophosphamide, 5,580 mg ifosfamide, and 504 mg platinum-containing drugs were handled. The excretion of 5 and 9 micrograms cyclophosphamide/l urine was measured in two samples, respectively. An elevated level of urinary platinum was found in one pharmacist (22.3 ng/g creatinine) in comparison with a nonexposed control group. Mean frequencies of MN and SCE did not differ significantly between the drug exposed group and control group. The employees who had incorporated chemotherapeutic agents were part of the follow-up group and, thus, particularly cautious and sensitive to a possible hazard. The results emphasize the necessity of improving personal protection of hospital pharmacy personnel occupationally exposed to cytostatic drugs and support the importance of biological monitoring. In an ongoing project in our department the sources of contamination are being investigated parallel to biological monitoring so as to determine critical situations and improve personal protection.

Degradation Signals for Ubiquitin System Proteolysis in Saccharomyces Cerevisiae

Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes.

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) is Required for Immediate-early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10DeltaPK). ICP10DeltaPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10DeltaPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10DeltaPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.

Technical Report: Automated Classification of First and Second Cycle Metaphases

Based on the technical configuration of the Metafer2 metaphase finder (Metasystems, Altlussheim, Germany), an automated system was developed that is able to discriminate first/M1 or second cycle/M2 metaphases. For an evaluation of the system in fluorescence plus Giemsa stained preparations of human lymphocytes, a learning sample and an independent test sample was used. Between 8 and 14 separated chromosomes per metaphase were sufficient to achieve correct classification rates between 95 and 100%. For practical application the system can be most efficiently used for biodosimetry of human radiation exposure which requires scoring of thousands of metaphases exclusively in M1. An application for selection of M2 for SCE scoring in mutagenicity testing was possible, but does not provide a comparable advantage.

Genetic Heterogeneity in a Prostatic Carcinoma and Associated Prostatic Intraepithelial Neoplasia As Demonstrated by Combined Use of Laser-microdissection, Degenerate Oligonucleotide Primed PCR and Comparative Genomic Hybridization

We combined laser-assisted microdissection from H&E-stained paraffin sections, degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR), and comparative genomic hybridization (CGH) to analyse chromosomal imbalances in small tumour areas consisting of 50-100 cells. This approach was used to investigate intratumour genetic heterogeneity in a case of metastatic prostatic adenocarcinoma and chromosomal changes in areas of prostatic intraepithelial neoplasia (PIN) adjacent to the invasive tumour. In four microdissected invasive tumour areas with different histological patterns (acinar, cribriform, papillary and solid) marked intratumour heterogeneity was found by CGH. Recurrent chromosomal imbalances detected in at least two microdissected tumour areas were gains on 1p32-->p36, 2p22, 3q21, 7, 8q21-->q24, 11q12-->q13, 16p12-->p13, 17, 19 and loss on 16q23. Additional chromosomal changes were found in only one of the microdissected areas (gains on 16q21-->q23, 20q22 and losses on 8p21-->p23, 12p11-->q12, 12q21-->q26, 13q21-->q34, 16q12, and 18q22). In PIN, gains on chromosomes 8q21-->q24 and 17 were found in both samples investigated (low and high grade PIN), while gains on chromosomes 7, 11q, 12q, 16p, and 20q and losses on 2p, 8p21-->p23, 12q were found only in one PIN area. Controls to ensure reliable CGH results consisted in CGH analyses of (i) approximately 80 microdissected normal epithelial cells, which showed no aberrations after DOP-PCR and (ii) larger cell numbers (approximately 10(5) or 10(7) cells) of the primary tumour investigated without DOP-PCR and partially displaying the chromosomal imbalances (gain on 16p12-->p13, losses on 2p25, 8p21-->p23, 12p11-->p12, 12q21-->q26, 18q22) found in the small microdissected areas. Microsatellite and FISH analyses further confirmed our CGH results from microdissected cells. The combined approach of laser-assisted microdissection, DOP-PCR and CGH is suitable to identify early genetic changes in PIN and chromosomal imbalances associated with the particular histological patterns of invasive prostatic adenocarcinoma.

Upper Gastrointestinal Bleeding and Intestinal Perforation Due to Multiple Duodenojejunal Metastases from a Silent Bronchogenic Adenosquamous Carcinoma

Immunohistochemical Localisation of Tenascin in Invasive Ductal Carcinoma of the Breast

The association of TN expression, tumour grading and expression of commonly used histopathologic prognostic factors (p53 and estrogen receptor) was examined in 62 cases of invasive ductal carcinoma of the breast.

Heat-induced Cell Cycle Arrest of Saccharomyces Cerevisiae: Involvement of the RAD6/UBC2 and WSC2 Genes in Its Reversal

The Saccharomyces cerevisiae RAD6 (UBC2 ) gene encodes a ubiquitin-conjugating enzyme that is involved in a wide range of cellular processes including DNA repair, sporulation and N-end rule protein degradation. Under mild heat stress conditions (37-38 degrees C) rad6 null and rad6-149 mutant cells are unable to grow. The molecular basis for this failure to grow is unknown. Here we show that the heat sensitivity of rad6 mutants is not due to cell death but to an inability to progress in the cell cycle. The temperature-induced cell cycle arrest of these mutants is due to a block in a branch of the RAD6 pathway distinct from the DNA repair and the N-end rule protein degradation pathways. Wild-type cells heated to 38 degrees C arrest transiently in the late G1 phase and then resume growth. At 38 degrees C rad6 mutant cells arrest in late G1 but, unlike wild-type cells, are unable to resume cell cycle progression. In both wild-type and in rad6 mutant cells, CLN1 and CLN2 transcript levels fall sharply upon temperature increase. In wild-type cells levels of these transcripts recover rapidly, whereas in the rad6 mutant they recover slowly. As rad6 cells remain arrested even after CLN1 and CLN2 mRNAs regain their preheat stress levels, factors additional to reduced G1 cyclin gene expression must cause the temperature-induced cell cycle block of the mutant. To identify genes involved in the relief of the cell cycle arrest under heat stress, we screened a multicopy yeast genomic library for clones that restore the growth of the rad6-149 mutant. A plasmid was isolated carrying the WSC2 gene, which is closely related to WSC1/SLG1/HCS77, a putative membrane heat sensor. Overexpression of WSC2 reverses the heat-induced cell cycle arrest of rad6-149 but not of rad6 null mutants. Taken together the findings point to the existence of an unidentified heat stress-activated cell cycle checkpoint pathway, which is antagonized by Rad6p by a mechanism also involving Wsc2p.

Follow-up Analysis of Translocation and Dicentric Frequencies, Measured by FISH-chromosome Painting in Breast Cancer Patients After Partial-body Radiotherapy with Little Bone Marrow Exposure

Follow-up translocation and dicentric measurements in blood lymphocytes of five breast cancer patients were performed by FISH using painting probes for chromosomes 1, 4, and 12 simultaneously with a pancentromeric DNA probe, during 14 months after fractionated photon therapy affecting only small areas of the bone marrow (about 5%). The analysis of individual time-courses for translocations and dicentrics revealed a significant temporal decline of the yields with comparable half-times, both for these stable and unstable aberration types in two patients. In three patients, the aberration yields remained fairly unchanged during the observation period. Regarding retrospective biodosimetry for cases with partial-body exposures or large dose inhomogeneity, it follows that even FISH chromosome painting is limited in assessing initial doses correctly in terms of stable translocations.

Tenascin Expression and Angiogenesis in Breast Cancers

The expression and the distribution of tenascin as well as the extent of blood vessel formation (angiogenesis) were investigated in 70 invasive human breast carcinomas. Formalin-fixed, paraffin-embedded specimens were stained with monoclonal antibody against tenascin-C (DAKO and Biogenex). Anti-CD31 antibody (Biogenex), an acknowledged marker of stromal angiogenesis, was used to detect endothelial cells. Tenascin immunostaining was positive in the tumours around the persisting normal ducts, around tumour-cell nests, in the neostroma, in some tumour cells, and it was found in or around vascular channels. Tumour vascularity was assessed by quantitative vascular grading (Chalkley point count) and was related to the localization and intensity of tenascin immunoreactivity. 19 tumours (27.1%) were scored as low, 35 (50%) as medium, and 16 (22.9%) as having a high vascular grade. The positive correlation between the vascular grade and the tenascin immunopositivity in tumour stroma was observed. Our results suggest that tenascin expression may be associated with endothelial cell activation and may play an important role in tumour angiogenesis.

World Health Organization (WHO) and the World Heart Federation (WHF) Pathobiological Determinants of Atherosclerosis in Youth Study (WHO/WHF PBDAY Study) 1986-1996. Histomorphometry and Histochemistry of Atherosclerotic Lesions in Coronary Arteries and the Aorta in a Young Population

The present work is a chapter in an investigation directed by the World Health Organization on the Pathobiological Determinants of Atherosclerosis In Youth (WHO-PBDAY). Our aim was to study the development of atherosclerotic lesions in a young population.

[Inadvertent Potassium Chloride Infusion in an Epidural Catheter]

In a 65 years old male patient 38 cc of a 7.45% potassium chloride-solution was inadvertently infused within 3 hours into an epidural catheter on the first postoperative day. The epidural potassium chloride administration resulted in a paresis and painful paraesthesia of the patient's legs and a level of sensory blockade to TH 11. Furthermore vegetative symptoms like hypertension and tachycardia were observed. For therapy a single bolus of 40 mg dexamethasone was administered intravenously followed by an epidural infusion of sodium chloride 0.9% 99 cc/h for several hours. About 6 hours after the start of infusion all symptoms had disappeared. It is proposed that the use of colour-coded epidural catheter devices and coloured electrolyte solutions as well as infusion-pumps with a larger reservoir that reduce the frequency of syringe changes would be helpful in avoiding such complications.

Dependence of CD8+ T-cell-mediated Suppression of HIV Type 1 on Viral Phenotypes and Mediation of Phenotype-dependent Suppression by Viral Envelope Gene and Not by Beta-chemokines

CD8+ T-cell-mediated HIV-1 suppressive activity has been shown against a number of strains of HIV-1 and HIV-2. In this study using a semiquantitative assay, we showed that CD8+ T cells from seropositive subjects and herpes virus saimiri transformed CD8+ T-cell clones from HIV-1-infected subjects exhibited 5 to 100-fold higher suppressive activity against slow replicating nonsyncytia-inducing strains (Slow/NSI) as compared to fast replicating syncytia-inducing strains (Fast/SI) of HIV-1. Such differential suppressive activity was not due to beta-chemokines as evidenced by the lack of blocking activity of antibodies to RANTES, MIP-1beta, and MIP-1alpha on the antiviral activities of CD8+ T cells. Moreover, there was no correlation between the level of CD8+ T-cell suppression and the level of these beta-chemokines in culture supernatant. Results from the CD8+ T-cell-mediated suppressive activity against two molecular cloned virus ME1 (Slow/NSI), ME46 (Fast/SI), and their interstrain recombinants indicate that the envelope gene carries a major genetic determinant responsible for this phenotypic-dependent differential suppressive activity.

A Novel Human Gene Similar to the Protein Kinase (PK) Coding Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Codes for a Serine-threonine PK and is Expressed in Melanoma Cells

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein that contains a serine-threonine protein kinase (PK) activity (Nelson, J. W., Zhu, J. , Smith, C. C., Kulka, M., and Aurelian, L. (1996) J. Biol. Chem. 271, 17021-17027). Phylogenetic analyses indicated that ICP10 PK belongs to a distinct subfamily of growth factor receptor serine-threonine PKs that are characterized by their ability to function with a limited number of conserved catalytic motifs (Hunter, J. C. R., Smith, C. C., and Aurelian, L. (1995) Int. J. Onc. 7, 515-522). Here, we report the isolation and characterization of a novel gene, designated H11, that contains an open reading frame of 588 nucleotides, which encodes a protein similar to ICP10 PK. The H11 protein has Mn(2+)-dependent serine-threonine-specific PK activity as determined with a GST-H11 fusion protein and by immununocomplex PK/immunoblotting assays of 293 cells transfected with a H11 eukaryotic expression vector. PK activity is ablated by mutation of Lys(113) within the presumtive catalytic motif II (invariant Lys). 293 cells stably transfected with H11 acquire anchorage-independent growth. Endogenous H11 RNA and the H11 phosphoprotein are expressed in melanoma cell lines and primary melanoma tissues at levels higher than in normal melanocytes and in benign nevi. Melanoma cell proliferation is inhibited by treatment with antisense oligonucleotides that inhibit H11 translation, suggesting that H11 expression is associated with cell growth.

Changes of Beta-adrenergic Signaling in Compensated Human Cardiac Hypertrophy Depend on the Underlying Disease

In human heart failure, desensitization of the beta-adrenergic signal transduction has been reported to be one of the main pathophysiological alterations. However, data on the beta-adrenergic system in human compensated cardiac hypertrophy are very limited. Therefore, we studied the myocardial beta-adrenergic signaling in patients suffering from hypertrophic obstructive cardiomyopathy (HOCM, n = 9) or from aortic valve stenosis (AoSt, n = 8). beta-Adrenoceptor density determined by [(125)I]iodocyanopindolol binding was reduced in HOCM and AoSt compared with nonhypertrophied, nonfailing myocardium (NF) of seven organ donors. In HOCM the protein expression of stimulatory G protein alpha-subunit (G(s)alpha) measured by immunoblotting was unchanged, whereas the inhibitory G protein alpha-subunit (Galpha(i-2)) was increased. In contrast, in AoSt, Galpha(i-2) protein was unchanged, but G(s)alpha protein was increased. Adenylyl cyclase stimulation by isoproterenol was reduced in HOCM but not in AoSt. Plasma catecholamine levels were normal in all patients. In conclusion, both forms of hypertrophy are associated with beta-adrenoceptor downregulation but with different changes at the G protein level that occur before symptomatic heart failure due to progressive dilatation of the left ventricle develops and are not due to elevated plasma catecholamine levels.

Human Immunodeficiency Virus Type 1 Shedding Pattern in Semen Correlates with the Compartmentalization of Viral Quasi Species Between Blood and Semen

High levels of human immunodeficiency virus (HIV) type 1 have been detected in semen at all stages of disease. However, it is not clear whether HIV-1 is shed in semen continuously or intermittently. In a prospective longitudinal study, viral RNA was measured weekly for 10 weeks in semen and blood of HIV-seropositive subjects. Results showed three different patterns of HIV-1 shedding in semen: none (28%), continuous (28%), and intermittent (44%). In contrast, there was no change in blood plasma virus load during the study period. Phylogenetic analysis of the envelope sequences of HIV-1 RNA in semen and blood revealed distinct virus populations in semen and blood of intermittent shedders but similar virus populations in the semen and blood of continuous shedder. These results indicate for the first time that HIV-1 is shed primarily in an intermittent manner and that shedding patterns of HIV-1 in semen are related to compartmentalization of HIV-1 between semen and blood.

Coronary Artery Plaque Disruption As Cause of Acute Myocardial Infarction During Cesarean Section with Spinal Anesthesia

A 31-year-old parturient delivered twins at 35 weeks' gestation by cesarean section with spinal anesthesia. Following anesthesia induction, hypotension and bradycardia occurred, and were immediately treated with theodrenaline plus cafedrin (Akrinor) and atropine. Blood pressure and heart rate increased to 180/100 mmHg and 140 beats per minute, respectively. Several minutes later, the patient developed a myocardial infarction (MI) that she survived after intensive care treatment without sequelae. Although the coronary angiography showed normal coronary vessels, an intravascular ultrasound study demonstrated an atheroma in the left main coronary artery with ruptured fibrous cap. Laboratory screening for risk factors of coronary artery disease (CAD) showed hypercholesterinemia, increased factor VII activity, and hyperfibrogenemia. Angiographically normal coronary vessels are frequently found in pregnant patients who suffered MI. In these patients, coronary spasms have been discussed as the major mechanism of disease. Our case demonstrates that a significant CAD may be present despite angiographically normal findings. Plaque rupture was triggered by hypertension and led to MI as the first symptom of disease. On the basis of these findings, we believe that MI during pregnancy is more often caused by plaque rupture than may be expected, according to the current literature.

Degradation Signals Recognized by the Ubc6p-Ubc7p Ubiquitin-conjugating Enzyme Pair

Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of beta-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Delta and sec61-2) did not inhibit the degradation of the beta-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p.

Primary Intimal Type Leiomyosarcoma with Rhabdomyosarcomatous Differentiation of the Thoracic Aorta

Modified Adenovirus Penton Base Protein (UTARVE) As a Non-replicating Vector for Delivery of Antisense Oligonucleotides with Antiviral And/or Antineoplastic Activity

Antisense oligonucleotides that selectively inhibit gene expression are a genetic approach for disease treatment and prevention. However, their use as therapeutic agents is complicated by their low rate of transport across cellular membranes and their sequestration within endocytic-like vesicles. We report that the adenovirus type-2 penton base protein modified to include the fusogenic peptide of the influenza virus hemagglutinin protein is a non-replicating vector (designated UTARVE) that improves delivery of antisense oligonucleotides. Approximately 10-18% of the input vector was internalized by A549 and HeLa cells as determined by immunoblotting. It was cleared by proteolysis within 48 h. The vector had endosome disruptive potential as evidenced by erythrocyte lysis activity at low pH and a primarily diffuse cytoplasmic distribution in treated cells. Despite concentration and time-dependent cell detachment, UTARVE was not cytotoxic in the dye release assay. We used R1T1, an antisense oligonucleotide that inhibits expression of the multifunctional herpes simplex virus type-2 (HSV-2) R1 protein, HSV-2 growth and the proliferation of R1 PK transformed cells to examine vector-mediated delivery. Conjugated FITC-labeled R1T1 was rapidly (15-30 min) internalized by all cells treated at low (80 nM) concentration and the oligomer was intracellularly dissociated from the vector. This compares to 65-83% of cells internalizing the unconjugated R1T1 when treated for 24 h. In antiviral assays, the IC50 and time required to inhibit HSV-2 growth were significantly lower for the conjugated (2 nM; 30 min) as compared to unconjugated (100 nM; 24 h) R1T1. The data indicate that the bioavailability and biological activity of R1T1 were significantly increased by its delivery with UTARVE.

Tenascin Expression in Primary and Recurrent Breast Carcinomas and the Effect of Tenascin on Breast Tumor Cell Cultures

Tenascin is generally classified as an anti-adhesive protein. Many cells do not adhere to tenascin or if they adhere they do not spread. In this study we analysed the stromal expression of tenascin-C in primary, second primary and recurrent breast carcinomas and the ability of tenascin-C to stimulate the focal adhesion plaques in MDA-MB-435 breast carcinoma cell line. To assess the tenascin-C expression formalin-fixed, paraffin-embedded specimens of 20 specially selected breast carcinomas and their recurrences (14) or a second primary breast cancer of the same patient (6) were examined with immunohistochemical methods. We also studied the effect of tenascin-C on focal adhesion plaques added to MDA-MB-435 breast carcinoma cell line. During a median 2,9-year patient follow up 14 local recurrences and 6-second primary breast carcinomas developed in the 20 patients. In 3 cases a second recurrence occurred. The presence of tenascin in tumor cells, in the proliferating and some normal ducts, near to the tumor cell nests, in the stroma and in ductal carcinoma in situ component of the invasive carcinoma may suggest the role of tenascin played in tumor cell migration. Soluble tenascin added to the cell culture had minimal or no effect on focal adhesion plaques. Tenascin only seems not to be of prognostic value in predicting the local recurrence of breast cancer.

Leiomyosarcomas of Great Vessels

Sarcomas of the great vessels are rare. Altogether 400 such cases have been described in the aorta, the pulmonary artery, and inferior vena cava. The clinical symptoms are generally related to embolic phenomena, aneurysm formation, and widespread metastases, especially to bones. With improved diagnostic modalities more cases are diagnosed and treated surgically. Resection of the tumor may prolong the patient s life. In this paper authors present two cases of such rare sarcomas. In our first case a tumor has developed in the thoracic aorta with symptoms of imminent aortic dissection. The tumorous nature of the lesion was revealed only histologically, since neither the operation, nor macroscopic picture gave any clue to its tumorous nature. The second case was a male patient with a huge retroperitoneal tumor arising from the inferior vena cava, which was clinically suspected to be a carcinomaarising in the adrenal gland.

Chromosomal Changes During Development and Progression of Prostate Adenocarcinomas

Chromosomal copy number changes were investigated in 16 prostate carcinomas, 12 prostatic intraepithelial neoplasias (PIN; 4 low-grade and 8 high-grade) adjacent to the invasive tumour areas, and 5 regional lymph node metastases. For this purpose, comparative genomic hybridization (CGH) was performed and a copy number karyotype for each histomorphological entity was created. CGH on microdissected cells from non-neoplastic glands was carried out on 3 different cases to demonstrate the reliability of the overall procedure. None of the non-neoplastic tissue samples revealed chromosome copy number changes. In PIN areas, chromosomal imbalances were detected on chromosomes 7, 8q, Xq (gains), and on 4q, 5q, 8p, 13q and 18q (losses). In the primary tumours, recurrent (at least 25% of cases) gains on chromosomes 12q and 15q, and losses on 2q, 4q, 5q, Xq, 13q and 18q became apparent. Losses on 8p and 6q as well as gains on 8q and of chromosome 7 were also detected at lower frequencies than previously reported. The pooled CGH data from the primary carcinomas revealed a novel region of chromosomal loss on 4q which is also frequently affected in other tumour entities like oesophageal adenocarcinomas and is supposed to harbour a new tumour suppressor gene. Gains on chromosome 9q and of chromosome 16 and loss on chromosome 13q were observed as common aberrations in metastases and primary tumours. These CGH results indicate an accumulation of chromosomal imbalances during the PIN-carcinoma-metastasis sequence and an early origin of tumour-specific aberrations in PIN areas.

A Novel Gene Expressed in Human Keratinocytes with Long-term in Vitro Growth Potential is Required for Cell Growth

The herpes simplex virus large subunit of ribonucleotide reductase differs from its counterparts in eukaryotic and prokaryotic cells and in other viruses in that it contains a unique domain that codes for a distinct serine-threonine protein kinase that activates the Ras/MEK/MAPK mitogenic pathway and is required for virus growth. Previous studies suggested that ribonucleotide reductase protein kinase was co-opted from a cellular gene. Cellular genes similar to ribonucleotide reductase protein kinase were not cloned, however, and their function is unknown. Here we report that a novel gene (H11) that codes for a protein similar to herpes simplex virus 2 ribonucleotide reductase protein kinase, is expressed in skin tissues, cultured keratinocytes, and the keratinocyte cell line A431. The protein is phosphorylated and it associates with the plasma membrane. H11 is expressed in keratinocytes with long-term in vitro growth potential and is coexpressed with high levels of adhesion molecules involved in signal transduction, such as beta1 integrin. Antisense oligonucleotides that inhibit H11 expression inhibit DNA synthesis and keratinocyte proliferation, suggesting that H11 expression is required for cell growth.

A Growth and Latency Compromised Herpes Simplex Virus Type 2 Mutant (ICP10DeltaPK) Has Prophylactic and Therapeutic Protective Activity in Guinea Pigs

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10DeltaPK) protects from fatal HSV-2 challenge in the mouse model (Aurelian L, Kokuba H, Smith CC. Vaccine potential of a Herpes Simplex Virus type 2 mutant deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10). Vaccine 1999;17:1951-1963). Here we report the results of our studies with ICP10DeltaPK in the guinea pig model of recurrent HSV-2 disease. ICP10DeltaPK was also compromised for growth and disease causation in this model. It was not isolated from latently infected ganglia by explant co-cultivation. The proportions of latently infected ganglia were significantly lower for ICP10DeltaPK than HSV-2 [3/25 (12%) and 7/10 (70%), respectively]. Similar results were obtained for the levels of viral DNA (8 x 10(3) and 2 x 10(5) molecules/ganglion for ICP10DeltaPK and HSV-2, respectively]. ICP10DeltaPK immunization caused a significant (P< or = 0.001) decrease in the proportion of animals with primary [1/14 (6%) and 16/16 (100%) for ICP10DeltaPK and PBS, respectively) and recurrent [1/14 (6%) and 11/14 (79%) for ICP10DeltaPK and PBS, respectively) HSV-2 skin lesions. It also protected from genital HSV-2 disease [1/10 and 10/10 for ICP10DeltaPK and PBS, respectively] and decreased the severity of the lesions in both models. Quantitative PCR (Q-PCR) with primers that distinguish between HSV-2 and ICP10DeltaPK indicated that immunization reduced the proportion of ganglia positive for HSV-2 DNA [8/25 (32%) and 7/10 (70%) for ICP10DeltaPK and PBS, respectively) and its levels [3 x 10(3) and 2 x 10(5) molecules/ganglion for ICP10DeltaPK and PBS, respectively]. The proportion of HSV-2 infected animals with recurrent disease was also significantly (P < or = 0.001) decreased by immunization with ICP10DeltaPK [1/15 (7%) and 11/14 (79%) with recurrent disease for ICP10DeltaPK and PBS, respectively], suggesting that ICP10DeltaPK has prophylactic and therapeutic activity in the guinea pig.

[Are There Certified Indications for the Use of Antithrombin III in Intensive Care]

The serine-protease-inhibitor antithrombin III (AT III) has often been recommended for the therapy of septic patients as it provides anticoagulant and antiinflammatory actions. In animal studies the prophylactic treatment with AT III in a dose > 250 U/kg prevented the development of disseminated intravascular coagulopathy and vital organ dysfunction during sepsis and lowered the mortality rate. In clinical studies with septic patients therapy usually was started several hours after the start of the disease in dosages much lower than those used in animal studies. In these patients AT III-therapy improved laboratory changes of disseminated intravascular coagulopathy but was unable to lower the mortality rate. Hereditary AT III deficiency, lack of heparin effect due to low AT III levels, disseminated intravascular coagulation disorders are indications for the use of AT III while beneficial effects of AT III in patients suffering from SIRS, sepsis or septic shock have not yet been demonstrated.

[Myocardial Infarction During Pregnancy]

Up to now 136 cases of myocardial infarction during pregnancy have been reported, and angiography revealed normal findings in 47%. In these cases coronary spasms have been discussed as the major mechanism of the disease. In isolated cases coronary artery dissection may also present with a normal coronary angiography. The case of a 31-year-old pregnant women who developed myocardial infarction during a caesarean section under spinal anaesthesia gives rise to the assumption that an early stage of coronary artery disease may be the third cause that has to be considered. Probably as a consequence of phases of tachycardia and hypertension during the course of anaesthesia, the patient developed a myocardial infarction that she survived without sequelae. While coronary angiography showed normal coronary vessels, an intravascular ultrasound study (IVUS) demonstrated an atheroma in the left main coronary artery with a ruptured fibrous cap. Laboratory screening for risk factors of coronary artery disease (CAD) showed hypercholesterinemia, increased factor VII activity and hyperfibrinogenemia. Platelet aggregation was not inhibited by acetylsalicylic acid. It was pointed out recently that even in asymptomatic patients, plaques may be present in coronary vessels indicating an early stage of CAD that cannot be diagnosed by angiography. Plaque rupture is often triggered by hypertension and may lead to myocardial infarction, instable angina pectoris, or sudden ischemic death. As IVUS is a new diagnostic tool that allows diagnoses of even early stages of CAD we believe that myocardial infarction during pregnancy is more often caused by plaque rupture than may be expected according to the current literature.

Technical Report: Application of the Metafer2 Fluorescence Scanning System for the Analysis of Radiation-induced Chromosome Aberrations Measured by FISH-chromosome Painting

The Metafer2 fluorescence scanning system was used for routine analysis of radiation-induced exchange aberrations measured by fluorescence in situ hybridisation (FISH) chromosome painting in human peripheral lymphocytes. The system enables a rapid and unbiased fully-automated finding and image acquisition of fluorescently stained metaphase spreads. The chromosome aberration analysis is performed interactively from stored digitised processed gallery images, presented on a screen. Appropriate software image filters are available to further improve these pictures by background correction, noise reduction and fluorescence signal enhancement. Data sets generated by computer-assisted and manual scoring of radiation-induced reciprocal translocations (2B) and total 2B (2B+related 'one-way' types) or complete dicentrics (2A) and total 2A (2A+related 'one-ways') involving painted target chromosomes 2, 3 or 4 were compared and no significant differences were found.A linear-quadratic dose-response curve for total translocations (2B+'one-ways'+complex-derived types) based on computer-assisted analysis of 27,741 metaphases with chromosome 4 painting was compared to a curve obtained earlier for manually scored translocations in a set of target chromosomes 1, 4 and 12. After extrapolation to the whole genome, no significant difference between both curves was found. From our results it can be derived that computer-assisted aberration analysis using the Metafer2 system is a reliable alternative to manual analysis. Since time saving for computer-assisted translocation analysis is about 50% compared to manual scoring, this system is highly promising for a practical application in retrospective biodosimetry of human radiation exposure.

Leiomyosarcoma of the Female Breast

Leiomyosarcomas of the breast are rare tumors. Less than 15 such cases have been reported in the literature so far. In this paper authors describe a case of leiomyosarcoma of a female breast presenting as a firm lobulated mass, mimicking a phylloid tumor radiographically. By fine needle aspiration biopsy, on the smears discohesive malignant looking cells were conclusive to a poorly differentiated invasive ductal carcinoma of the breast. The mastectomy specimen contained a lobulated mass, microscopically showing a partly epithelioid spindle cell tumor, immunoreactive for vimentin, desmin, smooth muscle actin antibodies, and negative for epithelial markers, hormone and growth factor receptors. Axillary lymph nodes were free of tumor. A primary leiomyosarcoma of the breast was diagnosed.

[Prevention of "post-sevoflurane Delirium" with Midazolam]

In a randomized double-blind placebo-controlled trial in children 2-7 years of age, we investigated the effect of a single prophylactic midazolam bolus (0.1 mg/kg b.w.) prior to the termination of anaesthesia, on the incidence and severity of agitation occurring after sevoflurane administration. Compared to the placebo group, midazolam prophylaxis significantly decreased the incidence of postanaesthetic delirium. However, the incidence of severe agitation requiring treatment was not different between the groups (placebo: n = 6; midazolam: n = 4). The mean severity of agitation was significantly lower in patients with midazolam prophylaxis. When midazolam was administered for the treatment of severe agitation it reduced the severity but did not abolish agitation. All patients were discharged from the recovery room after the 2 h observation period. From our study we conclude that a small prophylactic midazolam bolus is able to reduce the incidence and severity of agitation after sevoflurane anesthesia in some patients but is insufficiently effective in patients with severe agitation. Thus, the prophylactic administration of midazolam extenuates but does not solve the problem of post-sevoflurane agitation.

Clonidine Prevents Sevoflurane-induced Agitation in Children

In a double-blinded trial, 40 male children (age 2-7 yr) undergoing circumcision were randomly assigned to receive clonidine 2 microg/kg IV or placebo after anesthetic induction. For induction and maintenance of anesthesia, we used sevoflurane as the sole anesthetic. For pain treatment, a penile block was performed before surgery. After surgery the incidence and severity of agitation was measured during an observation period of 2 h. Severe agitation was treated with midazolam. In 16 placebo and 2 clonidine-treated patients agitation was observed (P < 0.001). In 6 patients of the Placebo group, agitation was graded as severe, whereas none of the patients in the Clonidine group developed severe agitation (P = 0.02). During the postoperative period heart rate and blood pressure were significantly decreased in clonidine treated patients (P < 0.05). We conclude that clonidine effectively prevents agitation after sevoflurane anesthesia. IMPLICATIONS: The recovery from sevoflurane anesthesia may be complicated by the presence of agitation in pediatric patients. Clonidine 2 microg/kg IV after anesthetic induction effectively reduces the incidence of agitation without resulting in clinically relevant bradycardia and hypotension.

Mast Cells and Nitric Oxide: Control of Production, Mechanisms of Response

Mast cells are involved in numerous activities ranging from control of the vasculature, to tissue injury and repair, allergic inflammation and host defences. They synthesize and secrete a variety of mediators, activating and modulating the functions of nearby cells and initiating complex physiological changes. Interestingly, NO produced by mast cells and/or other cells in the microenvironment appears to regulate these diverse roles. This review outlines some of the pathways central to the production of NO by mast cells and identifies many of the tightly controlled regulatory mechanisms involved. Several cofactors and regulatory elements are involved in NO production, and these act at transcriptional and post-translational sites. Their involvement in NO production will be outlined and the possibility that these pathways are critically important in mast cell functions will be discussed. The effects of NO on mast cell functions such as adhesion, activation and mediator secretion will be examined with a focus on molecular mechanisms by which NO modifies intracellular signalling pathways dependent or independent of cGMP and soluble guanylate cyclase. The possibility that NO regulates mast cell function through effects on selected ion channels will be discussed. Metabolic products of NO including peroxynitrite and other reactive species may be the critical elements that affect the actions of NO on mast cell functions. Further understanding of the actions of NO on mast cell activities may uncover novel strategies to modulate inflammatory conditions.

Regulation of CD8 Expression in Mast Cells by Exogenous or Endogenous Nitric Oxide

We recently reported a novel CD8 molecule on rat alveolar macrophages and peritoneal mast cells (PMC). However, little is known about the regulation of CD8 expression and function on these cells. We investigated the regulation of CD8 expression on PMC by NO, because NO can regulate inflammatory responses and also because anti-CD8 Ab stimulates inducible NO synthase and NO production by PMC and alveolar macrophages. Ligation of CD8alpha on PMC with Ab (OX8) induced CD8alpha mRNA expression after 3-6 h, and flow cytometry demonstrated that OX8 treatment increased CD8alpha protein expression compared with PMC treated with isotype control IgG1. To test whether NO mediates the up-regulation of CD8alpha, we used the NO donor S-nitrosoglutathione (500 microM) and NO synthase inhibitors (N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine methyl ester; 100 microM). S-nitrosoglutathione up-regulated both mRNA and protein expression of CD8alpha in PMC compared with that in sham-treated cells, while NO synthase inhibitors down-regulated OX8 Ab-induced CD8alpha expression. To investigate how NO regulates CD8 expression on PMC, we examined the cGMP-dependent pathway using 8-bromo-cGMP (2 mM) and the guanylate cyclase inhibitor, 1H-oxadiazoloquinoxalin-1-one (20 microM). 8-Bromo-cGMP up-regulated CD8 expression, whereas 1H-oxadiazoloquinoxalin-1-one down-regulated its expression. Thus, ligation of CD8 up-regulates CD8 expression on PMC, a response mediated at least in part by NO through a cGMP-dependent pathway. The significance of this up-regulation of CD8alpha on mast cells (MC) is unclear, but since ligation of CD8 on MC with OX8 Ab can alter gene expression and mediator secretion, up-regulation of CD8 may enhance the MC response to natural ligation of this novel form of CD8.

Expression and Functional Characterization of CFTR in Mast Cells

Mast cell activation requires Cl(-) flux, which maintains the driving force for entry of extracellular calcium and initiates release of mediators such as histamine. However, chloride channel expression in mast cells has been poorly understood. For the first time, reverse transcriptase-polymerase chain reaction shows that rat-cultured mast cells (RCMC) and peritoneal mast cells (PMC) contain mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR), an important chloride channel. Immunostaining with an anti-CFTR antibody indicates expression of CFTR in PMC and RCMC. Mast cell CFTR is a functional Cl(-) channel because it is capable of mediating Cl(-) flux in response to elevated cAMP. An inhibitor of CFTR-dependent Cl(-) flux, diphenylamine-2-carboxylate down-regulates mast cell mediator release. These results show that rat mast cells express a functional CFTR, which might be important in mediator release.

A Polymerase Chain Reaction-based Method for the Detection of Hepatitis A Virus in Produce and Shellfish

Outbreaks of gastroenteritis that are suspected to be of viral origin are on the rise. Thus, there is a need for regulatory agencies entrusted with food safety to develop adequate techniques for the detection of viruses in foods. We have established a general procedure for the detection of hepatitis A virus (HAV) in shellfish that, with minor modifications, is also applicable to fresh produce such as cilantro. Total RNA was isolated from shellfish or cilantro, followed by isolation of poly(A)-containing RNA. Because HAV genomic RNA contains a poly(A) tail, the isolation of poly(A)-containing RNA also enriches HAV genomic RNA. Reverse transcription was used to convert the RNA to cDNA, and then amplification was carried out by polymerase chain reaction (PCR). Reamplification with internal primers was used to improve the quality and the quantity of amplified DNA, allowing for post-PCR analysis such as sequence identification of the viral strain. With this procedure, multiple samples could be analyzed in four working days by a single trained individual. The nominal sensitivity of detection of the procedure was 0.15 TCID50 (50% tissue culture infective dose) per 0.62 g of tissue with a test virus. The direct RNA isolation protocol avoided pitfalls associated with whole-virus purification procedures by replacing virus precipitation steps involving polyethylene glycol and Procipitate with phenol extraction. The method is straightforward and reliable. We successfully used this procedure to detect naturally occurring HAV in clams involved in a gastroenteritis outbreak, as well as in cilantro artificially contaminated with a test virus.

Difficult Airway Management in a Patient with Severe Aortic Stenosis, Coronary Artery Disease, and Heart Failure

We report a patient with aortic stenosis, coronary artery disease, and heart failure in whom awake fiberoptic intubation was performed because the patient's mouth opening was insufficient as a result of disc prolapse of the mandibular joint. We planned to perform this procedure during conscious sedation, spontaneous respiration, and with stable hemodynamic parameters (no tachycardia, hypertension, or hypotension). After oral premedication with dikaliumclorazepate, the patient received clonidine. Fiberoptic intubation was performed while the patient breathed spontaneously. Throughout the procedure, no adverse events occurred. Clonidine is a valuable adjunct for conscious sedation and can be used safely for sympathicolysis, even in patients with aortic stenosis.

Characteristics of Nasal T/NK-cell Lymphoma Among Brazilians

Nasal T/NK-cell lymphomas are highly associated with Epstein-Barr virus (EBV). They are more frequent in Asia than in Western countries. In Central and South America there are few studies about nasal T/NK-cell lymphoma and they have shown a strong predominance of this phenotype in Native American descents, supporting the hypothesis of a racial predisposition for the disease. We studied the lymphomas involving midline facial region at a Brazilian institution. T/NK cell lymphomas (16/25) were more frequently found compared to B lymphomas (9 cases, all B large cell). T/NK cell lymphomas involved predominantly the nasal region. Histologically they showed angioinvasion and necrosis. All of them were positive for CD3 and CD56 and showed numerous tumor cells labeled by EBER-1. Although disease was localized in 61% at diagnosis, there was no tendency to cure. The racial distribution of patients with T/NK-cell phenotype was similar to that found in B-cell lymphomas. EBV was more frequently found in adenoids than in palatine tonsils. In inflammatory lesions of the nasal and palatal regions EBV was not found. In the present study the relative frequency of T/NK versus B cell sinonasal lymphomas was high and similar to that observed in other Latin American countries. However, there was not any racial association with T/NK-cell phenotype and the tumor showed an agressive behavior similar to that reported in Asia. The high frequency of EBV-positive lymphocytes in nasopharyngeal lymphoid tissue (adenoids) suggests that they could serve as a reservoir for the virus.

Extracellular Matrix Components in Breast Carcinomas

A malignant process interferes with the normal 'programme' of extracellular matrix biosynthesis and can modify extensively the structure and composition of the matrix. This effect appears to be attributable to several processes such as direct production of some selected matrix macromolecules by malignant cells or indirectly by the production of factors by malignant cells interfering with the regulation of normal matrix production. Other possibilities may also exist, such as the direct action of an environmental carcinogen on otherwise normal mesenchymal cells. The result is a more or less profound modification of tissue structure and composition with possible feedback effects on the malignant process. Some examples will be discussed such as elastin production by some tumours as well as the biosynthesis of some other selected matrix macromolecules as tenascin and osteopontin by breast tumours. Although the detailed mechanisms of these specific matrix productions is not yet completely elucidated, the rapidly increasing knowledge on the regulation of specific matrix production process and deranged matrix production might represent a new area of crosstalk between cancer research and matrix biology.

Psychological Reactions to Terrorist Attacks: Findings from the National Study of Americans' Reactions to September 11

The terrorist attacks of September 11, 2001, represent an unprecedented exposure to trauma in the United States.

[Role of Osteopontin in the Formation of Microcalcifications in Breast Cancer]

Osteopontin, a non-collagen, Ca-binding phosphoprotein, plays roles in both physiological and pathological mineralisation processes. Its expression may be severely altered in malignant tumours.

Mast Cells Express Chloride Channels of the ClC Family

The expression of specific Cl- channels in mast cells (MC) is poorly understood. Because the largest and most ubiquitously expressed family of Cl- channels is the ClC, we studied MC mRNA and protein expression for members of the ClC family.

Heat Balance Limits in Football Uniforms How Different Uniform Ensembles Alter the Equation

Football season becomes a danger zone when warm weather collides with the need for protective gear. Researchers determined critical heat balance limits in non-heat-acclimatized men who wore various football uniform ensembles. The air temperature and humidity limits that they identified are helpful in implementing strategies to prevent heat-related illness.

[Delayed Negative Pressure Pulmonary Edema]

A young athletic male adult (smoker) developed a pulmonary edema 30 min after the end of anaesthesia. Extubation was complicated by a laryngospasm. After artificial ventilation for 12 h the patient recovered completely.A negative pressure pulmonary edema (NPPE) develops after deep inspiratory efforts with an occluded airway. Such a maneuver leads to negative intrapleural pressures of -50 to -100 mmHg. This pressure gradient causes damage to the pulmonary capillaries, a transcapillary volume shift into the interstitium,and hemodynamic changes that increase the intrapulmonary blood volume. As a NPPE may occur with a delay of up to 1 h it is crucial to provide adequate monitoring for patients at risk. Symptomatic therapy usually leads to complete recovery within several hours.

Fibrinolytic Activity and Bleeding After Cardiac Surgery with Cardiopulmonary Bypass and Low-dose Aprotinin Therapy

Low-dose aprotinin inhibits hyperfibrinolysis in cardiac surgery. However, excessive postoperative bleeding and increased fibrinolysis may occur despite low dose-aprotinin administration. We investigated (i) whether fibrinolytic activity significantly rises under low-dose aprotinin administration, and (ii) whether this is associated with excessive postoperative bleeding (> or = 1000 ml/24 h). In a prospective single-blind trial, 120 consecutive patients were randomized to receive 280 mg aprotinin or no aprotinin before skin incision. D-dimer levels increased significantly to the end of surgery, reaching higher levels in the control group. The risk for excessive bleeding was lower in the aprotinin group (12 versus 37%, = 0.001). Fifteen minutes after heparin reversal, patients were at risk for excessive bleeding, when enhanced fibrinolysis was documented (aprotinin group, D-dimer > or = 1.0 micro g/ml, odds ratio = 9.1, = 0.047; control group, D-dimer > or = 3.0 micro g/ml, odds ratio = 4.6, = 0.014). Ninety-seven per cent of the aprotinin group and 81% of control group patients had no excessive bleeding when the D-dimer plasma level was below these values. We conclude that (i) fibrinolytic activity significantly rises under low-dose aprotinin administration, and (ii) plasma D-dimer levels at end of surgery may help to identify patients who are unlikely to develop excessive postoperative bleeding.

The Dynamic and Complex Role of Mast Cells in Allergic Disease

Mast cells (MCs) are found widely distributed in tissues and contribute to regulation of inflammatory responses and ongoing modulation of the tissues. Although MCs are important in a variety of processes, including innate immunity, their role in allergic disease has received increasing attention in the past decade. MCs are located throughout the human body and, upon allergen exposure, they are stimulated via the immunoglobulin E (IgE) receptor (Fc(epsilon)RI) to release several pro-inflammatory mediators such as tumor necrosis factor (TNF), reactive oxygen species such as nitric oxide (NO), proteases, and lipid-derived mediators. However, we now recognize that MCs can be activated by a variety of mechanisms and that mediator release is a consequence of several intra- and extracellular signals. Some of these mechanisms, such as Fc receptor aggregation and proteinase-activated receptor (PAR)-mediated activation facilitate and augment local inflammatory responses. Other mechanisms, such as interferon gamma (IFN-gamma) induction of NO, may inhibit MC function and downregulate inflammatory responses. Increased understanding of these complex pathways has encouraged the development of therapies for allergic inflammation that target specific MC functions and mediators. Some novel strategies include oligonucleotides that induce or inhibit the production of specific mediators. Such approaches may yield useful therapies for allergic individuals in the near future.

The Cytopathic 18f Strain of Hepatitis A Virus Induces RNA Degradation in FrhK4 Cells

The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

Activation of AMP-activated Protein Kinase Reduces CAMP-mediated Epithelial Chloride Secretion

AMP-activated protein kinase (AMPK) is activated in response to fluctuations in cellular energy status caused by oxidative stress. One of its targets is the cystic fibrosis transmembrane conductance regulator (CFTR), which is the predominant Cl- secretory channel in colonic tissue. The aim of this study was to determine the role of AMPK in the modulation of colonic chloride secretion under conditions of oxidative stress and chronic inflammation. Chloride secretion and AMPK activity were examined in colonic tissue from adult IL-10-deficient and wild-type 129 Sv/Ev mice in the presence and absence of pharmacological AMPK inhibitors and activators, respectively. Apical levels of CFTR were measured in brush-border membrane vesicles. Cell culture studies in human colonic T84 monolayers examined the effect of hydrogen peroxide and pharmacological activation of AMPK on forskolin-stimulated chloride secretion. Inflamed colons from IL-10-deficient mice exhibited hyporesponsiveness to forskolin stimulation in association with reductions in surface CFTR expression and increased AMPK activity. Inhibition of AMPK restored tissue responsiveness to forskolin, whereas stimulation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) induced tissue hyporesponsivness in wild-type mice. T84 cells exposed to hydrogen peroxide demonstrated a time-dependent increase in AMPK activity and reduction of forskolin-stimulated chloride secretion. Inhibition of AMPK prevented the reduction in chloride secretion. Treatment of cells with the AMPK activator, AICAR, resulted in a decreased chloride secretion. In conclusion, AMPK activation is linked with reductions in cAMP-mediated epithelial chloride flux and may be a contributing factor to the hyporesponsiveness seen under conditions of chronic inflammation.

Pathological Work-up of Sentinel Lymph Nodes in Breast Cancer. Review of Current Data to Be Considered for the Formulation of Guidelines

Controversies and inconsistencies regarding the pathological work-up of sentinel lymph nodes (SNs) led the European Working Group for Breast Screening Pathology (EWGBSP) to review published data and current evidence that can promote the formulation of European guidelines for the pathological work-up of SNs. After an evaluation of the accuracy of SN biopsy as a staging procedure, the yields of different sectioning methods and the immunohistochemical detection of metastatic cells are reviewed. Currently published data do not allow the significance of micrometastases or isolated tumour cells to be established, but it is suggested that approximately 18% of the cases may be associated with further nodal (non-SN) metastases, i.e. approximately 2% of all patients initially staged by SN biopsy. The methods for the intraoperative and molecular assessment of SNs are also surveyed.

Primary Systemic Therapy (PST) of Locally Advanced Breast Cancer Using Doxorubicin/Docetaxel Combination

PST of locally advanced breast carcinomas causes tumour shrinkage and down-staging, therefore, optimal circumstances for surgery.

Distribution of Radiation-induced Exchange Aberrations in All Human Chromosomes

To investigate the DNA-proportional distribution of radiation-induced chromosome aberrations for all chromosomes of a male and a female human karyotype.

[New TNM Classification of Breast Tumors]

The TNM classification of breast carcinomas has substantially changed. Both the newly implemented Hungarian breast cancer screening programme and the national cancer registry require the adequate reporting of the disease with particular emphasis on its extent.

[Coding of Core Biopsies of the Breast: B1-B5]

Porphyrins As Radiosensitizing Agents for Solid Neoplasms

The biological effects of radiation affect both neoplastic and normal tissues. The nature and extent of such effects, however, depend on selected biological parameters (e.g., oxygen supply, cell cycle) and can be modified by chemical agents such as radiosensitizers, radioprotectors and chemotherapeutic agents. A precise control of the mode of action of the radiation is important in order to achieve the maximum effect on tumor tissue, while minimizing the effect on normal tissues. Most of the known and routinely used radiosensitizers are neither selective nor tumor specific. This article reviews a new selective and specific modality that increases the sensitivity of solid tumor tissue, especially of radio resistant, hypoxic tumor cells, to radiation. This modality is currently under early clinical evaluation and encompasses the application of Photofrin II, which is already used as a photosensitizer in photodynamic therapy (PDT) at predetermined times prior to irradiation.

Photofrin As a Radiosensitizer in an in Vitro Cell Survival Assay

Chemical modifiers (radiosensitizers) are used in order to increase the efficacy of radiotherapy. The use of Photodynamic Therapy for tumor treatment, especially with Photofrin II, is also known. At present, no chemical modifier has been found to act as a selective radiosensitizer. Experiments using several series of cell lines were performed; human bladder cancer cell line (RT4), colon adenocarcinoma cells (HT-29), and the glioblastoma cells (U-373 MG) were investigated, with and without incubation with Photofrin II, before irradiation. The irradiation was performed using doses ranging from 0 to 8Gy. Colony forming tests were applied to determine the efficiency of Photofrin II as a radiation sensitizer in comparison to irradiation alone. Two of the cell lines tested, cultures of the RT4 and U-373 MG, treated with Photofrin II prior to radiation, showed cell survival lower than cultures untreated with Photofrin II but irradiated under identical conditions. For the HT-29 cells, the results did not differ between the two groups (with and without Photofrin). The results of this study showed that Photofrin II can act, under certain conditions as a tumor radiosensitizer.

[Tenascin-C Expression in Human Breast Tumors]

Increased amounts of extracellular matrix proteins have been described in breast tumours, which are normally present in embryonal connective tissue, in adults however, these occur only in pathological conditions: one of which is tenascin. Within the tenascin family the most examined and most controversial member is tenascin-C. It has been suggested that tenascin-C decreases cell adhesion, promotes invasion and metastases, and may play role in pathological angiogenesis.

[Botulinum Toxin A in the Treatment of Stiff Man Syndrome]

Stiff-Person Syndrome (SPS) is a very rare disorder characterised by progressive fluctuating muscle rigidity and episodic spasm. So far, only two reports have demonstrated a significant clinical improvement in the patients with SPS when muscle were injected with Botulinum Toxin A (BTA). We investigated the effectiveness of intramuscular injections of BTA in a patient with clinical, biochemical and electrophysiological evidence of SPS. A 41-year-old woman with coexisting epilepsy and insulin-dependent diabetes mellitus was hospitalised in our Department because of stiffness and paroxysmal spasm of trunk and proximal limbs muscles. Because of not sufficient results of the pharmacological treatment the injections of BTA into involved muscles were done. Clinical observations included measure of pain, frequency of spasm, well-being and selection's activities were performed at baseline and in 1, 2, 7, 11, 16, 20, weeks. Significant improvement started one week after injections and lasting about 4 months was observed. Using BTA injections into involved muscles for the treatment of SPS can be followed by marked functional improvement and reducing the need for systemic drugs.

NADPH Oxidase is Not Required for Spontaneous and Staphylococcus Aureus-induced Apoptosis of Monocytes

Reactive oxygen intermediates (ROI) generated in the respiratory burst reaction are crucial for the killing of bacteria and fungi in phagocytes. The key enzyme for the respiratory burst reaction is the NADPH oxidase. Reactive oxygen intermediates have additionally been proposed to be of general importance for the expression of FAS and soluble FAS ligand (sFASL) and the subsequent induction of apoptosis. This conclusion has been drawn from the observation that neutrophils with an inborn lack of the NADPH oxidase as well as cell lines and monocytes with artificially blocked NADPH oxidase exhibit impaired apoptosis. Being one of the few centers caring for patients with chronic granulomatous disease (CGD) who exhibit an inborn lack of NADPH oxidase, we had the unique opportunity to determine the role of the NADPH oxidase for apoptosis in monocytes with otherwise unmanipulated cells of these patients (CGD monocytes). We compared the expression of FAS on monocytes and the concentration of sFASL in the supernatant between CGD monocytes and healthy donors undergoing spontaneous apoptosis. Neither the expression of FAS nor the concentration of sFASL was decreased in CGD monocytes. We further compared spontaneous apoptosis and apoptosis occurring after the phagocytosis of Staphylococcus aureus in CGD monocytes to monocytes of healthy controls. In these experiments we could not determine any significant impairment of apoptosis in CGD monocytes. Our data indicate for the first time that in an unmanipulated human model a functional NADPH oxidase is not crucial for the apoptosis of monocytes and disprove a general role of ROI for the induction of apoptosis in phagocytes.

Use of Reverse Transcription and PCR to Discriminate Between Infectious and Non-infectious Hepatitis A Virus

Hepatitis A virus (HAV) is a major cause of infectious hepatitis worldwide. Detection of HAV in contaminated food or water is a priority research area in laboratories worldwide. Our laboratory has reported previously the development of reverse transcription-polymerase chain reaction (RT-PCR) based detection and typing methods for HAV in contaminated shellfish and produce. It is commonly held that RT-PCR can detect viral genome, but cannot distinguish between infectious and inactivated virus. Therefore, signals obtained after PCR should be considered as false positives unless it can be shown that the sample contains virus capable of infecting a suitable host cell line in culture. We present data to show that this general assumption is not valid. Evidence is provided that demonstrate that signals generated after RT-PCR amplification of viral genome correlated well with the presence of infectious virus in the sample. Viral samples inactivated by heat or UV treatment produced significantly lower signal strength that paralleled infectivity of the sample in cultured cells. The loss of signal strength is most likely the result of damage to the viral RNA that renders it unsuitable for RT-PCR. The correlation between PCR signal and infectivity was better following UV inactivation than heat treatment. The procedure may be adapted to other viruses and inactivating agents.

Mid-IR Synchrotron Radiation for Molecular Specific Detection in Microchip-based Analysis Systems

Microstructures constructed from SU-8 polymer and produced on CaF(2) base plates have been developed for microchip-based analysis systems used to perform FTIR spectroscopic detection using mid-IR synchrotron radiation. The high brilliance of the synchrotron source enables measurements at spot sizes at the diffraction limit of mid-IR radiation. This corresponds to a spatial resolution of a few micrometers (5-20 microm). These small measurement spots are useful for lab-on-a-chip devices, since their sizes are comparable to those of the structures usually used in these devices. Two different types of microchips are introduced here. The first chip was designed for time-resolved FTIR investigations of chemical reactions in solution. The second chip was designed for chip-based electrophoresis with IR detection on-chip. The results obtained prove the operational functionality of these chips, and indicate the potential of these new devices for further applications in (bio)analytical chemistry.

Discrepancies in Current Practice of Pathological Evaluation of Sentinel Lymph Nodes in Breast Cancer. Results of a Questionnaire Based Survey by the European Working Group for Breast Screening Pathology

To evaluate aspects of the current practice of sentinel lymph node (SLN) pathology in breast cancer via a questionnaire based survey, to recognise major issues that the European guidelines for mammography screening should address in the next revision.

Consistency of Staining and Reporting of Oestrogen Receptor Immunocytochemistry Within the European Union--an Inter-laboratory Study

To assess the variability of oestrogen receptor (ER) testing using immunocytochemistry, centrally stained and unstained slides from breast cancers were circulated to the members of the European Working Group for Breast Screening Pathology, who were asked to report on both slides. The results showed that there was almost complete concordance among readers (kappa=0.95) in ER-negative tumours on the stained slide and excellent concordance among readers (kappa=0.82) on the slides stained in each individual laboratory. Tumours showing strong positivity were reasonably well assessed (kappa=0.57 and 0.4, respectively), but there was less concordance in tumours with moderate and low levels of ER, especially when these were heterogeneous in their staining. Because of the variation, the Working Group recommends that laboratories performing these stains should take part in a external quality assurance scheme for immunocytochemistry, should include a tumour with low ER levels as a weak positive control and should audit the percentage positive tumours in their laboratory against the accepted norms annually. The Quick score method of receptor assessment may also have too many categories for good concordance, and grouping of these into fewer categories may remove some of the variation among laboratories.

Activation of Mast Cells by Double-stranded RNA: Evidence for Activation Through Toll-like Receptor 3

Although mast cells (MCs) have been clearly implicated in innate immune responses involving bacteria, their ability to respond to viral infection is less clear.

Apoptosis Induced by a Cytopathic Hepatitis A Virus is Dependent on Caspase Activation Following Ribosomal RNA Degradation but Occurs in the Absence of 2'-5' Oligoadenylate Synthetase

We have presented previously evidence that the cytopathogenic 18f strain of hepatitis A virus (HAV) induced degradation of ribosomal RNA (rRNA) in infected cells [Arch. Virol. 148 (2003) 1275-1300]. In contrast, the non-cytopathogenic parent virus HM175 clone 1 had no effect on rRNA integrity. We present here data showing that rRNA degradation is followed by apoptosis accompanied by characteristic DNA laddering in the cytoplasm of 18f infected cells. The DNA laddering coincided with the detection of caspase 3 and PARP-1 cleavage and was dependent upon activation of the caspase pathway, since treatment with Z-VAD-FMK, a pan-caspase inhibitor, inhibited both events. RNase L mRNA was present in both virus-infected and uninfected cells. Messenger RNA for the interferon inducible enzyme 2'-5' oligoadenylate synthetase (2'-5' OAS), which polymerizes ATP into 2'-5' oligo adenylate (2-5A, the activator of RNase L) in the presence of double-stranded RNA, was not detected following virus infection. 2'-5' OAS mRNA was induced by treatment of the cells with interferon-beta (IFN-beta). IFN-beta mRNA was marginally induced following infection. However, phosphorylated STAT 1, a key regulator of interferon-stimulated gene transcription was not detected in virus infected cells. STAT 1 phosphorylation in response to IFN treatment was lower in virus-infected cells, compared to uninfected cells treated with interferon, suggesting that 18f virus infection interferes with interferon signaling. The results suggest that 18f infection causes the induction of a 2-5A independent RNase L like activity.

Adaptive Doses of Irradiation-an Approach to a New Therapy Concept for Bladder Cancer?

Radiation adaptive response in terms of induced radioresistance or hyperradiosensitivity, has been studied in HCV29 (human bladder epithelium) and RT4 (human bladder carcinoma) cell lines. After pre-irradiation doses of 0.05 Gy or 0.1 Gy, HCV29 cells showed induced radioresistance, whereas after pre-irradiation doses of 0.05 Gy, 0.1 Gy, 0.2 Gy, and 0.5 Gy, the RT4 cells clearly showed hyperradiosensitivity. On the basis of these results, an approach has been developed that may lead to a concept for a new radiotherapeutic regimen of bladder cancer that includes protection of normal cells, on the one hand, and the potential of tumor cell damage, on the other hand. These findings need to be confirmed in further studies for the benefit of the patients.

Suspected Central Anticholinergic Syndrome in a 6-week-old Infant

A 6-wk-old male infant became unresponsive after an uneventful general anesthetic for hernia repair. His symptoms were consistent with central anticholinergic syndrome. He appeared to awaken after treatment with IV physostigmine in a dose of 0.04 mg/kg. Because of the recurrence of sedation, a second physostigmine infusion was administered, which again led to transient arousal. Finally, the patient awoke spontaneously after 24 h and recovered uneventfully.

Persistent HIV Type 1 Infection in Semen and Blood Compartments in Patients After Long-term Potent Antiretroviral Therapy

HIV-1 RNA levels in semen and blood compartments decrease below detection limits during highly active antiretroviral therapy. Despite these therapeutic effects, it is clear that persistent, latent HIV-1 reservoirs are capable of rebounding in the absence of drug treatment or by evolution of escape mutants remain. The current study was designed to examine the presence of latent virus in semen and blood compartments and its evolution following potent combination therapy with indinavir (protease inhibitor) and efavirenz [nonnucleoside reverse transcriptase (RT) inhibitor]. Using an ultrasensitive in situ hybridization assay HIV-1 mRNA was detected in cultured seminal and blood mononuclear cells in all patients up to 1789 days posttherapy. Higher levels of HIV-1 mRNA were consistently detected in seminal mononuclear cells as compared to peripheral blood mononuclear cells (PBMC) in all time points analyzed posttherapy. Analysis of viral RNA from cultured PBMC before and after therapy displayed no evidence of therapy-induced drug resistance in the viral polymerase gene in the majority of patients. However, distinct envelope populations were detected in these viral RNA populations following therapy, indicating possible selection of quasispecies. The observed ongoing replication and evolution in the PBMC viral envelope sequences likely occurred in the seminal compartment HIV populations, given that the seminal cells showed the ability to express HIV-1 mRNA following cultivation. This together with our previous studies (Gupta P, et al.: J Infect Dis 2000;182:79-87) suggest that the genital and blood compartments likely serve as distinct reservoirs harboring latent HIV-1 during prolonged drug therapy.

High-resolution Tracking of Cell Division Demonstrates Differential Effects of TH1 and TH2 Cytokines on SCF-dependent Human Mast Cell Production in Vitro: Correlation with Apoptosis and Kit Expression

T-helper 1 (TH1) (interferon-gamma [IFN-gamma]) and TH2 (interleukin-4 [IL-4] and IL-5) cytokines have been variably reported to alter human mast cell numbers in complex culture systems. The effects of these cytokines on the kinetics of cell division and cell death are unknown, and their effect on mast cell behavior is relevant to anticipate the consequences of in vivo strategies that alter cytokine levels. To determine the effect of these cytokines on stem cell factor (SCF)-dependent human mast cell production, we used high-resolution tracking of cell division and correlated the results with cell apoptosis, expression of Kit, and mast cell degranulation. When IFN-gamma, IL-5, or IL-4 was administered over 8 weeks, we found each cytokine decreased the mast number through a different mechanism. IFN-gamma inhibited early progenitor cell division, IL-4 down-regulated early Kit expression, and IL-5 blocked later cell division. Further, IL-4 and IFN-gamma had the greatest suppressive effect on degranulation and FcepsilonRI expression. When these cytokines were administered to mature mast cells, IFN-gamma and IL-5 had no effect on degranulation and cell division, but IL-4 induced division and potentiated FcepsilonRI-mediated degranulation. Thus, exposure of human mast cells to IL-4, IL-5, and IFN-gamma during growth and differentiation generally down-regulated mast cell number and function, whereas IL-4 increased mature mast cell division and degranulation.

Claudin-1, -3 and -4 Proteins and MRNA Expression in Benign and Malignant Breast Lesions: a Research Study

We compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions.

The Application of Photofrin II As a Sensitizing Agent for Ionizing Radiation--a New Approach in Tumor Therapy?

Radiosensitizers represent an enticing concept in tumor therapy. As ionizing radiation affects both neoplastic and normal tissues, its effects are generally non-specific. The aim of applying a radiosensitizing agent is to achieve a maximum effect on tumor tissue, while minimizing the damage to normal tissues. A variety of parameters such as the oxygen supply and the state in the cell cycle, need to be taken into account when evaluating a potential radiosensitizer. Most of the previously known radiosensitizers are neither selective nor tumor specific. In this article, we review the properties and radiosensitizing potential of Photofrin II. Photofrin II is well-known as a photosensitizing agent in photodynamic therapy. In recent years, a radiosensitizing potential of the substance has been demonstrated, specifically increasing the sensitivity of solid tumor tissues, especially of radio-resistant, hypoxic tumor cells, to radiation. This radiosensitizing effect has been demonstrated both by in vitro studies and by animal experiments. Several studies with tissue cultures have demonstrated a radiosensitizing effect of Photofrin II in glioblastoma (U-373MG) and bladder cancer cell lines (RT-4). No effect was noted in colon carcinoma cell lines (HT-29). Unpublished data of additional cell lines will be mentioned in the review. Animal experiments with Lewis sarcoma and with bladder cancer have moreover demonstrated an in vivo effect of Photofrin II as a radiosensitizer. The mechanism of this radiosensitizing effect is not completely understood. In vitro data, however, support the hypothesis that the radiosensitizing action involves OH-radicals in addition to a potential impairment of repair mechanisms after sublethal damage of ionizing radiation. Moreover, early results of a phase I trial are available and document the potential feasibility of the application of Phototofrin II as a radiosensitizing agent in clinical practice.

SKY and FISH Analysis of Radiation-induced Chromosome Aberrations: a Comparison of Whole and Partial Genome Analysis

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.

Differential Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Interferon Gamma in Mast Cells and Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel in epithelial cells; recently, we identified it in mast cells. Previous work that we confirmed showed that interferon gamma (IFNgamma) down-regulated CFTR expression in epithelial cells (T84), but by contrast, we found that IFNgamma up-regulated CFTR mRNA and protein expression in rat and human mast cells. IFNgamma up-regulation of CFTR in mast cells was inhibited by p38 and extracellular signal-regulated kinase (ERK) kinase inhibitors but not a Janus tyrosine kinase (JAK)2 inhibitor, whereas in T84 cells IFNgamma-mediated down-regulation of CFTR was JAK2-dependent and ERK- and p38-independent. Furthermore, IFNgamma down-regulation of CFTR in T84 epithelial cells was STAT1-dependent, but up-regulation of CFTR in mast cells was STAT1-independent. Thus, differential regulatory pathways of CFTR expression in mast cells and epithelial cells exist that depend upon either p38/ERK or JAK/STAT pathways, respectively. Surprisingly, IFNgamma treatment of mast cells inhibited Cl(-) efflux, in contrast to up-regulation of CFTR/mRNA and protein expression. However, down-regulation of Cl(-) flux correlated with IFNgamma-mediated inhibition of mediator secretion. This and other work suggests that the effect of IFNgamma on CFTR expression in mast cells is important for their function.

Claudin Expression in Breast Tumors

[Oncocytic Tumor of the Adrenal Gland]

Oncocytic tumor of the adrenal gland. The authors describe a case of a rare tumor arising in the adrenal gland. The tumor 6 cm in diameter, connected to the right adrenal gland, was found incidentally in a healthy young man of 34 years of age, who suffered an accident and had a rib fracture. No pathological hormone production was detected on laboratory tests. The right adrenal gland with the tumor was removed by laparoscopic surgery. Histopathological examination revealed an oncocytic adenoma of the adrenal cortex. The authors describe the morphology and the differential diagnosis of this rare tumor of the adrenal gland.

The Expression of Five Different Claudins in Invasive Breast Carcinomas: Comparison of PT1pN1 and PT1pN0 Tumors

The evaluation of the role of claudins (CLDNs) in breast carcinogenesis has recently begun. We investigated the expression of CLDNs 1, 2, 3, 4, and 7 in pT1pN0 and pT1pN1 invasive ductal breast carcinomas. Tissue arrays of 30-30 pT1pN0 and pT1pN1 invasive ductal breast carcinomas of different grades were constructed, and the expression of CLDN 1, 2, 3, 4, and 7 proteins was analyzed using standard and immunofluorescent immunohistochemistry. The results were evaluated by light and confocal microscopy. Regarding CLDN 1, 4, and 7 expressions, differences were noted between normal and tumor cells and also between tumors of different grades, while no remarkable differences were noted between pT1pN0 and pT1pN1 tumors. CLDNs 1 and 7 were found to be downregulated in tumor cells compared to the normal epithelium, while CLDN 4 expression was decreased in grade 1 tumors. CLDN 7 protein was abundant in normal epithelia, and the staining decreased in grade 3 tumors. There were no differences between normal and neoplastic cells regarding CLDN 2 and 3 expressions. As a preliminary result, our observations suggest that the analyzed CLDNs do not promote tumor metastasis. On the basis of our findings, it seems that CLDN 1, CLDN 4, and CLDN 7 may rather have an important role in tumorigenesis or in cell-to-cell adhesion.

[The Results of Cetuximab Treatment of a Patient with Multiple Pretreated, Irinotecan Resistant Metastatic Colorectal Cancer]

Aim: The case of a patient with heavily pretreated, irinotecan-resistant, metastatic colorectal cancer is presented by the authors, and they would like to point to the effectiveness of a new therapeutic option, cetuximab treatment. Additionally, they would like to call attention to the need of performing EGFR determination from all of the biopsy samples (primary tumor, lymph nodes and metastases) to start the treatment. A weak EGFR-positivity of the primary tumor in fact does not mean the contraindication of cetuximab treatment, since the metastases, which are usually treated, could be strongly positive. The therapeutic answer could be excellent in this case, as it occurred in our case.

[Her2/neu Receptor: One of the Basic Factors Rendering Tailored Therapy Feasible in Breast Cancer Patients]

In a considerable part of breast carcinomas the amount of cell surface Her2/neu receptors has been increased such that it becomes the main factor defining the behaviour of the tumor. At the same time, in such amount, this receptor is easily detectable by simple, routine pathological methods. If we are aware of the exact biological characteristics of a given tumor, targeted therapy may be used since an antibody against the Her2 receptor is already available. This drug, called trastuzumab (Herceptin), has been used in advanced breast carcinoma as adjuvant or primary treatment. In clinical trials its advantage in early stage breast carcinomas has been investigated so far.

TLR3 Activation Inhibits Human Mast Cell Attachment to Fibronectin and Vitronectin

Mast cells are involved in both the genesis of allergic inflammation and in host defense; and reside in tissues where their location and responsiveness is regulated in part by adhesion to extracellular matrix proteins (ECM). We have reported that human mast cells (huMC) express TLR1-7, and 9 and respond to toll-like receptors (TLR) ligands by releasing cytokines and leukotriene C4. To determine if TLR ligation could similarly affect mast cells via an influence on adhesion, we employed huMC; and as substrates, fibronectin (FN) and vitronectin (VN). huMC were thus treated with double-stranded RNA (dsRNA) and adhesion to ECM was quantified. FcvarepsilonRI dependent mast cell degranulation was assessed. Adhesion molecule expression and activation was measured by flow cytometry. Activation of huMC through TLR3 with increasing amounts of polyI:C inhibited mast cell adhesion in a dose-dependent manner. This decrease in adhesion was accompanied by a similar decrease in IgE-mediated mast cell degranulation. Activation of TLR3 on huMC resulted in a change in the conformation of CD29, the receptor for FN, to an inactive form. Thus, TLR3 activation decreases mast cell attachment to VN and FN through an active process and one, which would abrogate mast cell attachment dependent potentiation of IgE-mediated responses.

Mast Cells, Which Interact with Escherichia Coli, Up-regulate Genes Associated with Innate Immunity and Become Less Responsive to Fc(epsilon)RI-mediated Activation

Mast cells, which are associated with T helper cell type 2-dependent inflammation, have now been implicated in the innate immune response. To further characterize how mast cells are programmed to respond to infectious organisms, we used expression profiling using DNA microarray analysis of gene expression by human mast cells (huMC) during ingestion of Escherichia coli and examined immunoglobulin E (IgE)-mediated degranulation. Analysis of data revealed that specific groups of genes were modulated, including genes encoding transcription factors, cell signaling molecules, cell cycle regulators, enzymes, cytokines, novel chemokines of the CC family, adhesion molecules, and costimulatory molecules. Enzyme-linked immunosorbent assay analysis confirmed the production of tumor necrosis factor and the chemokines CC chemokine ligand (CCL)-1/I-309, CCL-19/macrophage-inflammatory protein-3beta (MIP-3beta), and CCL-18/MIP-4; flow cytometry confirmed the up-regulation of carcinoembryonic antigen-related cell adhesion molecule 1, the integrin CD49d, and CD80. Coincubation with E. coli down-regulated Fc receptor for IgE I (FcepsilonRI) expression and FcepsilonRI-mediated huMC degranulation. These data are consistent with the concept that bacterial exposure directs mast cell responses toward innate immunity and away from IgE-mediated effects.

Towards Biochemical Reaction Monitoring Using FT-IR Synchrotron Radiation

A lab-on-a-chip device made of CaF2 windows and SU-8 polymer was used for fluid lamination to achieve rapid mixing of two streamlines with a cross section of 300 x 5 microm each. Time resolved measurements of the induced chemical reaction was achieved by applying constant feeding low flow rates and by on-chip measurement at defined distances after the mixing point. Synchrotron IR microscopic detection was employed for direct and label-free monitoring of (bio)chemical reactions. Furthermore, using synchrotron IR microscopy the measurement spot could be reduced to the diffraction limit, thus maximizing time resolution in the experimental set-up under study. Based on computational fluid dynamic simulations the principle of the set-up is discussed. Experimental results on the basic hydrolysis of methyl chloroacetate proved the working principle of the experimental set-up. First results on the interaction between the antibiotic vancomycin and a tripeptide (Ac2KAA) involved in the build up of the membrane proteins of gram-positive bacteria are presented.

Use of Frozen-thawed Cervical Tissues in the Organ Culture System to Measure Anti-HIV Activities of Candidate Microbicides

Cervical tissue-based organ culture system has been used to test the cytotoxicity and antiviral activity of microbicides. One of the problems of using current organ culture methods for routine microbicide testing is the need to continually obtain fresh tissue, which can be limited in access and supply. Use of frozen tissue, stored when available and thawed when needed, would alleviate the need for constant access to new tissue. This study was designed to explore the possibility of using frozen-thawed cervical tissue to test microbicides for their anti-HIV activity. We provided biochemical, histological, and quantitative immunohistochemical data to demonstrate the integrity of the frozen-thawed organ culture system. Significant levels of HIV-1 mucosal transmission were noted with both fresh and frozen-thawed tissue, regardless of the coreceptor usage of the virus isolate. Furthermore, candidate microbicides UC781, beta-cyclodextrin, and octylglycerol inhibited HIV-1 transmission across the mucosa of frozen-thawed tissues with a level of efficiency similar to that of fresh tissues. Therefore, frozen-thawed cervical tissue in the organ culture system provides a practical and convenient model to screen topical microbicides for their ability to block sexual transmission of HIV-1, and reduces the problems associated with procurement of the numerous tissues required for evaluation and comparison of microbicide candidates among different laboratories.

Automated Sample Preparation and Analysis Using a Sequential-injection-capillary Electrophoresis (SI-CE) Interface

A fully automated sequential-injection-capillary electrophoresis (SI-CE) system was developed using commercially available components as the syringe pump, the selection and injection valves and the high voltage power supply. The interface connecting the SI with the CE unit consisted of two T-pieces, where the capillary was inserted in one T-piece and a Pt electrode in the other (grounded) T-piece. By pressurising the whole system using a syringe pump, hydrodynamic injection was feasible. For characterisation, the system was applied to a mixture of adenosine and adenosine monophosphate at different concentrations. The calibration curve obtained gave a detection limit of 0.5 microg g(-1) (correlation coefficient of 0.997). The reproducibility of the injection was also assessed, resulting in a RSD value (5 injections) of 5.4%. The total time of analysis, from injection, conditioning and separation to cleaning the capillary again was 15 minutes. In another application, employing the full power of the automated SIA-CE system, myoglobin was mixed directly using the flow system with different concentrations of sodium dodecyl sulfate (SDS), a known denaturing agent. The different conformations obtained in this way were analysed with the CE system and a distinct shift in migration time and decreasing of the native peak of myoglobin (Mb) could be observed. The protein samples prepared were also analysed with off-line infrared spectroscopy (IR), confirming these results.

Feasibility of Photofrin II As a Radiosensitizing Agent in Solid Tumors--preliminary Results

Photofrin II has been demonstrated to serve as a specific and selective radiosensitizing agent in in vitro and in vivo tumor models. We aimed to investigate the feasibility of a clinical application of Photofrin II.

Cytokinins Inhibit Epiphyllous Plantlet Development on Leaves of Bryophyllum (Kalanchoë) Marnierianum

When leaves of Bryophyllum marnierianum are detached from the plant, plantlets develop from primordia located at their margins. Leaves excised with a piece of stem attached do not produce plantlets. Severing the major leaf veins overcomes the inhibitory effect of the attached stem, indicating that the control agent is transmitted through the vascular system. A possible mechanism is that an inhibitory substance, possibly a known plant hormone, transported from the stem to the leaf, suppresses plantlet development. A number of hormones were tested for their ability to inhibit plantlet primordium development in whole isolated leaves. Auxins had no effect, indicating that apical dominance is not involved. The cytokinins zeatin, kinetin, and benzylaminopurine (BAP) strongly inhibited plantlet development, suggesting that they may be the or a factor involved in maintenance of plantlet primordium dormancy when the leaf is attached to the plant. This hypothesis was strongly supported by the finding that treatment of leaves attached to stems with a cytokinin antagonist (purine riboside) released the primordia from inhibition. In contrast to whole leaves, plantlet primordium development on leaf explants incubated on Murashige Skoog medium containing 3% sucrose was strongly stimulated by cytokinins. A possible explanation of these observations is that in whole leaves the cytokinin signal is transduced into an inhibitory signal whereas in the isolated primordium cytokinin has a direct stimulatory effect. The inhibitory cytokinin pathway must be dominant as long as the leaf is attached to the plant. A model is proposed which could explain these findings. This study points to a novel role of cytokinins in the maintenance of foliar plantlet primordium dormancy.

[Evaluation of Care Load for Children and Teenagers with Severe and Multiple Handicaps. A Quantitative Empirical Study]

The aim of the study was to gain differentiated and plausible data on care load for severely and multiply disabled children and teenagers. Those data are required for public health as well as for forensic reasons.

Detection of HER-2/neu Gene Amplification in Breast Carcinomas Using Quantitative Real-time PCR - a Comparison with Immunohistochemical and FISH Results

The aim of our study was to evaluate the value of quantitative real-time-PCR (qPCR) in the determination of HER-2/neu amplification status of human breast carcinomas by comparing qPCR, FISH and immunohistochemistry results from the same samples. A total of 210 breast carcinomas were examined. Ready-to-use CB11 antibody was applied to detect HER-2/neu oncoprotein expression. In 76 out of 210 cases FISH was performed, and 162 cases were investigated with qPCR. Seventy-five tumors were 2+ or 3+ positive with immunohistochemistry, while 135 samples were either completely negative or 1+. In 45 cases results from all three methods were available. Out of these, in twenty negative and sixteen positive cases both FISH and qPCR led to similar results. The mean qPCR amplification ratio in the concordant positive cases was 5.424 while in the qPCR+/FISH- group the mean ratio was 2.765. Out of 121 samples with scores of 0 or 1+ immunohistochemical result, analyzed also with qPCR, 26 showed HER-2/neu gene amplification. In these cases the mean amplification ratio was 2.53. Comparison of FISH and qPCR together with immunohistochemistry shows that qPCR is more sensitive to detect HER-2/neu gene amplification in tumors scored as 2+ with immunohistochemistry, but the diagnostic cut-off ratio should be defined above 2.7 to avoid high number of false positive cases. Amongst the immunohistochemistry score 2+ cases, 10 of 18 showed gene amplification by qPCR while 10 of 26 by FISH. In conclusion, a well calibrated HER-2/neu qPCR assay may serve as useful alternative to FISH in breast cancer patients.

Divergences in Diagnosing Nodular Breast Lesions of Noncarcinomatous Nature

Nodular breast lesions of noncarcinomatous origin are often of fibroepithelial origin. They may cause classification problems when they are hypocellular or hypercellular; the latter setting may also raise the differential diagnosis of phyllodes tumors. Thirty equivocal nodular breast lesions were collected and one hematoxylin and eosin slide from each was assessed by six pathologists with special interest in breast pathology. The overall reproducibility of classifying these lesions into categories of fibroadenoma, phyllodes tumor or anything else was moderate (kappa value: 0.48). The lack of a uniform nomenclature was not felt disturbing for hypocellular lesions, but the discordant diagnosis of tumors resembling or representing phyllodes tumors was acknowledged to require intervention, such as more obvious implication of guidelines and quality assurance programs aiming at assessing diagnoses and prognostic parameters.

Difficulties in the Diagnosis of Intracystic Tumors of the Female Breast

The objective of the current study was to determine the accuracy of radiological and cytological diagnoses of intracystic and papillary lesions in distinguishing between true papillary benign and malignant lesions. Seventy cytological reports of intracystic proliferations were selected from our cytopathological database at the Breast Health Corporation, Budapest, Hungary, dating back to the last 7 years. Retrospective analysis of the diagnostic approaches--mammography, ultrasonography, clinical examination and cytology--was performed in selected cases. The results of imaging and cytological examination are routinely reported on a categorical scale ranging from 1 to 5. 44 patients underwent surgical excision: histology showed benign lesions in 21 and malignant lesions in 23 cases. Twelve patients, who did not undergo biopsy and presented a stable disease at follow-ups, were also included in the group of benign lesion. Fifteen patients were not available for follow-up. Concerning the total investigated cases the mean categorical values of mammography, ultrasonography and cytology were 2.24, 2.78 and 3.05 respectively. The malignant and benign groups significantly differ from each other concerning the mean age of the patients (p=0.0216), the distribution of the coded mammographical results (p= 0.0171) the cytological results (p=0.0001), and average tumor size measured on mammogram images (p=0.0199). The two group does not significantly differ over the distribution of mammographical density patterns (p=0.1075), radiomorphological appearance (p=0.1101), average tumor size measured on ultrasonography (p=0.2665), and patient complaints (p=0.2634). The evaluation of ultrasonography shows borderline significance (Pearson Chi-square test: p=0.0616, M-L Chi-square test: p=0.0404) between the malignant and benign groups. Differential diagnosis between malignant and benign intracystic and papillary lesions is feasible using common radiological diagnostics. However, more efficient teamwork is needed with the cooperation of a well-trained cytologist and radiologist, who are able to produce precise images of the lesions, and guides the aspiration of the adequate samples for cytology, which is the most valuable examination.

Sentinel Lymph Node Biopsy in Staging Small (up to 15 Mm) Breast Carcinomas. Results from a European Multi-institutional Study

Sentinel lymph node (SLN) biopsy has become the preferred method for the nodal staging of early breast cancer, but controversy exists regarding its universal use and consequences in small tumors. 2929 cases of breast carcinomas not larger than 15 mm and staged with SLN biopsy with or without axillary dissection were collected from the authors' institutions. The pathology of the SLNs included multilevel hematoxylin and eosin (HE) staining. Cytokeratin immunohistochemistry (IHC) was commonly used for cases negative with HE staining. Variables influencing SLN involvement and non-SLN involvement were studied with logistic regression. Factors that influenced SLN involvement included tumor size, multifocality, grade and age. Small tumors up to 4 mm (including in situ and microinvasive carcinomas) seem to have SLN involvement in less than 10%. Non-SLN metastases were associated with tumor grade, the ratio of involved SLNs and SLN involvement type. Isolated tumor cells were not likely to be associated with further nodal load, whereas micrometastases had some subsets with low risk of non-SLN involvement and subsets with higher proportion of further nodal spread. In situ and microinvasive carcinomas have a very low risk of SLN involvement, therefore, these tumors might not need SLN biopsy for staging, and this may be the approach used for very small invasive carcinomas. If an SLN is involved, isolated tumor cells are rarely if ever associated with non-SLN metastases, and subsets of micrometastatic SLN involvement may be approached similarly. With macrometastases the risk of non-SLN involvement increases, and further axillary treatment should be generally indicated.

[Quality Control of HER2 Immunohistochemistry--results from a Hungarian Study]

Whether a breast carcinoma is HER2-positive or -negative has a significant impact on its treatment. The most common method of evaluating the HER2 status of breast tumors is immunohistochemistry. Preliminary data suggest that the proportion of HER2-positive tumors in Hungary shows a wide range from laboratory to laboratory, and overall it just reaches the bottom levels of the incidence reported in the literature (12% 3+ and 11% 2+ cases). A 3-round quality-control test was implemented on a voluntary and anonymous basis. Participating laboratories had to immunostain and evaluate 4 tumor samples per circulation, according to their daily routine. The authors of the present article gave an expert opinion in all cases, and this was compared with the individual laboratories' evaluation and the real FISH-controlled HER2 status of the samples. On the basis of the participants' and experts' evaluation 22/218 and 21/218 HER2 3+ cases were underscored, corresponding to an underevaluation rate of 10%. As most samples were from HER2-positive tumors, overscoring was less common (1%), but reached 5% (individual laboratories' evaluation) or 7% (expert evaluation) when the 2+ scoring of negative cases was also considered. Each case was discussed interactively with the participants and technical advise was also given when deemed necessary. The evaluation of the HER2 status of breast cancers gives reliable results only if adequate quality control measures are implemented, and this study was an important step in this respect.

Codeine Induces Human Mast Cell Chemokine and Cytokine Production: Involvement of G-protein Activation

Activation of mast cells and the systemic release of histamine are common side effects of opiates such as codeine and morphine. In some individuals, codeine not only elicits a sizable early response due to mast cell degranulation, but can also lead to late cutaneous allergic inflammation possibly through the production of chemokines. However, individuals who exhibit a late phase reaction to codeine often do not react to its synthetic analog, meperidine. The goal of this study was to test whether codeine and meperidine induce secretion of inflammatory mediators in human mast cells.

Particulate Air Pollution and Vascular Reactivity: the Bus Stop Study

Particulate air pollution is associated with cardiovascular morbidity but mechanisms are not well understood. We tested the effects on vascular reactivity of exposure to fine particulates matter mass (PM(2.5)), number of particles

Sentinel Lymph Node Biopsy and Non-sentinel Node Involvement in Special Type Breast Carcinomas with a Good Prognosis

This study aimed at identifying factors related to sentinel lymph node (SLN) involvement in patients with tubular, cribriform, mucinous or papillary breast carcinoma and those related to non-SLN metastases if an SLN was positive. Multivariate analyses involved logistic and stepwise regressions. The SLNs harboured metastases in 85 of 572 cases, 78 of whom underwent axillary dissection; 19 presented non-SLN positive disease. Lack of lymphovascular invasion, a tumour size < or = 10 mm and a single SLN removed were the factors predicting an SLN metastasis rate <10%, and patients with these features could be candidates for no surgical axillary staging. A positive SLN proportion of < or = 50% and no lymphovascular invasion were associated with a <10% rate of non-SLN invasion; patients with a positive SLN and these features could be candidates for the omission of completion axillary dissection. The opposite presentation of these factors would mandate SLN biopsy and axillary dissection, respectively.

Validation of Clinical Prediction Rules for a Low Probability of Nonsentinel and Extensive Lymph Node Involvement in Breast Cancer Patients

Two recently developed clinical prediction rules aim to anticipate the lack of nonsentinel lymph node metastases and the involvement of less than 4 lymph nodes in breast cancer patients with positive sentinel lymph nodes (SLNs).

The Psychological Risks of Vietnam: the NVVRS Perspective

In recent years, controversy concerning the psychological consequences of service in the Vietnam war has rearisen. In this article, the Co-Principal Investigators of the National Vietnam Veterans Readjustment Study (NVVRS) provide a perspective on new findings reported by B. P. Dohrenwend et al. (2006) that addresses criticisms of the NVVRS PTSD (posttraumatic stress disorder) prevalence findings, and on a perspective that was provided by R. J. McNally (2006) in an accompanying commentary. They find that Dohrenwend et al.'s study, which evaluated empirically a variety of the critics' alternative explanations and found little support for any of them, represents a landmark contribution to the trauma field. However, they found that McNally's commentary misrepresented the history and context of the NVVRS, and then misinterpreted Dohrenwend et al.'s findings and their importance.

Patients with Advanced Stage Breast Carcinoma Immunoreactive to Biotinylated Herceptin Are Most Likely to Benefit from Trastuzumab-based Therapy: an Hypothesis-generating Study

Biotin-labeled trastuzumab (BiotHER) can be used to test for HER2 by immunohistochemistry. We previously showed that BiotHER immunoreactivity is highly correlated with HER2 amplification and indicated that it could be associated with better clinical outcome in advanced breast cancer patients receiving trastuzumab.

Neuropeptides Activate Human Mast Cell Degranulation and Chemokine Production

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcepsilonRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34(+) progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-gamma (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte-macrophage colony-stimulating factor, but not IL-4, interferon-gamma or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases.

Exhaled Nitric Oxide Concentration is Affected by Age, Height, and Race in Healthy 9- to 12-year-old Children

The fractional concentration of exhaled nitric oxide (Feno) is a useful indicator of airway inflammation in children and adults with asthma.

Lymphoepithelioma-like Carcinoma of the Breast: Not Epstein-Barr Virus-, but Human Papilloma Virus-positive

Lymphoepithelioma-like carcinoma of the breast is a rare tumor, with fewer than 20 cases documented in the literature. None of the published cases was Epstein-Barr virus positive, and our case was also Epstein-Barr virus negative. However, in our case, human papilloma virus (HPV) types 18 and 33 DNA could be demonstrated within the tumor tissue. Many years previously, the patient underwent hysterectomy for cervical carcinoma in situ which showed the presence of HPV-33. To the best of our knowledge, this is the first report on lymphoepithelioma-like carcinoma of the breast where high-risk HPV infection may be suggested as an etiological factor in a patient with a previous history of cervical carcinoma in situ.

Antiangiogenic Therapy with Mammalian Target of Rapamycin Inhibitor RAD001 (Everolimus) Increases Radiosensitivity in Solid Cancer

Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth.

Radial Scar-significant Diagnostic Challenge

The prevalence of radial scar (RS) is 0.04% in asymptomatic women participating in population screening for breast cancer. It is important to differentiate RS from concomitant malignancies, which occur in 20-30% of patients, or from small stellate carcinomas which give similar radiomorphology. The aim of our study was to evaluate the effectivity of current breast diagnostic methods in distinguishing between real RS, concomitant malignancy and carcinomas imitating RS. Diagnosis of RS was set up in 61 cases by mammography. Forty-four patients underwent surgical excision: histology showed benign or malignant lesions in 28 and 16 cases, respectively. A series of negative results at follow-up proved the benign nature of the lesion in further 11 cases. Six patients were not available for follow-up. Results of mammography, physical examination, ultrasonography and cytology were evaluated and were compared in 39 benign and 16 malignant cases. Results of examinations were reported on the BI-RADS scale ranging from 1 to 5. The mean categorical scores of all diagnostic processes were around the level of borderline lesions: mammography: 3.49, ultrasonography: 3.06, cytology: 2.47 and physical examination: 1.67. The average age of the patients in the benign and malignant groups were the same: 58 years. The two groups did not differ significantly over either distribution of coded mammographical results (p = 0.2092), or the distribution of mammographical parenchyma density patterns (p = 0.4875). However, the malignant and benign groups differed significantly from each other over the distribution of coded ultrasonographic (p = 0.0176) and cytological (p < 0.0001) results. In conclusion, in the preoperative diagnosis of asymptomatic "black-stars", mammography detects the non-palpable lesions, and ultrasonography together with cytology proved better in the analysis, provided FNAB is US guided. Due to the complex diagnostic approach the nature of the "black stars" is known in the majority of cases prior to the surgical biopsy.

The Effect of Radio-adaptive Doses on HT29 and GM637 Cells

The shape of the dose-response curve at low doses differs from the linear quadratic model. The effect of a radio-adaptive response is the centre of many studies and well known inspite that the clinical applications are still rarely considered.

The Role of Radiotherapy in the Conservative Treatment of Ductal Carcinoma in Situ of the Breast

Breast-conserving surgery (BCS) followed by radiotherapy (RT) has become the standard of care for the treatment of early-stage (St. I-II) invasive breast carcinoma. However, controversy exists regarding the value of RT in the conservative treatment of ductal carcinoma in situ (DCIS). In this article we review the role of RT in the management of DCIS. Retrospective and prospective trials and meta-analyses published between 1975 and 2007 in the MEDLINE database, and recent issues of relevant journals/handbooks relating to DCIS, BCS and RT were searched for. In retrospective series (10,194 patients) the 10-year rate of local recurrence (LR) with and without RT was reported in the range of 9-28% and 22-54%, respectively. In four large randomised controlled trials (NSABP-B-17, EORTC-10853, UKCCCR, SweDCIS; 4,568 patients) 50 Gy whole-breast RT significantly decreased the 5-year LR rate from 16-22% (annual LR rate: 2.6-5.0%) to 7-10% (annual LR rate: 1.3-1.9%). In a recent meta-analysis of randomised trials the addition of RT to BCS resulted in a 60% risk reduction of both invasive and in situ recurrences. In a multicentre retrospective study, an additional dose of 10 Gy to the tumour bed yielded a further 55% risk reduction compared to RT without boost. To date, no subgroups have been reliably identified that do not benefit from RT after BCS. In the NSABP-B-24 trial, the addition of tamoxifen (TAM) to RT reduced ipsilateral (11.1% vs. 7.7%) and contralateral (4.9% vs. 2.3%) breast events significantly. In contrast, in the UKCCCR study, TAM produced no significant reduction in all breast events. Based on available evidence obtained from retrospective and prospective trials, all patients with DCIS have potential benefit from RT after BCS. Further prospective studies are warranted to identify subgroups of low-risk patients with DCIS for whom RT can be safely omitted. Until long-term results of ongoing studies on outcomes of patients treated with BCS alone (with or without TAM or aromatase inhibitors) are available, RT should be routinely recommended after BCS for all patients except those with contraindication.

Hormonal Control of Root Development on Epiphyllous Plantlets of Bryophyllum (Kalanchoe) Marnierianum: Role of Auxin and Ethylene

Epiphyllous plantlets develop on leaves of Bryophyllum marnierianum when they are excised from the plant. Shortly after leaf excision, plantlet shoots develop from primordia located near the leaf margin. After the shoots have enlarged for several days, roots appear at their base. In this investigation, factors regulating plantlet root development were studied. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) abolished root formation without markedly affecting shoot growth. This suggested that auxin transport from the plantlet shoot induces root development. Excision of plantlet apical buds inhibits root development. Application of indole-3-acetic acid (IAA) in lanolin at the site of the apical buds restores root outgrowth. Naphthalene acetic acid (NAA), a synthetic auxin, reverses TIBA inhibition of plantlet root emergence on leaf explants. Both of these observations support the hypothesis that auxin, produced by the plantlet, induces root development. Exogenous ethylene causes precocious root development several days before that of a control without hormone. Ethylene treatment cannot bypass the TIBA block of root formation. Therefore, ethylene does not act downstream of auxin in root induction. However, ethylene amplifies the effects of low concentrations of NAA, which in the absence of ethylene do not induce roots. Ag(2)S(2)O(3), an ethylene blocker, and CoCl(2), an ethylene synthesis inhibitor, do not abolish plantlet root development. It is therefore unlikely that ethylene is essential for root formation. Taken together, the experiments suggest that roots develop when auxin transport from the shoot reaches a certain threshold. Ethylene may augment this effect by lowering the threshold and may come into play when the parent leaf senesces.

Pigmented Papillary Carcinoma: a Rare Tumor of the Male Breast

Primary melanin pigment containing tumors of the breast are rare. We report a pigmented papillary carcinoma of a 60-year-old male patient who presented a firm mass 1.7 cm in diameter with an ill defined border on ultrasonography behind the mamilla. To the best of our knowledge this is the third case report of this type of tumor in male breast.

Effect of Lipopolysaccharide (LPS) and Peptidoglycan (PGN) on Human Mast Cell Numbers, Cytokine Production, and Protease Composition

Human mast cell (HuMC) maturation occurs in tissues interfacing with the external environment, exposing both mast cell progenitors and mature mast cells, to bacteria and their products. It is unknown, however, whether long- or short-term exposure to bacteria-derived toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS) or peptidoglycan (PGN), influences HuMC biology.

Variations in Sentinel Node Isolated Tumour Cells/micrometastasis and Non-sentinel Node Involvement Rates According to Different Interpretations of the TNM Definitions

Breast cancers with nodal isolated tumour cells (ITC) and micrometastases are categorised as node-negative and node-positive, respectively, in the tumour node metastasis (TNM) classification. Two recently published interpretations of the TNM definitions were applied to cases of low-volume sentinel lymph node (SLN) involvement and their corresponding non-SLNs for reclassification as micrometastasis or ITC. Of the 517 cases reviewed, 82 had ITC and 435 had micrometastasis on the basis of one classification, and the number of ITC increased to 207 with 310 micrometastases on the basis of the other. Approximately 24% of the cases were discordantly categorised. The rates of non-SLN metastases associated with SLN ITCs were 8.5% and 13.5%, respectively. Although the second interpretation of low-volume nodal stage categories has better reproducibility, it may underestimate the rate of non-SLN involvement. The TNM definitions of low-volume nodal metastases need to be better formulated and supplemented with visual information in the form of multiple sample images.

Expression and Localisation of Claudin-1, -2, -3, -4, -5, -7 and -10 Proteins in the Normal Canine Mammary Gland

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumour development. The aim of the present study was to analyse the expression of claudin-1, -2, -3, -4, -5, -7 and -10 in normal canine mammary glands. Samples from the inguinal mammary regions of 20 non-castrated, 1-13 years old female dogs were studied. Immunohistochemical analysis was performed on conventional specimens and tissue microarrays. The results of the immunohistochemical reactions detecting claudins in tissue sections were photodocumented. The immunoreactivity of claudins was quantitatively analysed on digital images using Leica QWin morphometry software. Intense membranous immunolabelling was found for claudin-1, -3 and -7, intense membranous with non-granular cytoplasmic immunolabelling for claudin-2, moderate membranous immunolabelling for claudin-4 and -5, and weak membranous immunolabelling for claudin-10. The occurrence of tight junctions was confirmed by ultrathin section electron microscopy. The available data suggested that claudins might be proteins preserved throughout the evolution of mammals. The results of our study support the concept that they are indeed preserved, since the same type of claudins, in identical distribution, could be detected in our canine mammary tissue samples as could be found in human mammary tissue.

[Combined Surgery and Radiotherapy in the Treatment of Ductal Carcinoma in Situ of the Breast: Preliminary Results of the Hungarian Multicenter Prospective Randomised Study]

The aim of this work is to report the preliminary results of the Hungarian multicentric randomised DCIS study. Between 2000 and 2007, 278 patients with ductal carcinoma in situ (DCIS) treated by breast-conserving surgery were randomised according to predetermined risk groups. Low/intermediate-risk patients (n=29) were randomised to 50 Gy whole-breast irradiation (WBI) or observation. High-risk cases (n=235) were allocated to receive 50 Gy WBI vs. 50 Gy WBI plus 16 Gy tumour bed boost. Very high-risk patients (patients with involved surgical margins; n=14) were randomised to 50 Gy WBI plus 16 Gy tumour bed boost or reoperation (reexcision plus radiotherapy or mastectomy alone). Immunohistochemistry (IHC) was performed to detect the expression of potential molecular prognostic markers (ER, PR, Her2, p53, Bcl-2 and Ki-67). At a median follow-up of 36 months no recurrence was observed in the low/intermediate- and very high-risk patient groups. In the high-risk group, 4 (1.7%) local recurrences and 1 (0.4%) distant metastasis occurred. No patient died of breast cancer. In the high-risk group of patients, the 3- and 5-year probability of local recurrence was 1.1% and 3.1%, respectively. The positive immunostaining for Her2 (38%), p53 (37%) and Ki-67 (44%) correlated with a high nuclear grade. Significant inverse correlation was found between the expression of ER (77%), PR (67%), Bcl-2 (64%) and grade. Preliminary results suggest that breast-conserving surgery followed by radiotherapy yields an annual local recurrence rate of less than 1% in patients with DCIS. IHC of molecular prognostic markers can assist to gain insight into the biologic heterogeneity of DCIS.

Expression of Claudin-1, -2, -3, -4, -5 and -7 Proteins in Benign and Malignant Canine Mammary Gland Epithelial Tumours

Claudins are tight junction proteins expressed by epithelial and endothelial cells. The present study has evaluated the expression of claudin-1, -2, -3, -4, -5 and -7 in 115 hyperplastic and neoplastic lesions of the canine mammary gland and compared this expression with that of normal mammary epithelium. The lesions studied included lobular hyperplasia (n=15), simple adenoma (n=20), non-infiltrating carcinoma in situ (n=20) and infiltrating carcinomas of histological grades I, II and III (n=20 of each). There was strong expression of claudin-1, -3, -4, -5 and -7 by epithelia within examples of lobular hyperplasia and simple adenoma, and strong expression of claudin-3 and -4 by non-infiltrating carcinomas and all three grades of infiltrating carcinoma. By contrast, there was reduced expression of claudin-5 and -7 by non-infiltrating and infiltrating carcinomas and the expression of these two molecules was inversely correlated with histological grade. Claudin-1 was expressed focally within carcinoma in situ, but this molecule was not detected in any invasive carcinoma. Claudin-2 was weakly expressed within areas of lobular hyperplasia and by simple adenomas, but was not expressed by any carcinoma cells. These results suggest that loss or reduction of expression of claudin-1, -2, -5 and -7 may lead to cellular disorientation, detachment and invasion in canine mammary neoplasia.

Retrospective Evaluation of Clinical Outcomes in Patients with HER2-positive Advanced Breast Cancer Progressing on Trastuzumab-based Therapy in the Pre-lapatinib Era

Patients with HER2-positive breast cancer whose disease has become resistant to the anti-HER2 monoclonal antibody trastuzumab can benefit from lapatinib, a dual epidermal growth factor receptor/HER2 tyrosine kinase (TK) inhibitor. Before the availability of this compound, trastuzumab was often continued beyond disease progression, usually in addition to further chemotherapy, an approach which was not based on randomized studies. We sought to retrospectively compare the clinical outcomes of patients who, upon progression during an initial trastuzumab-based regimen, stopped or continued trastuzumab in addition to further chemotherapy.

Evaluation of Microvessel Density (MVD) in Canine Mammary Tumours by Quantitative Immunohistochemistry of the Claudin-5 Molecule

In our recent investigation, angiogenesis was evaluated and quantified by immunohistochemical evaluation of microvessel density (MVD) using claudin-5 (CLDN-5) as a marker for vascular endothelium in 67 canine mammary gland tumours. Computer image analysis was used to measure the intratumoural MVD. Higher intratumoural MVD was detected in malignant simple neoplasms compared with benign tumours. Furthermore, the results of MVD were correlated with histological grade, higher grades being accompanied by higher MVD. In simple adenomas and grade I tubular-tubulopapillary simple carcinomas the intratumoural microvessels were wide and regular in shape with evident erythrocytes in their lumen. In grade III solid carcinomas the microvessels were smaller, less regular and had irregular shape, often without a distinct lumen, and isolated endothelial cells were frequently present. In the complex carcinomas MVD was low and the intratumoural microvessels were mostly irregular in shape without a distinct lumen. The evaluation of MVD by CLDN-5 immunohistochemistry may give useful additional information on the angiogenic potential of breast cancers in dogs.

Expression of Tight Junction Protein Claudin-4 in Basal-like Breast Carcinomas

Claudins (CLDN) are tight junction proteins which contribute to the paracellular transport and ionic permeability of various epithelia. In recent years they came into focus for their suggested role in carcinogenesis and possible role in cancer therapy. According to our previous studies, in breast tissue CLDN4 is also related to the level of cellular differentiation. Thirty-eight estrogen (ER) and progesterone receptor (PgR) negative, HER2/neu negative, but cytokeratin 5/6 positive basal-like-mainly grade 3-breast carcinomas were compared with twenty-one grade 1, twenty-five grade 2 and twenty grade 3 non-basal-like invasive breast carcinomas, in respect to their CLDN4 expression. The immunohistochemical reactions were evaluated both semiquantitatively and by morphometrical analysis. Statistically significant difference (p = 0.001) was observable regarding CLDN4 expression in the basal-like group as compared to grade 1 and 2 cancers. Further, CLDN4 expression was significantly higher (p = 0.017) in the basal-like compared with the non-basal-like grade 3 carcinomas. Our results suggest that basal-like carcinomas are a subset of breast cancer with high level of CLDN4 protein expression. The finding is in accordance with our former observation that CLDN4 is indeed related to cellular differentiation. This observation may be seen as a further proof that basal-like carcinomas represent a separable group amongst grade 3 breast carcinomas.

Triple Test Score for the Evaluation of Invasive Ductal and Lobular Breast Cancer

The aim of our study was to compare the preoperative sum score diagnostics of invasive ductal and lobular cancers using three or four diagnostic methods. The novelty of this study is the examination of this phenomenon based on sum score, no such papers can be found in the literature. Ductal cancers have higher score values indicating easier diagnostics, but the difference in distribution of the scores was significant (p = 0.0086) only in case of the triple-test. The score values give appropriate opportunity to create their order of diagnostic power which was the same by both histologic types and in their subgroups with low sum-score: the strongest was cytology, followed by mammography, ultrasound and physical examination. No significant difference was found between the two histologic group in their mammographic appearances-stellate, circumscribed, assymmetric distortion or microcalcification-(p = 0.0694). In low score subgroup besides the occult forms, structural distortion and indeterminate microcalcifications overweighed the stellate and circumscribed lesions typical for the whole groups. In symptomless cases of both histologic groups only one strongly malignant diagnostic test result warrants the right diagnosis. Summarizing the score distribution of the results in case of four diagnostic tools the higher scores-indicating malignancy-were more frequent in the ductal group compared to the lobular ones. Extra attention has to be paid to rare radiomorphologic appearances and to the most deterministic examination, namely cytology.

WHO 1st Consultation on the Development of a Global Biodosimetry Laboratories Network for Radiation Emergencies (BioDoseNet)

The World Health Organization (WHO) held a consultation meeting at WHO Headquarters, Geneva, Switzerland, December 17-18, 2007, to develop the framework for a global biodosimetry network. The WHO network is envisioned to enable dose assessment using multiple methods [cytogenetics, electron paramagnetic resonance (EPR), radionuclide bioassays, etc.]; however, the initial discussion focused on the cytogenetic bioassay (i.e., metaphase-spread dicentric assay). Few regional cytogenetic biodosimetry networks have been established so far. The roles and resources available from United Nations (UN) agencies that provide international cooperation in biological dosimetry after radiological emergencies were reviewed. In addition, extensive reliance on the use of the relevant International Standards Organization (ISO) standards was emphasized. The results of a WHO survey of global cytogenetic biological dosimetry capability were reported, and while the survey indicates robust global capability, there was also a clear lack of global leadership and coordination. The expert group, which had a concentrated focus on cytogenetic biodosimetry, formulated the general scope and concept of operations for the development of a WHO global biodosimetry laboratory network for radiation emergencies (BioDoseNet). Follow-on meetings are planned to further develop technical details for this network.

Stromal Matrix Protein Expression Following Preoperative Systemic Therapy in Breast Cancer

Stromal alterations are observed following preoperative systemic therapy in breast cancer. The aim of the present study was to analyze the qualitative and quantitative changes of representative tumor stroma proteins in the context of neoadjuvant therapy and the response of patients undergoing preoperative systemic therapy.

The Effective Duration of Analgesia After Intrathecal Morphine in Patients Without Additional Opioid Analgesia: a Randomized Double-blind Multicentre Study on Orthopaedic Patients

To know whether the application of patient-controlled analgesia devices could be avoided if intrathecal morphine is given in combination with spinal anaesthesia.

Activation of the 2-5OAS/RNase L Pathway in CVB1 or HAV/18f Infected FRhK-4 Cells Does Not Require Induction of OAS1 or OAS2 Expression

The latent, constitutively expressed protein RNase L is activated in coxsackievirus and HAV strain 18f infected FRhK-4 cells. Endogenous oligoadenylate synthetase (OAS) from uninfected and virus infected cell extracts synthesizes active forms of the triphosphorylated 2-5A oligomer (the only known activator of RNase L) in vitro and endogenous 2-5A is detected in infected cell extracts. However, only the largest OAS isoform, OAS3, is readily detected throughout the time course of infection. While IFNbeta treatment results in an increase in the level of all three OAS isoforms in FRhK-4 cells, IFNbeta pretreatment does not affect the temporal onset or enhancement of RNase L activity nor inhibit virus replication. Our results indicate that CVB1 and HAV/18f activate the 2-5OAS/RNase L pathway in FRhK-4 cells during permissive infection through endogenous levels of OAS, but contrary to that reported for some picornaviruses, CVB1 and HAV/18f replication is insensitive to this activated antiviral pathway.

Dexamethasone and FK506 Inhibit Expression of Distinct Subsets of Chemokines in Human Mast Cells

Mast cells produce a large amount of several chemokines after cross-linking of FcepsilonRI and participate in the pathogenesis of allergic diseases. The objective of this study was to comprehensively investigate FcepsilonRI-mediated chemokine induction in human mast cells and the effect of a corticosteroid (dexamethasone) and a calcineurin inhibitor (FK506). Human peripheral blood-derived mast cells were stimulated with anti-IgE Ab in the presence of dexamethasone or FK506. Gene expression profiles were evaluated using GeneChip and confirmed by real-time PCR, and chemokine concentrations were measured by cytometric bead arrays and ELISA. Expression of eight chemokines was significantly induced in mast cells by anti-IgE stimulation. Induction of CCL2, CCL7, CXCL3, and CXCL8 by anti-IgE was significantly inhibited by dexamethasone but was enhanced by FK506. In contrast, induction of CCL1, CCL3, CCL4, and CCL18 was significantly inhibited by FK506 but, with the exception of CCL1, was enhanced by dexamethasone. Combination of dexamethasone and FK506 suppressed production of all chemokines by anti-IgE stimulation. Studies using protease inhibitors indicate that mast cell proteases may degrade several of the chemokines. These results suggest that corticosteroids and calcineurin inhibitors inhibit expression of distinct subsets of chemokines, and a combination of these drugs almost completely suppresses the induction of all chemokine genes in human mast cells in response to FcepsilonRI-dependent stimulation. This implies that a combination of a corticosteroid and a calcineurin inhibitor may be more effective than each single agent for the treatment of allergic diseases in which mast cell-derived chemokines play a major role.

Secondary Tumoural Valvulopathy in a Dog

This short report describes a case of tricuspid valvular metastasis of canine disseminated histiocytic sarcoma in a 9-year-old female Rottweiler. Immunohistochemically the malignant neoplastic cells gave a strong reaction for vimentin and lysozyme, and showed negativity for serotonin, CD3, CD79a and cytokeratin. The intratumoural microvessels were detected by immunohistochemistry using CD31 and claudin-5. This appears to be the first report of a valvular metastasis of canine malignant histiocytosis.

Claudin-5 Protein is a New Differential Marker for Histopathological Differential Diagnosis of Canine Hemangiosarcoma

Claudin-5 protein is an endothel-specific claudin, present in tight junctions. To evaluate its usefulness as a differential diagnostic marker of canine hemangiosarcomas, the expression of claudin-5 molecule was studied in different canine tumours of vascular origin.

Differences in the Expression of Histamine-related Genes and Proteins in Normal Human Adrenal Cortex and Adrenocortical Tumors

Histamine is involved in the pathogenesis of several tumors; however, there are no data on its possible involvement in human adrenocortical tumorigenesis. The expression of genes and proteins involved in the biosynthesis (histidine decarboxylase, HDC), action (histamine receptors: HRH1-HRH4), and metabolism of histamine is largely unknown both in the normal human adrenal cortex and in adrenocortical tumors. In this study, we examined the expression of histamine-related genes and proteins and histamine content in normal adrenal cortex, benign adrenocortical adenomas, and malignant adrenocortical cancer (ACC). Fifteen normal adrenals and 43 tumors were studied. mRNA expression was examined by real time RT-PCR. Western-blotting and immunohistochemistry were used for the study of proteins. Tissue histamine content was determined by enzyme-linked immunosorbent assay. We found that all proteins involved in histamine biosynthesis and action are present both in the normal adrenal cortex and in the tumors studied. HDC expression and histamine content was highest in the normal tissues and lower in benign tumors, whereas it was significantly less in ACCs. HRH3 expression was significantly higher in ACC samples than in the other groups. Adrenocortical tumorigenesis might, thus, be characterized by reduced histamine biosynthesis; furthermore, different adrenocortical tumor subtypes may show unique histamine receptor expression profiles.

Cutaneous Mast Cell Tumour Within a Lipoma in a Boxer

This report describes a case of a canine cutaneous grade I mast cell tumour which developed within a lipoma in the right axillar region of an 8-year-old male Boxer. Immunohistologically, the neoplastic mast cells were positive for serotonin, CD45 vimentin and p53, and negative for lysozyme, CD3 and CD79a expression. The proliferation index of the mast cell tumour based on the Ki-67 antigen was 6.1%. Between the benign neoplastic lipocytes and mastocytoma tumour cells intratumoural microvessels were detected by immunohistochemical staining using CD31 and claudin-5 as markers for vascular endothelium.

Human Mast Cells Synthesize and Release Angiogenin, a Member of the Ribonuclease A (RNase A) Superfamily

ANG is a plasma protein with angiogenic and ribonucleolytic activity implicated in tumor growth, heart failure, wound healing, asthma, and the composition of the adult gut microflora. Human mast cells (HuMC) are similarly associated with modulation of vascular permeability, angiogenic processes, wound healing, and asthma. We hypothesized that HuMC express and secrete ANG in response to divergent stimuli. ANG expression was evaluated in the LAD2 HMC, the HMC-1, and CD34+-derived HuMC, following exposure to live Escherichia coli, TLR ligands, or neuropeptides and following FcepsilonRI aggregation. Expression and production of ANG were determined by microarray analysis, qRT-PCR, confocal microscopy, and ELISA. Microarray analysis showed that ANG is up-regulated by LAD2 cells exposed to live E. coli. qRT-PCR analysis revealed that LAD2, HMC-1, and HuMC constitutively expressed ANG mRNA and that it was up-regulated by exposure to E. coli. Activation of HuMC by FcepsilonRI aggregation resulted in release of small amounts of ANG (<100 pg/mL), whereas compound 48/80, NGF, LPS, PGN, and flagellin activated HuMC to secrete >160 pg/mL ANG. These observations demonstrate that HuMC store and secrete ANG to a variety of stimuli and suggest that MC-derived ANG is available in the subsequent inflammatory response.

A Microarray Based Approach for the Identification of Common Foodborne Viruses

An oligonucleotide array (microarray) incorporating 13,000 elements representing selected strains of hepatitis A virus (HAV), human coxsackieviruses A and B (CVA and CVB), genogroups I and II of Norovirus (NV), and human rotavirus (RV) gene segments 3,4,10, and 11 was designed based on the principle of tiling. Each oligonucleotide was 29 bases long, starting at every 5th base of every sequence, resulting in an overlap of 24 bases in two consecutive oligonucleotides. The applicability of the array for virus identification was examined using PCR amplified products from multiple HAV and CV strains. PCR products labeled with biotin were hybridized to the array, and the biotin was detected using a brief reaction with Cy3-labeled streptavidin, the array subjected to laser scanning, and the hybridization data plotted as fluorescence intensity against each oligonucleotide in the array. The combined signal intensities of all probes representing a particular strain of virus were calculated and plotted against all virus strains identified on a linear representation of the array. The profile of the total signal intensity identified the strain that is most likely represented in the amplified cDNA target. The results obtained with HAV and CV indicated that the hybridization profile thus generated can be used to identify closely related viral strains. This represents a significant improvement over current methods for virus identification using PCR amplification and amplicon sequencing.

Cytogenetic Damage Analysed by the Dicentric Assay

Biological dosimetry, based on the analysis of dicentric chromosomes, is an internationally established, independent method applied in the area of radiation protection. The biodosimetry is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment, especially after unclear or suspected radiation dose exposures. However, a new biodosimetrical challenge has emerged in recent years in the form of a possible large scale radiation accident potentially involving large numbers of exposed persons. In order to be prepared to act in an efficient manner in such an accident, the established cytogenetic laboratories have increased their cooperation at the national and international level. General experience and results of intercomparisons are reported and future options to increase sample throughput are outlined.

Expression of the Claudin-4 Molecule in Benign and Malignant Canine Hepatoid Gland Tumours

Claudins are integral membrane proteins of the tight junction structures expressed by epithelial and endothelial cells. The present study has evaluated the expression of claudin-4 in 10 normal canine hepatoid glands and in 67 hepatoid glands with hyperplastic and neoplastic lesions. The lesions studied included normal hepatoid glands (n = 10), nodular hyperplasias (n = 10), adenomas (n = 12), epitheliomas (n = 15), differentiated carcinomas (n = 15) and anaplastic carcinomas (n = 15). There was an intensive expression of claudin-4 in normal canine hepatoid glands as well as in hyperplasias and adenomas. Claudin-4 was detected as a well-localised linear circumferential membranous staining pattern of epithelial cells (mature hepatoid cells) in normal hepatoid glands, perianal gland hyperplasias and adenomas. In nodular hyperplasia and adenoma, the reserve cells showed membrane positivity for the claudin-4 molecule. There was a weaker expression in hepatoid gland epitheliomas. In the epitheliomas, the basaloid reserve cells never expressed the claudin-4 molecule. The multiple small parts of epitheliomas in which the cells exhibited typical hepatoid features showed a well-localised linear circumferential membranous staining pattern for claudin-4. The numerical score for cellular expression of claudin-4 was higher in differentiated carcinomas than in epitheliomas, but moderately lower than in adenomas. The anaplastic, poorly differentiated hepatoid gland carcinomas showed an overexpression of claudin-4. These results suggest that low claudin-4 expression in epitheliomas is a molecular characteristic indicative of increasing cellular disorientation, detachment motility and invasion by tumour cells, and claudin-4 seems to be helpful in distinguishing undifferentiated carcinomas from differentiated carcinomas and epitheliomas of the hepatoid gland. In addition, claudin-4 can help distinguish epithelioma from differentiated carcinoma of the canine hepatoid gland.

The Potential of Natural Products As Effective Treatments for Allergic Inflammation: Implications for Allergic Rhinitis

The impact of natural products on human health has been enormous, and the study of natural products continues to influence research in the fields of chemistry, biology, and ecology. Historically, the majority of our medicines originate from natural products and their synthetic derivatives, many of which have taught us valuable lessons about biology. While advances in synthetic and combinatorial chemistry have given rise to notable successes in the development of new drugs, the perceived value of natural products in the treatment of allergic disease has yet to be fully explored. The immune system is a highly complex, intricately regulated group of cells whose integrated function is essential to health. Cells of the immune system may interact in a cell-cell manner and may also respond to intercellular messages including hormones, cytokines, and effector molecules produced by various cells. These effector molecules include histamine, kinins, leukotrienes, prostaglandins, and serotonin. The immune system can be modified by diet, pharmacologic agents, environmental pollutants, and naturally occurring food chemicals, such as vitamins and flavonoids. Allergic inflammation is mediated by several types of immune cells all of which can be effected by these naturally occurring bioactive compounds but this review will focus on mast cells and their mediators since these cells are the focal point of allergic reactions such as allergic rhinitis. The molecular mechanisms and scientific validity of some herbal remedies currently used clinically in the treatment of allergic rhinitis will be explored.

Changes Induced by Two Strains of Vibrio Splendidus in Haemocyte Subpopulations of Mya Arenaria, Detected by Flow Cytometry with LysoTracker

Flow-cytometric characterisation of bivalve haemocytes is usually performed by light-scatter profiles based on size and complexity of the cells. Additional means of characterisation such as specific fluorescent dyes are not commonly used to discriminate cell subpopulations in challenged and unchallenged haemocytes. In the present study, we characterise the changes in haemocyte subpopulations of soft-shell clam Mya arenaria induced by in vivo challenge with 2 strains of Vibrio splendidus by using a fluorescent probe. Responses were measured 24 h after infection with either a local wild strain (7SHRW) or a modification (LGP32-GFP) of a strain associated with oyster mortalities in France (LGP32). Changes in haemocyte subpopulations were analysed using flow cytometry based on 2-parameter scatter profiles and lysosomal content reflected by LysoTracker staining. Forward and side-scatter profiles revealed 2 haemocyte subpopulations: hyalinocytes and granulocytes. Granulocytes exhibited significantly higher levels of lysosomal staining (p < 0.01). Following infection with LGP32-GFP, both subpopulations merged into a single continuous group and their lysosomal content significantly decreased (p < 0.05). Independent modifications after infection were observed in the proportions of subpopulations established by their lysosomal content. While the subpopulation of hyalinocytes had lower levels of lysosomal content after infection, especially with LGP32-GFP (p < 0.001), the subpopulation of granulocytes had similar levels of lysosomes after infection with 7SHRW and significantly decreased levels after infection with LGP32-GFP (p = 0.001). Our data suggest specific modulation of bivalve responses against pathogenic bacteria that would include degranulation.

[Immunohistochemical Phenotype of Breast Carcinomas Predicts the Effectiveness of Primary Systemic Therapy]

The purpose of the study was to identify breast cancer subtypes by immunohistochemistry likely to respond to neoadjuvant chemotherapy and to analyze the used chemotherapy regimen and the range of response rates. Analysis of a collected database was performed. Ninety-two patients were identified in our files who received neoadjuvant chemotherapy between 1998 and 2009. We used immunohistochemical profiles (ER, PgR, HER2, Ki-67 and p53) of NCB, FNAB and surgical breast specimens to subclassify the tumors. Pathological response rates were assessed following surgical removal of tumors by using the Chevallier classification. DFS and OS was measured in 88 cases from the date of definitive surgery to the date of last follow-up or death. Pathological complete or near-complete remission (pCR = Chevallier I and II) was observed in 13 of 92 cases (14.1%). According to the preoperative characteristics of the 13 tumors achieving pCR, 9 of the cases were triple negative, one of 13 was ER-/HER2+ and three of 13 ER+/HER2+. Twenty-four of 92 patients received taxane based neoadjuvant chemotherapy, 30 of 92 anthracycline based neoadjuvant chemotherapy, 33 of 92 taxane + anthracycline regimen and 2 of 92 CMF regimen. In the taxane treated group of patients the pCR rate was 29.1%, in the anthracycline group 6.6% and in the taxane + anthracycline treated group 12.1%. Concerning DFS, significant difference was observed between the Chevallier III and IV groups (p=0.006), and less events were observed in the pCR group (not significant). pCR was associated with significantly better OS (p=0.050). It seems that even limited, routinely used immunohistochemical profiling of tumors is able to predict the likelihood of pCR to neoadjuvant chemotherapy. Patients with triple negative and HER2-positive cancers are likely to achieve pCR after neoadjuvant chemotherapy.

Beta-catenin Expression and Claudin Expression Pattern As Prognostic Factors of Prostatic Cancer Progression

To investigate the patterns of expression of the junctional proteins beta-catenin and claudins in different prognostic groups of patients with prostatic cancer, to determine their value as prognostic markers.

Expression of Claudin-1, -3, -4, -5 and -7 Proteins in Low Grade Colorectal Carcinoma of Canines

The aim of the present study was to characterise the expression pattern of claudin-1, -3, -4, -5 and -7 tight junction proteins in canine normal colorectum and in the low-grade, tubulopapillary colorectal carcinoma in canines. Methods and results: The biopsy samples included 10 canine normal colorectal tissues and 20 canine low grade colorectal carcinomas (CLGCCs). The canine normal colorectal mucosa was negative for claudin-1. Claudin-1 was detected as a non-diffuse intense membrane labelling of neoplastic epithelial cells in low grade colorectal cancer in canines. Fifty five per cent of all tumours showed a weak cytoplasmic pattern of staining for claudin-1 protein. The normal colorectal mucosa showed diffuse punctate positivity for claudin-3. Claudin-3 was detected as an intense lateral membrane labelling of tumour cells in CLGCCs. Claudin-4 expression in surface and crypt epithelial cells of the intact colorectal mucosa in canines was punctate. Claudin-4 molecule was detected as a lateral membrane labelling of neoplastic cells in CLGCCs. The epithelium of the CLGCCs and the low grade colorectal carcinoma were negative for claudin-5. The surface and crypt epithelial cells of the canine normal colorectal mucosa showed a diffuse lateral membranous pattern of staining for claudin-7. Claudin-7 molecule was detected as an intense membrane labelling of neoplastic cells in CLGCCs. Seventy per cent of all tumours showed weak cytoplasmic positivity for claudin-7. Conclusion: Consequently, we hypothesize that claudin-1 plays a role in the progression of CLGCCs. Further functional studies are needed to clarify the biological role of the mislocalization of the claudin-1 molecule from cell membrane to the cytoplasm in CLGCCs. Lower claudin-4 expression suggests that reduced expression of claudin-4 molecule may lead to cellular disorientation, detachment and invasion of CLGCCs. Further functional studies are needed to clarify the biological role of overexpression and mislocalisation of claudin-7 in CLGCCs.

[Pathological Aspects of in Situ Carcinoma/intraepithelial Neoplasia of the Breast]

Dramatic development has happened in the field of classical and molecular breast pathology in the last three decades. Introduction of systematic screening programs advanced our knowledge in the field of classical surgical pathology, while molecular technical revolution resulted in dramatic improvement of our understanding of molecular pathology of breast tumors and precancerous lesions. This continuous increase of our knowledge results in the change of our concepts, classifications and approach. In this review, I would like to share the new and recently adapted views regarding intraepithelial neoplastic lesions of the breast.

Nodal-stage Classification in Invasive Lobular Breast Carcinoma: Influence of Different Interpretations of the PTNM Classification

PURPOSE Application of current nodal status classification is complicated in lobular breast carcinoma metastases. The aim of this study was to define the optimal interpretation of the pTNM classification in sentinel node (SN) -positive patients to select patients with limited or with a high risk of non-SN involvement. PATIENTS AND METHODS SN metastases of 392 patients with lobular breast carcinoma were reclassified according to interpretations of the European Working Group for Breast Screening Pathology (EWGBSP) and guidelines by Turner et al, and the predictive power for non-SN involvement was assessed. Results Reclassification according to definitions of EWGBSP and Turner et al resulted in different pN classification in 73 patients (19%). The rate of non-SN involvement in the 40 patients with isolated tumor cells according to Turner et al and with micrometastases according to EWGBSP was 20%, which is comparable to the established rate for micrometastases. The rate of non-SN involvement in the 29 patients with micrometastases according to Turner et al and with macrometastases according to EWGBSP was 48%, which is comparable to the established rate for macrometastases. Therefore, the EWGBSP method to classify SN tumor load better reflected the risk of non-SN involvement than the Turner et al system. CONCLUSION Compared with the guidelines by Turner et al, the EWGBSP definitions better reflect SN metastatic tumor load and allow better differentiation between patients with lobular breast carcinoma who have a limited or a high risk of non-SN metastases. Therefore, we suggest using the EWGBSP definitions in these patients to select high-risk patients who may benefit from additional local and/or systemic therapy.

Substance P Downregulates Expression of the High Affinity IgE Receptor (FcepsilonRI) by Human Mast Cells

The effect of the neuropeptide substance P (SP) on human mast cell (MC) phenotype is poorly understood. In this study, SP effects on human MC expression of the high affinity IgE receptor (FcepsilonRI) were characterized. SP downregulated expression of FcepsilonRI mRNA and protein by approximately 50% and in a concentration dependent manner, the effect was partially mediated by engagement of the neurokinin-1 receptor (NK1R) and resulted in reduced mast cell activation. Sensitization of MC with IgE prior to SP exposure protected MC from SP-mediated FcepsilonRI downregulation. SP release may inhibit MC responses to allergens and these results may have implications in neuroinflammatiion and stress.

Expression of Claudin-5 in Hepatoid Gland Biopsies

Claudins are integral membrane proteins involved in the structure of the tight junctions found in epithelial and endothelial cells. This study evaluated the expression of claudin-5 in 67 hyperplastic and neoplastic lesions of canine hepatoid glands. These included normal hepatoid glands (n = 10), nodular hyperplasia (n = 10), adenomas (n = 12), epitheliomas (n = 15), differentiated carcinomas (n = 15) and anaplastic carcinomas (n = 15). There was intense lateral membrane expression of claudin-5 on epithelial cells from normal hepatoid glands, nodular hyperplasia and adenomas, but expression was weaker in hepatoid gland epitheliomas. Basal reserve cells from normal glands, nodular hyperplasia, adenomas and epitheliomas never expressed claudin-5. There was membrane-bound immunoreactivity for claudin-5 in selected areas of the epitheliomas where the cells exhibited typical hepatoid features. The weak expression of claudin-5 molecule in epitheliomas may nevertheless lead to cellular disorientation, detachment and invasion. Claudin-5 expression seemed to be helpful in distinguishing poorly differentiated carcinomas, differentiated carcinomas and epitheliomas of the hepatoid glands. Increased claudin-5 expression by invasive anaplastic carcinomas may facilitate invasion and metastasis through the activation of matrix metalloproteinases.

Syndecan-4 Promotes Cytokinesis in a Phosphorylation-dependent Manner

During mitosis, cells detach, and the cell-matrix interactions become restricted. At the completion of cytokinesis, the two daughter cells are still connected transiently by an intercellular bridge (ICB), which is subjected to abscission, as the terminal step of cytokinesis. Cell adhesion to the matrix is mediated by syndecan-4 (SDC4) transmembrane heparan sulfate proteoglycan. Our present work demonstrated that SDC4 promotes cytokinesis in a phosphorylation-dependent manner in MCF-7 breast adenocarcinoma cells. The serine179-phosphorylation and the ectodomain shedding of SDC4 changed periodically in a cell cycle-dependent way reaching the maximum at G2/M phases. On the contrary, the phospho-resistant Ser179Ala mutant abrogated the shedding. The phosphorylated full-length and shed remnants enriched along the mitotic spindles, and subsequently in the ICBs, however, proper membrane insertion was necessary for midbody localization. Expression of phosphomimicking Ser179Glu SDC4 resulted in incomplete abscission, whereas expression of the phospho-resistant SDC4 led to giant, multinucleated cells.

The Role of G Protein-coupled Receptors in Mast Cell Activation by Antimicrobial Peptides: is There a Connection?

Antimicrobial peptides (AMPs) are ancient and essential elements of the host defense system, which are found in a wide variety of species. They show antimicrobial activity against a wide range of pathogenic microorganisms. In addition, AMPs are expressed by different immune cells and have a important function in host innate immune response against pathogens by mechanisms that are different from those involved in direct microbial cytolysis. One host innate immune response that is directly activated by AMPs involves induction of localized inflammation through interaction with mast cells. Activation of mast cells releases pre-formed mediators, cytokines, chemokines and eicosaniods, which influence recruitment, survival, phenotype and functions of many immune cells. Mast cells can respond to AMPs independent of antigen and Fc epsilon receptor 1 stimulation. One of these pathways involves G protein-coupled receptor signaling, which can lead to mast cell degranulation. Whether AMPs activate G proteins in mast cells through a receptor-dependent or a receptor-independent mechanism remains poorly understood and there are a great many questions that have yet to be answered. In this review, we will discuss the possible involvement and role of GPCRs in mast cells activation by AMPs and the gaps in our current understanding of this important interaction.

[Comparison of Breast Cancer in Young and Old Women Based on Clinicopathological Features]

The two far ends of the age at the diagnosis of breast cancer are the age of younger than 35, and that of older than 70. Most probably, these two groups of patients differ in many ways. The aim of our present study was to underline the fact that age at the diagnosis of breast cancer is indeed a prognostic factor. Between October 1995 and March 2009, 80 old and 51 young breast cancer patients were treated at the Department of Diagnostic Radiology and Oncotherapy, Semmelweis University, Budapest. The prognostic and predictive factors of the tumors were analysed together with the disease-free and overall survival data. There were statistically significant differences between the two groups concerning the menstrual and reproductive factors, histological characteristics and immunophenotype of the tumors. Tumor size, nodal status and the Nottingham Prognostic Index did not show statistically significant differences. A trend to a shorter disease-free survival, higher rate of distant metastases and disease-specific death was seen in the group of young patients, but it was not significant. Overall survival was significantly shorter in the group of young patients. Therefore, we can state that young patients have a more aggressive disease and worse outcome. There is an increased importance of self examination in these groups, since both age groups are beyond the age limits of the screening population in Hungary. The media and primary school education as well should be involved in educating women concerning this aspect. The individual follow-up of young patients with positive family history should also be established.

Substance P Primes Lipoteichoic Acid- and Pam3CysSerLys4-mediated Activation of Human Mast Cells by Up-regulating Toll-like Receptor 2

Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and potentiating signalling pathways associated with TLR. These results suggest that neuronal responses may influence innate host defence responses.

Claudin-7 Protein Differentiates Canine Cholangiocarcinoma from Hepatocellular Carcinoma

The aim of the present study was to characterise the expression pattern of claudin-7 tight junction protein in canine normal liver, hyperplastic and primary neoplastic lesions of the canine liver and whether this tight junction protein can help differentiate canine cholangiocarcinomas from canine hepatocellular carcinomas. METHODS AND RESULTS: Necropsy samples included 15 canine normal liver tissue samples, 10 hepatocellular nodular hyperplasias, 6 hepatocellular adenomas, 15 well-differentiated and 6 poorly differentiated hepatocellular carcinomas, 6 cholangiocellular hyperplasias, 10 cholangiocellular adenomas, 15 well-differentiated and 6 poorly differentiated cholangiocarcinomas, 6 normal extrahepatic bile ducts, 8 normal gall bladder tissue samples, and 5 cystic mucinous hyperplasias of the gall bladder. In all canine normal liver tissue samples the hepatocytes were negative for claudin-7 and the normal biliary epithelial cells showed intense basolateral membrane claudin-7 positivity. In all cholangiocellular hyperplasia samples and in all cholangiocellular adenoma samples the benign cholangiocytes showed intense basolateral membrane positivity for claudin-7. In all samples of the well-differentiated and poorly differentiated cholangiocarcinomas, the malignant neoplastic biliary epithelial cells showed intense basolateral membrane positivity for claudin-7. Neither the hyperplastic nodules of the liver cells nor the hepatocellular adenomas reacted with claudin-7. The well-differentiated and poorly differentiated hepatocellular cancers were negative for claudin-7. The epithelial cells of canine normal extrahepatic bile ducts, gall bladder and cystic mucinous hyperplasias of the gall bladder showed intense basolateral membrane positivity for claudin-7. Differences in the intensity of claudin-7 reaction were not apparent among different types of proliferative lesions of cholangiocytes or degrees of cellular differentiation of neoplastic biliary epithelial cells. CONCLUSION: Consequently, we hypothesize that claudin-7 is an excellent immunohistochemical marker of the cholangiocellular differentiation in canines and can be used to detect benign and malignant proliferative lesions of the canine biliary tract. It can also help to differentiate canine cholangiocarcinomas from hepatocellular carcinomas.

Exercising During Learning Improves Vocabulary Acquisition: Behavioral and ERP Evidence

Numerous studies have provided evidence that physical activity promotes cortical plasticity in the adult brain and in turn facilitates learning. However, until now, the effect of simultaneous physical activity (e.g. bicycling) on learning performance has not been investigated systematically. The current study aims at clarifying whether simultaneous motor activity influences verbal learning compared to learning in a physically passive situation. Therefore the learning behavior of 12 healthy subjects (4 male, 19-33 years) was monitored over a period of 3 weeks. During that time, behavioral and electrophysiological responses to memorized materials were measured. We found a larger N400 effect and better performance in vocabulary tests when subjects were physically active during the encoding phase. Thus, our data indicate that simultaneous physical activity during vocabulary learning facilitates memorization of new items.

[Treatment of Pregnancy Associated Breast Cancer]

Pregnancy-associated breast cancer (PABC) is defined as cancer of the breast diagnosed during pregnancy and up to 1 year postpartum. The crude incidence is 1/3000 pregnant women. As women delay childbearing the incidence of PABC increases with age. Young patients with PABC do not have worse prognosis compared with those with non-PABC; however, pregnancy can contribute to a delay in breast cancer diagnosis, evaluation, and treatment. Primary care physicians and gynecologists should be careful in the thorough workup of breast symptoms in the pregnant population to expedite diagnosis and allow multidisciplinary treatment as early as possible following the established diagnosis. Authors report a case of a 30-year-old pregnant woman, who detected inflammatory signs of her right breast and a palpable axillary mass at the 21st week of gestation. Her symptoms did not improve with administration of antibiotics. Therefore fine needle aspiration biopsy of the axillary lump was performed, with the result of unequivocal diagnosis of metastatic invasive carcinoma. The patient was referred to the multidisciplinary tumor board of our Department at the 27st week of gestation with the symptoms of inflammatory breast cancer, palpable right axillary and supraclavicular lymph nodes. Core biopsy showed an ER and PR negative, Her-2 positive, grade 3, infiltrating ductal carcinoma of the breast. After multidisciplinary team consultation the patient declined any kind of therapy during her pregnancy. On the 30th week of gestation caesarean section was performed. The premature baby girl was treated in the Neonatal Intensive Care Unit. Imaging modalities revealed no evidence of distant metastases short after the delivery. After 6 cycles of chemotherapy (docetaxel-doxorubicin-cycclophosphamid) the patient underwent right mastectomy and axillary lymph node dissection. Complete pathological response was diagnosed, since no residual tumor was found in the surgical resection specimen. After radiotherapy, trastuzumab medication was initiated. To date, there is no evidence of local recurrence or distant metastases during her 24 months follow-up. The newborn is on close neurohabilitation follow-up due to the evidence of generalized muscle dystonia. Had the patient accepted chemotherapy, the damage of the newborn baby would have been avoidable.

Claudin-5-positive Angioleiomyoma in the Uterus of a Degu (Octodon Degus )

A 5-year-old female degu (Octodon degus ) showed the clinical sign of metrorrhagia. During ovariohysterectomy a circumscribed tumoural lesion was found in the right uterine horn. The histopathological diagnosis of this soft tissue mass was primary benign cavernous angioleiomyoma of the uterus. During immunohistochemical analysis the neoplastic endothelial cells of this mixed mesenchymal tumour showed strong membrane positivity for the endothelial marker claudin-5 but were negative for CD31 (another endothelial marker). The endothelial cells of the internal positive control tissues such as intact peritumoural vessels were positive for claudin-5 but negative for the CD31 endothelial marker. As it has been described also in other species, it seems that claudin-5 is a better endothelial marker than CD31 for the detection of normal and neoplastic endothelial cells in different tissues of degus.

Isolation of Tissue Mast Cells

Located primarily in tissues, mast cells are one of the principal effector cells in allergic inflammation. Mast cells derive from mononuclear precursor cells which undergo their final phase of differentiation in the tissues. Mast cells express a unique set of proteases and display functional diversity depending on the tissue in which they differentiate--a phenomenon often referred to as mast cell heterogeneity. Enzymatic digestion and density centrifugation have often been used to isolate human mast cells from tissues such as lung and skin, frequently resulting in cells with low viability and purity. Here, we describe a protocol that combines gentle enzymatic digestion with positive selection techniques to isolate reasonably viable and substantially enriched preparations of tissue mast cells.

MicroRNA Expression Profiling in Benign (sporadic and Hereditary) and Recurring Adrenal Pheochromocytomas

MicroRNAs are involved in the pathogenesis of several tumors, however, there have been no data on microRNA expression in pheochromocytomas to date. The objective of our study was to perform microRNA expression profiling in sporadic and hereditary benign, and recurring adrenomedullary tumors. Furthermore, the applicability of formalin-fixed paraffin-embedded tissue samples for the analysis of microRNA expression in pheochromocytomas was examined. MicroRNA expression data of three matched frozen and formalin-fixed paraffin-embedded samples were correlated. A total of 21 formalin-fixed paraffin-embedded samples (sporadic benign, multiple endocrine neoplasia 2, von Hippel-Lindau disease, sporadic recurring) were subjected to microRNA expression profiling using microarrays. MicroRNAs with significant differences in expression were validated and sample sizes were extended including tumors from neurofibromatosis type 1 patients by real-time quantitative reverse-transcription PCR (n=33). MicroRNA target prediction was carried out by TargetScan and MicroCosm Targets. Pathway analysis of targets was performed by Ingenuity Pathway Analysis and DIANA mirPath. Furthermore, microRNA expression profiles of a malignant pheochromocytoma and a pair of primary and recurrent tumors were studied by TaqMan Human MicroRNA Cards. MicroRNA expression correlated well between frozen and formalin-fixed paraffin-embedded samples (70-92%). Microarray analysis revealed 16 significantly differentially expressed microRNAs. Five of these were validated by real-time RT-PCR. miR-139-3p, miR-541 and miR-765 were significantly differentially expressed between sporadic benign and von Hippel-Lindau-related pheochromocytomas. Significantly higher expression of miR-885-5p and miR-1225-3p was found in multiple endocrine neoplasia type 2 and sporadic recurring pheochromocytomas, respectively. Pathway analysis revealed the possible involvement of Notch- and G-protein-coupled receptor signaling in tumor recurrence. MicroRNA expression profiles in the primary recurrent and recurring malignant comparisons have been similar. In conclusion, we have proved that formalin-fixed paraffin-embedded samples can be used for the analysis of microRNA expression in pheochromocytomas. MicroRNA expression patterns differ between various sporadic, hereditary and recurring tumors and miR-1225-3p may be useful for identifying recurring pheochromocytomas.

Influence of Mitochondrial Genome Rearrangement on Cucumber Leaf Carbon and Nitrogen Metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO(2) assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH(4) (+) assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu.

[Pathologic Diagnosis and Histopathology Record of Breast Cancer]

Immunohistochemical Detection of Arteriolar Hyperplasia in Canine Liver Biopsy Samples Using the Claudin-5 Antibody

Claudins are key tight junctional proteins between adjacent epithelial, mesothelial or endothelial cells, which are responsible for the permeability of the paracellular space. This paper describes that the endothelial cells of normal hepatic arterioles, portal venules and portal lymphatics as well as the endothelium of sinusoids from dogs show strong membranous claudin-5 cross-reactivity. In 25 liver biopsy samples taken from dogs with portal vein hypoperfusion, an increased number of arterioles was detected in the portal areas (PAs) by the use of humanised anti-claudin-5 antibody. The increased number of hyperplastic hepatic arterioles per PA was 5-6, 8-12 and 15-20 in the case of small, medium-sized and large PAs, respectively. It is suggested that the claudin-5 marker can improve the detection of hepatic arteriolar proliferation in the PAs of liver samples.

[Pathology Background of Targeted Therapy; Quality Control in Pathology]

The administration of targeted therapy of gastric carcinoma is a very important recent improvement of its treatment and prognosis. The basis of the successful treatment is the excellent quality of pathology, now including HER2 testing: the use of validated methods and strict criteria. This is especially important if we consider that many gastric cancers are diagnosed in small biopsy material, in which HER2 testing is challenging. This requires standardized, validated methods and experienced pathologists. Being of diagnostic and predictive significance, high quality of both the technique and the interpretation of the test is mandatory. In order to achieve general high quality in this field, technical and interpretation external quality control of HER2 testing is necessary. Hungarian pathologists with the help of Roche Hungary Ltd. completed an external quality control round which showed that most of the participating laboratories are able. Kulka J, Szirtes I, Szász AM, Kupcsulik P, Kenessey I, Lotz G, Tímár J. Pathology background of targeted therapy; quality control in pathology.

Validation of Continuous Particle Monitors for Personal, Indoor, and Outdoor Exposures

Continuous monitors can be used to supplement traditional filter-based methods of determining personal exposure to air pollutants. They have the advantages of being able to identify nearby sources and detect temporal changes on a time scale of a few minutes. The Windsor Ontario Exposure Assessment Study (WOEAS) adopted an approach of using multiple continuous monitors to measure indoor, outdoor (near-residential) and personal exposures to PM₂.₅, ultrafine particles and black carbon. About 48 adults and households were sampled for five consecutive 24-h periods in summer and winter 2005, and another 48 asthmatic children for five consecutive 24-h periods in summer and winter 2006. This article addresses the laboratory and field validation of these continuous monitors. A companion article (Wheeler et al., 2010) provides similar analyses for the 24-h integrated methods, as well as providing an overview of the objectives and study design. The four continuous monitors were the DustTrak (Model 8520, TSI, St. Paul, MN, USA) and personal DataRAM (pDR) (ThermoScientific, Waltham, MA, USA) for PM₂.₅; the P-Trak (Model 8525, TSI) for ultrafine particles; and the Aethalometer (AE-42, Magee Scientific, Berkeley, CA, USA) for black carbon (BC). All monitors were tested in multiple co-location studies involving as many as 16 monitors of a given type to determine their limits of detection as well as bias and precision. The effect of concentration and electronic drift on bias and precision were determined from both the collocated studies and the full field study. The effect of rapid changes in environmental conditions on switching an instrument from indoor to outdoor sampling was also studied. The use of multiple instruments for outdoor sampling was valuable in identifying occasional poor performance by one instrument and in better determining local contributions to the spatial variation of particulate pollution. Both the DustTrak and pDR were shown to be in reasonable agreement (R² of 90 and 70%, respectively) with the gravimetric PM₂.₅ method. Both instruments had limits of detection of about 5 μg/m³. The DustTrak and pDR had multiplicative biases of about 2.5 and 1.6, respectively, compared with the gravimetric samplers. However, their average bias-corrected precisions were <10%, indicating that a proper correction for bias would bring them into very good agreement with standard methods. Although no standard methods exist to establish the bias of the Aethalometer and P-Trak, the precision was within 20% for the Aethalometer and within 10% for the P-Trak. These findings suggest that all four instruments can supply useful information in environmental studies.

Prognostic Significance of Claudin Expression Changes in Breast Cancer with Regional Lymph Node Metastasis

Adherent and tight junction molecules have been described to contribute to carcinogenesis and tumor progression. Additionally, the group of claudin-low tumors have recently been identified as a molecular subgroup of breast carcinoma. In our study, we examined the expression pattern of claudins, beta-catenin and E-cadherin in invasive ductal (IDCs) and lobular (ILCs) carcinomas and their corresponding lymph node metastases (LNMs). Tissue microarrays of 97 breast samples (60 invasive ductal carcinomas, 37 invasive lobular carcinomas) and their corresponding LNMs have been analyzed immunohistochemically for claudin-1, -2, -3, -4, -5, -7, beta-catenin and E-cadherin expression. The stained slides were digitalized with a slide scanner and the reactions were evaluated semiquantitatively. When compared to LNMs, in the IDC group beta-catenin and claudin-2, -3, -4 and -7 protein expression showed different pattern while claudin-1, -2, -3, -4 and -7 were differently expressed in the ILC group. Lymph node metastases developed a notable increase of claudin-5 expression in both groups. Decrease or loss of claudin-1 and expression of claudin-4 in lymph node metastases correlated with reduced disease-free survival in our patients. According to our observations, the expression of epithelial junctional molecules, especially claudins, is different in primary breast carcinomas compared to their lymph node metastases as demonstrated by immunohistochemistry. Loss of claudin junctional molecules might contribute to tumor progression, and certain claudin expression pattern might be of prognostic relevance.

Distinction of Isolated Tumour Cells and Micrometastasis in Lymph Nodes of Breast Cancer Patients According to the New Tumour Node Metastasis (TNM) Definitions

Isolated tumour cells and micrometastases represent two different staging categories and are often dealt with differently when identified in sentinel lymph nodes of breast cancer patients. The reproducibility of these categories was found to be suboptimal in several studies. The new edition of the TNM (Tumour Node Metastasis) is expected to improve the reproducibility of these categories. Fifty cases of possible low-volume nodal involvement were represented by one to four digital images and were analysed by members of the European Working Group for Breast Screening Pathology (EWGBSP). The kappa value for interobserver agreement of the pN (TNM) staging categories and of the isolated tumour cells category were 0.55 and 0.56 reflecting moderate reproducibility, and the kappa of the micrometastatic category (0.62) reflected substantial reproducibility. This is an improvement over the results gained on the basis of the previous edition of the TNM. Maximal adherence to the category definitions supplemented by explanatory texts in the staging manual should result in more homogeneous nodal staging of breast cancer.

The WST Survival Assay: an Easy and Reliable Method to Screen Radiation-sensitive Individuals

An easy, fast and reliable method was developed to screen hundreds of Epstein-Barr virus-transformed cell lines (lymphoblastoid cell lines, LCLs) for radiation sensitivity that were generated from lymphocytes isolated from young lung cancer patients. The WST-1 test explores the metabolic activity of the mitochondria as an indicator for the vital status of cells. Cell proliferation as well as indirect cell death can be quantified by this method on a large scale in microtiter plates. Cell survival was measured at 24- and 48-h post-irradiation with 10 Gy ((137)Cs source) by the WST-1 assay and Trypan blue staining. To set up the experimental screening conditions and to establish a positive and a negative control, an ATM-mutated cell line from a radiation-sensitive ATM patient and an ATM proficient cell line from a healthy brother were compared. An optimal differentiation between the two cell lines was demonstrated for 10 Gy and 24- and 48-h cell growth after irradiation. Upon screening 120 LCLs of young lung cancer patients under these conditions, 5 of them were found to be radiation sensitive to a high degree of statistical significance. The results have been confirmed by a second laboratory by means of Trypan blue testing. The WST-1 test represents an efficient and reliable method by means of screening for radiation-sensitive cell lines.

Review of Retrospective Dosimetry Techniques for External Ionising Radiation Exposures

The current focus on networking and mutual assistance in the management of radiation accidents or incidents has demonstrated the importance of a joined-up approach in physical and biological dosimetry. To this end, the European Radiation Dosimetry Working Group 10 on 'Retrospective Dosimetry' has been set up by individuals from a wide range of disciplines across Europe. Here, established and emerging dosimetry methods are reviewed, which can be used immediately and retrospectively following external ionising radiation exposure. Endpoints and assays include dicentrics, translocations, premature chromosome condensation, micronuclei, somatic mutations, gene expression, electron paramagnetic resonance, thermoluminescence, optically stimulated luminescence, neutron activation, haematology, protein biomarkers and analytical dose reconstruction. Individual characteristics of these techniques, their limitations and potential for further development are reviewed, and their usefulness in specific exposure scenarios is discussed. Whilst no single technique fulfils the criteria of an ideal dosemeter, an integrated approach using multiple techniques tailored to the exposure scenario can cover most requirements.

Estrogen Receptor Negative and Progesterone Receptor Positive Breast Carcinomas-how Frequent Are They?

Estrogen receptor (ER) testing has become an important part of breast cancer reporting as the ER status is a predictor of hormonal treatment efficacy. Progesteron receptors (PR) are often tested in parallel, and the best response to hormonal manipulations can be expected in tumors positive for both receptors. The existence of breast cancers with an ER negative and PR positive phenotype is controversial. A series of cases with this phenotype were reevaluated to clarify the existence and the frequency of this entity. A total of 205/6587 (3.1%; range of the rate per department: 0.3-7.1%.) cases reported to have the ER-negative and PR-positive status by immunohistochemistry were collected from 9 Hungarian departments. After careful reevaluation of the tumor slides and control tissues with a 1% cut-off for positivity and restaining of the questionable cases, all but 1 of the reevaluable 182 cases changed their original phenotype. Most cases converted to dual positives (n = 124) or dual negatives (n = 31) or unassessable / questionable. ER-negative and PR-positive breast cancers are very rare if existing. Such a phenotype should prompt reassessment.

Expression of Claudin-5 in Canine Pancreatic Acinar Cell Carcinoma - An Immunohistochemical Study

Claudin-5 is an endothelium-specific tight junction protein. The aim of the present study was to detect the expression pattern of this molecule in intact pancreatic tissues and in well-differentiated and poorly differentiated pancreatic acinar cell carcinomas from dogs by the use of cross-reactive humanised anticlaudin-5 antibody. The necropsy samples taken from dogs included 10 nonneoplastic pancreatic tissues, 10 well-differentiated pancreatic acinar cell carcinomas, 10 poorly differentiated pancreatic acinar cell carcinomas, 5 intrahepatic metastases of well-differentiated and 5 intrahepatic metastases of poorly differentiated acinar cell carcinomas. A strong lateral membrane claudin-5 positivity was detected in exocrine cells in all intact pancreas samples. The endocrine cells of the islets of Langerhans and the epithelial cells of the ducts were negative for claudin-5. The endothelial cells of vessels and lymphatic channels in the stroma of the intact pancreas showed strong membrane positivity for this claudin. All well-differentiated exocrine pancreas carcinomas and all poorly-differentiated pancreatic acinar cell carcinoma samples showed a diffuse loss of claudin-5 expression. The claudin-5-positive peritumoural vessels and lymphatic channels facilitated the detection of vascular invasion of the claudin-5-negative cancer cells. In liver metastasis samples, the pancreatic carcinomas were negative for claudin-5. It seems that the loss of expression of claudin-5 may lead to carcinogenesis in canine exocrine pancreatic cells.

Claudin-7-positive Synchronous Spontaneous Intrahepatic Cholangiocarcinoma, Adenocarcinoma and Adenomas of the Gallbladder in a Bearded Dragon (Pogona Vitticeps)

In this study, synchronous spontaneous, independent liver and gallbladder tumours were detected in a Bearded dragon (Pogona vitticeps). The multiple tumours consisted of intrahepatic cholangiocarcinoma as well as in situ adenocarcinoma and two adenomas of the gallbladder. The biliary epithelial cells and the cholangiocarcinoma showed membranous cross-immunoreactivity for claudin-7. The gallbladder epithelial cells, its adenoma and adenocarcinoma showed basolateral cross-reactivity for claudin-7. We think that the humanised anti-claudin-7 antibody is a good marker for the detection of different primary cholangiocellular and gallbladder tumours in Bearded dragons. The cholangiocytes, the cholangiocarcinoma, the endothelial cells of the liver and the epithelial cells and gallbladder tumours all showed claudin-5 cross-reactivity. The humanised anti-cytokeratin AE1-AE3 antibody showed cross-reactivity in the biliary epithelial cells, cholangiocarcinoma cells, epithelial cells and tumour cells of the gallbladder. It seems that this humanised antibody is a useful epithelial marker for the different neoplastic lesions of epithelial cells in reptiles. The humanised anti-α-smooth muscle actin (α-SMA) antibody showed intense cross-reactivity in the smooth muscle cells of the hepatic vessels and in the muscle layer of the gallbladder. The portal myofibroblasts, the endothelial cells of the sinusoids and the stromal cells of the cholangiocarcinoma and gallbladder tumours were positive for α-SMA. The antibovine anti-vimentin and humanised anti-Ki-67 antibodies did not show crossreactivity in the different samples from the Bearded dragon.

Windsor, Ontario Exposure Assessment Study: Design and Methods Validation of Personal, Indoor, and Outdoor Air Pollution Monitoring

The Windsor, Ontario Exposure Assessment Study evaluated the contribution of ambient air pollutants to personal and indoor exposures of adults and asthmatic children living in Windsor, Ontario, Canada. In addition, the role of personal, indoor, and outdoor air pollution exposures upon asthmatic children's respiratory health was assessed. Several active and passive sampling methods were applied, or adapted, for personal, indoor, and outdoor residential monitoring of nitrogen dioxide, volatile organic compounds, particulate matter (PM; PM < or = 2.5 microm [PM2.5] and < or = 10 microm [PM10] in aerodynamic diameter), elemental carbon, ultrafine particles, ozone, air exchange rates, allergens in settled dust, and particulate-associated metals. Participants completed five consecutive days of monitoring during the winter and summer of 2005 and 2006. During 2006, in addition to undertaking the air pollution measurements, asthmatic children completed respiratory health measurements (including peak flow meter tests and exhaled breath condensate) and tracked respiratory symptoms in a diary. Extensive quality assurance and quality control steps were implemented, including the collocation of instruments at the National Air Pollution Surveillance site operated by Environment Canada and at the Michigan Department of Environmental Quality site in Allen Park, Detroit, MI. During field sampling, duplicate and blank samples were also completed and these data are reported. In total, 50 adults and 51 asthmatic children were recruited to participate, resulting in 922 participant days of data. When comparing the methods used in the study with standard reference methods, field blanks were low and bias was acceptable, with most methods being within 20% of reference methods. Duplicates were typically within less than 10% of each other, indicating that study results can be used with confidence. This paper covers study design, recruitment, methodology, time activity diary, surveys, and quality assurance and control results for the different methods used.

Windsor, Ontario Exposure Assessment Study: Design and Methods Validation of Personal, Indoor, and Outdoor Air Pollution Monitoring

The Windsor, Ontario Exposure Assessment Study evaluated the contribution of ambient air pollutants to personal and indoor exposures of adults and asthmatic children living in Windsor, Ontario, Canada. In addition, the role of personal, indoor, and outdoor air pollution exposures upon asthmatic children's respiratory health was assessed. Several active and passive sampling methods were applied, or adapted, for personal, indoor, and outdoor residential monitoring of nitrogen dioxide, volatile organic compounds, particulate matter (PM; PM-2.5 pm [PM2.5] and < or =10 microm [PM10] in aerodynamic diameter), elemental carbon, ultrafine particles, ozone, air exchange rates, allergens in settled dust, and particulate-associated metals. Participants completed five consecutive days of monitoring during the winter and summer of 2005 and 2006. During 2006, in addition to undertaking the air pollution measurements, asthmatic children completed respiratory health measurements (including peak flow meter tests and exhaled breath condensate) and tracked respiratory symptoms in a diary. Extensive quality assurance and quality control steps were implemented, including the collocation of instruments at the National Air Pollution Surveillance site operated by Environment Canada and at the Michigan Department of Environmental Quality site in Allen Park, Detroit, MI. During field sampling, duplicate and blank samples were also completed and these data are reported. In total, 50 adults and 51 asthmatic children were recruited to participate, resulting in 922 participant days of data. When comparing the methods used in the study with standard reference methods, field blanks were low and bias was acceptable, with most methods being within 20% of reference methods. Duplicates were typically within less than 10% of each other, indicating that study results can be used with confidence. This paper covers study design, recruitment, methodology, time activity diary, surveys, and quality assurance and control results for the different methods used.

Detection and Identification of Common Food-borne Viruses with a Tiling Microarray

Microarray hybridization based identification of viral genotypes is increasingly assuming importance due to outbreaks of multiple pathogenic viruses affecting humans causing wide-spread morbidity and mortality. Surprisingly, microarray based identification of food-borne viruses, one of the largest groups of pathogenic viruses, causing more than 1.5 billion infections world-wide every year, has lagged behind. Cell-culture techniques are either unavailable or time consuming for routine application. Consequently, current detection methods for these pathogens largely depend on polymerase chain reaction (PCR) based techniques, generally requiring an investigator to preselect the target virus of interest. Here we describe the first attempt to use the microarray as an identification tool for these viruses. We have developed methodology to synthesize targets for virus identification without using PCR, making the process genuinely sequence independent. We show here that a tiling microarray can simultaneously detect and identify the genotype and strain of common food-borne viruses in a single experiment.

Traffic-related Air Pollution and Acute Changes in Heart Rate Variability and Respiratory Function in Urban Cyclists

Few studies have examined the acute health effects of air pollution exposures experienced while cycling in traffic.

Expression of Claudin-4 Molecule in Canine Exocrine Pancreatic Acinar Cell Carcinomas

Claudins, integral membrane proteins are components of the tight junction structures between epithelial and endothelial cells. These transmembrane proteins create a primary barrier to prevent paracellular transport of solutes, and also restrict the lateral diffusion of membrane lipids and proteins to maintain the cellular polarity. The aim of the present study was to characterise the expression pattern of claudin-4 tight junction molecule in canine normal pancreatic tissues and in the well-differentiated and poorly-differentiated pancreatic acinar cell carcinomas in canines.

Identification of a Claudin-4 and E-cadherin Score to Predict Prognosis in Breast Cancer

The elevated expression of claudins (CLDN) and E-cadherin (CDH-1) was found to correlate with poor prognostic features. Our aim was to perform a comprehensive analysis to assess their potential to predict prognosis in breast cancer. The expression of CLDN-1, -3-5, -7, -8, -10, -15, -18, and E-cadherin at the mRNA level was evaluated in correlation with survival in datasets containing expression measurements of 1809 breast cancer patients. The breast cancer tissues of 197 patients were evaluated with tissue microarray technique and immunohistochemical method for CLDN-1-5, -7, and E-cadherin protein expression. An additional validation set of 387 patients was used to test the accuracy of the resulting prognostic score. Based on the bioinformatic screening of publicly-available datasets, the metagene of CLDN-3, -4, -7, and E-cadherin was shown to have the most powerful predictive power in the survival analyses. An immunohistochemical protein profile consisting of CLDN-2, -4, and E-cadherin was able to predict outcome in the most effective manner in the training set. Combining the overlapping members of the above two methods resulted in the claudin-4 and E-cadherin score (CURIO), which was able to accurately predict relapse-free survival in the validation cohort (P = 0.029). The multivariate analysis, including clinicopathological variables and the CURIO, showed that the latter kept its predictive power (P = 0.040). Furthermore, the CURIO was able to further refine prognosis, separating good versus poor prognosis subgroups in luminal A, luminal B, and triple-negative breast cancer intrinsic subtypes. In breast cancer, the CURIO provides additional prognostic information besides the routinely utilized diagnostic approaches and factors.

Expression of Tight Junction Molecules in Breast Carcinomas Analysed by Array PCR and Immunohistochemistry

In the past few decades an enormous amount of data became known to clarify the molecular composition and architecture of tight junctions (TJs). Despite the efforts, the expression and function of several TJ genes and proteins in breast carcinoma are still not known and some of the data are contradictory. The expression of forty-four TJ associated genes was examined at mRNA level in eighteen invasive ductal breast carcinoma samples and corresponding normal breast tissues by using low density array PCR. Expressions of claudins (CLDNs) 5, 10, 16, 17, and 18, and ZO-1, ZO-2 were evaluated by immunohistochemistry as well. Using immunohistochemical phenotype as a surrogate for the genetic subtype, 11 luminal A, 3 luminal B, 3 triple negative and one HER2+ cases were included. Ten genes were significantly downregulated in tumors compared with normal breast tissues (CLDNs 5, 10, 16, 18, 19, CTNNAL1, JAM-B, ZO-1, ZO-2 and PARD3), whereas one gene (CLDN17) was significantly up-regulated in tumors when compared with normal breast. At protein level CLDNs 5, 10, 16, 18, ZO-1 and ZO-2 were downregulated in tumors as compared with normal breast tissue. CLDN17 showed variable expression in tumor tissues in comparison to normal breast. In the single HER2+ tumor when compared with the other subtypes CLDNs 5, 16, 17, 18, CTNNAL1, JAM-B, ZO-1, ZO-2 and PARD3 genes were found to be upregulated. We found altered TJ genes and proteins whose expression has not yet been associated with breast carcinoma. Our findings show a tendency of TJ genes and proteins to be downregulated in breast cancer. Further studies are necessary to examine whether the downregulation of the above mentioned TJ associated genes and proteins may contribute to the malignant progression of invasive ductal breast carcinomas.

Primary Systemic Therapy in Breast Cancer--an Update for Gynecologic Oncologists

Primary systemic - or neoadjuvant - chemotherapy (PST) is the standard of care in locally advanced breast cancer and it has also become an option in primary operable disease for patients who are candidates for adjuvant systemic chemotherapy. There are several advantages of administering PST: tumor downstaging--improving the chance of breast conserving surgery; in vivo assessment of tumor sensitivity to the chosen therapeutic regimen; and, early control of micrometastatic disease. On the other hand, the rate of tumor response can be used as a surrogate prognostic marker and for rapid screening of efficiency of new drugs. PST initially referred to systemic chemotherapy, but in recent years endocrine--and now multiple targeted therapies--are available in most of the countries within the confines of clinical trials.

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