C1-inhibitor for Prophylaxis of Xenograft Rejection After Pig to Cynomolgus Monkey Kidney Transplantation

Transplantation. Mar, 2002  |  Pubmed ID: 11907412

Early rejection of discordant porcine xenografts in primate recipients is initiated by the intragraft binding of either preformed (hyperacute xenograft rejection) or induced (acute vascular rejection) antiporcine recipient antibodies with subsequent complement activation via the classical pathway. We have investigated the efficacy of the supplemental administration of C1-inhibitor (C1-INH), a specific inhibitor of the classical complement activation pathway, for prophylaxis of xenograft rejection in a pig to primate kidney xenotransplantation setting.

Open Reading Frame UL26 of Human Cytomegalovirus Encodes a Novel Tegument Protein That Contains a Strong Transcriptional Activation Domain

Journal of Virology. May, 2002  |  Pubmed ID: 11967300

A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.

Vibrational Spectrum of M-benzyne: a Matrix Isolation and Computational Study

Journal of the American Chemical Society. Nov, 2002  |  Pubmed ID: 12405834

m-Benzyne (2) was generated in low-temperature matrices and IR spectroscopically characterized from four different precursors. To assign the IR absorptions, the perdeuterated derivative 2-d(4) was also investigated. By comparison with CCSD(T) calculations all vibrations between 200 and 2500 cm(-)(1) with a predicted relative intensity >2% could be assigned. All experimental and theoretical results are in accordance with a biradicaloid structure for 2, while there is no evidence for a bicyclic closed-shell structure. While benzyne 2 is stable under the conditions of matrix isolation at low temperature, flash vacuum pyrolysis at high temperatures or UV irradiation results in the rearrangement to cis-enediyne. A mechanism involving ring opening accompanied by hydrogen migration is proposed.

Inolimomab (OPi)

Current Opinion in Investigational Drugs (London, England : 2000). Oct, 2002  |  Pubmed ID: 12431019

OPi (formerly Orphan Pharma International) is developing inolimomab, an anti-interleukin-2 receptor (CD25) monoclonal antibody for the potential treatment of acute graft versus host disease. As of April 2001, inolimomab was undergoing phase II/III clinical trials.

One Century of Aryne Chemistry

Angewandte Chemie (International Ed. in English). Feb, 2003  |  Pubmed ID: 12569480

Arynes, which are formally derived from aromatic rings by abstraction of two hydrogen atoms, have been a focus of organic chemistry for 100 years. In contrast to ortho-benzyne, which is mentioned in almost every introductory textbook on organic reaction mechanisms as a reactive intermediate of nucleophilic aromatic substitution, the meta and para isomers were regarded as rather exotic until recently. This situation has changed dramatically with the discovery of the enediyne antibiotics, a promising new class of antitumor drugs, and has aroused the interest of research groups from all branches of chemistry. Nowadays, arynes and related compounds are among the most intensively studied systems in chemistry. However, many aspects of the chemistry of these reactive intermediates are not well understood yet. In this review we outline the historical developement with an emphasis on recent progress in this challenging field of research.

Intraportal Infusion of 99mtechnetium-macro-aggregrated Albumin Particles and Hepatocytes in Rabbits: Assessment of Shunting and Portal Hemodynamic Changes

Transplantation. Feb, 2003  |  Pubmed ID: 12589148

Partial correction of metabolic liver disease by hepatocyte transplantation requires infusion of a large number of cells into the portal vein. Uncontrolled infusion of cells leads to extrahepatic shunting. Obstruction of the sinusoidal space may result in hemodynamic changes and impairment of liver function.

A Nonconventional Nuclear Localization Signal Within the UL84 Protein of Human Cytomegalovirus Mediates Nuclear Import Via the Importin Alpha/beta Pathway

Journal of Virology. Mar, 2003  |  Pubmed ID: 12610148

The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.

Gene Silencing by Adenovirus-delivered SiRNA

FEBS Letters. Mar, 2003  |  Pubmed ID: 12650936

RNA interference is the process that double-stranded RNA induces the homology-dependent degradation of cognate mRNA mediated by 21-23 nucleotide short interfering RNA (siRNA). Here, we describe a simple virus vector for efficient delivery of siRNA into mammalian cells utilizing the well-defined H1-RNA promoter and conventional adenovirus. In this pilot study, p53 was targeted by this vector. Our results demonstrate efficient and specific knock-down of p53 in breast cancer MCF-7 and lung carcinoma A549 cells and indicate a prospective application of this siRNA expressing recombinant adenovirus system in functional genomics, cancer gene therapy and virus inhibition.

3,5-Pyridyne--a Heterocyclic Meta-benzyne Derivative

Journal of the American Chemical Society. May, 2004  |  Pubmed ID: 15137779

3,5-Pyridyne (3) has been generated by flash vacuum pyrolysis of 3,5-diiodopyridine (20) and 3,5-dinitropyridine (21) and characterized by IR spectroscopy in cryogenic argon matrices. The aryne can clearly be distinguished from other side products by its photolability at 254 nm, inducing a rapid ring-opening presumably to (Z)-1-aza-hex-3-ene-1,5-diyne. As byproducts of the pyrolysis, HCN and butadiyne were identified, together with traces of acetylene, cyanoacetylene, (E)-1-aza-hex-3-ene-1,5-diyne, and the 3-iodo-5-pyridyl radical (from 20). Several pathways for rearrangements and fragmentations of 3 and of the parent meta-benzyne (1) have been explored computationally by density functional theory and ab initio quantum chemical methods. The lowest energy decomposition pathway of biradicals 1 and 3 is a ring-opening process accompanied by hydrogen migration, leading to (Z)-hex-3-ene-1,5-diyne [(Z)-10] and (Z)-3-aza-hex-3-ene-1,5-diyne [(Z)-24], respectively. Both reactions require activation energies of 45-50 kcal mol(-1). Mechanisms leading from (Z)-24 or directly from 3 to the experimentally observed byproducts are discussed. Upon replacement of the C(5)H moiety by N in meta-benzyne, high-level calculations predict a modest shortening of the interradical distance by 5-7 pm and a reduction of the singlet-triplet energy splitting by 3 kcal mol(-1), in good agreement with isodesmic equations, according to which the singlet ground state of 3 is destabilized relative to 1 by 3-4 kcal mol(-1). In contrast to 3,5-borabenzyne (2), which is found to be doubly aromatic, nucleus-independent chemical shifts of 3 are almost identical to that of pyridine, indicating the absence of paramagnetic ring current effects that may be associated with "in-plane antiaromaticity". As compared with 1, the overall perturbation caused by the nitrogen atom in 3 is weak, and four electron, three center interaction is of minor importance in this molecule.

Functional Tissue Engineering of Autologous Tunica Albuginea: a Possible Graft for Peyronie's Disease Surgery

European Urology. Jun, 2004  |  Pubmed ID: 15149752

The aim of the present study was to generate a tissue engineered type of mechanically stable graft suitable for surgical replacement of the tunica albuginea penis.

Impact of Donor-recipient MHC Matching on Experimental Islet Allotransplant Survival in Naïve and Presensitized Lewis Rats

Transplantation. Jul, 2004  |  Pubmed ID: 15257057

In human islet transplantation (ITX), the impact of donor-recipient major histocompatibility complex (MHC) matching on transplant survival is currently unknown. Utilizing defined MHC mismatches, we have investigated the outcome of ITX in naïve and presensitized congenic Lewis rats. ITX into streptozotocin diabetic Lewis rats was performed under the kidney capsule. Presensitization by skin transplantation was performed on days 1, 28, and 56, followed by ITX on day 84. Survival was greatest in isolated MHC class I mismatches, followed by isolated MHC class II mismatches. The shortest transplant survival was observed following full MHC mismatched ITX (P<0.05 vs. isolated MHC class I or II). Following recipient presensitization, islets in general showed reduced survival compared to naïve recipients. In this congenic rat model, islet transplant survival was significantly influenced by the degree of donor-recipient MHC matching, as well as by recipient presensitization. These data suggest that MHC matching might be useful in human islet transplantation.

Human Cytomegalovirus Tegument Proteins PpUL82 (pp71) and PpUL35 Interact and Cooperatively Activate the Major Immediate-early Enhancer

Journal of Virology. Sep, 2004  |  Pubmed ID: 15308743

The tegument protein ppUL82 (pp71) of human cytomegalovirus (HCMV) has previously been shown to activate the immediate-early transcription of HCMV and to enhance the infectivity of viral DNA. This is concordant with its localization adjacent to promyelocytic leukemia oncogenic domains (PODs) immediately after infection. In a yeast two-hybrid screen, we identified the tegument protein ppUL35 as an interacting partner of ppUL82. The interaction could be confirmed in transfected and infected cells. The domain responsible for interaction was narrowed down to amino acids 447 to 516 within ppUL35, thus allowing both forms of ppUL35 to interact with ppUL82. Immunofluorescence experiments showed a relocalization of ppUL35 from a diffuse nuclear pattern when expressed alone to PODs when expressed together with ppUL82. In accordance with this observation and the role of ppUL82 as a transactivator, we observed a cooperative activation of the HCMV major immediate-early enhancer but not of heterologous viral enhancer elements. These results suggest an important role for this interaction in the stimulation of viral immediate-early gene expression and viral infection.

Tolerability and Steady-state Pharmacokinetics of Everolimus in Maintenance Renal Transplant Patients

Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association. Oct, 2004  |  Pubmed ID: 15316094

Current immunosuppressant regimens need to be improved to prevent acute and chronic graft rejection. The novel macrocyclic immunosuppressant everolimus (Certican, RAD) is currently in clinical development to address this issue.

Human Cytomegalovirus Tegument Protein PpUL35 is Important for Viral Replication and Particle Formation

Journal of Virology. Mar, 2005  |  Pubmed ID: 15709028

The tegument proteins ppUL35 and ppUL82 (pp71) of human cytomegalovirus (HCMV) physically interact and cooperatively activate the major immediate-early transcription. While an HCMV mutant lacking UL82 displayed a multiplicity of infection (MOI)-dependent growth, the biological significance of ppUL35 has not been addressed so far. We generated a mutant virus with a deletion of the UL35 gene. Using an MOI of 0.1, the progeny virus yield of this mutant was reduced by a factor of 1,000; however, when infected at a low MOI (0.01), the gene was essential. Characterization of the replication cycle showed that the mutant virus had two defects: when virus inoculum was standardized by the amount of viral DNA, a reduced immediate-early gene expression was observed, leading to a strongly delayed expression of lytic genes. A second defect was apparent in the virus assembly, as fewer enveloped particles and no dense bodies were present in cells infected with the mutant virus. However, the particles produced by wild-type and mutant viruses did not show significant ultrastructural differences. These results suggest an important role for ppUL35 in immediate-early gene expression and virus assembly.

Analysis of Pig-to-human Porcine Endogenous Retrovirus Transmission in a Triple-species Kidney Xenotransplantation Model

Transplant International : Official Journal of the European Society for Organ Transplantation. May, 2005  |  Pubmed ID: 15864489

Clinical pig-to-human xenotransplantation might be associated with the risk of transmission of xenozoonoses, especially porcine endogenous retroviruses (PERVs). We have established a pig-to-humanised-cynomolgus monkey xenotransplantation model allowing the analysis of potential PERV-transmission from normal or transgenic porcine organs to human vascular tissue. Pig-to-human kidney xenotransplantation was performed in cynomolgus monkeys. An interposition graft constructed from a human saphena vein replaced the porcine kidney vein. After graft rejection and/or death of the recipient (survival 2, 4, 6, 13, 16, 19 days), the human interposition grafts were removed. Human endothelial cells (huECs) were isolated from the interposition grafts and cultivated in vitro. Explanted human vascular tissue, isolated huECs, plasma and serum samples of the graft recipients were characterised by flow cytometry and immunohistochemistry and screened for indications of PERV transmission by quantitative polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RT-PCR) and RT assay. PERV-specific immune response of recipients was analysed by Western blot. No evidence of PERV infection or PERV-specific immune response was detected.

Increased Occurrence of Out-of-hospital Cardiac Arrest on Mondays in a Community-based Study

Chronobiology International. 2005  |  Pubmed ID: 15865325

Acute myocardial infarction and sudden cardiac death are more common on Mondays than other days of the week. The stress of returning to work at the beginning of the week has been postulated as a possible trigger factor. This project examined the weekly variation of out-of-hospital cardiac arrests of nontraumatic origin for the entire case series as well as for selected subgroups. A retrospective analysis of 1,498 incidences between January 1, 1995 and December 31, 1996 revealed a distinct Monday peak in occurrence irrespective of age, gender, presence of witnesses, primary survival, or primary ECG. This finding, however, was most pronounced in retired patients, subjects living alone, and persons found unconscious outside buildings or in public places. One important trigger of cardiac arrest is going to work after weekends; however, resumption of social and other activities on Mondays is another possible trigger. Other factors, such as endogenous biological rhythms, may contribute to an increased risk at this particular time even in elderly.

Influence of Everolimus on Steady-state Pharmacokinetics of Cyclosporine in Maintenance Renal Transplant Patients

Journal of Clinical Pharmacology. Jul, 2005  |  Pubmed ID: 15951468

To investigate possible interactions of the novel immunosuppressant everolimus with cyclosporine, a multicenter, randomized, double-blind, placebo-controlled, dose-escalating phase I study was performed. Everolimus regimens (0.75-10 mg/d) were administered for 28 days to stable renal allograft recipients receiving the microemulsion form of cyclosporine. Steady-state cyclosporine profiles were assessed at baseline on day 0 (cyclosporine alone) and on day 21 with everolimus on steady state. By day 21, mean dose-normalized cyclosporine AUC0-12 increased by 15% in patients receiving placebo. In everolimus-treated patients, mean increases in cyclosporine AUC0-12 ranged from 7% to 43%, which were not significantly different across all dosing cohorts including placebo. Linear regression of everolimus AUC on day 21 versus the increase in cyclosporine AUC0-12 yielded a slope not significantly different from a horizontal line (P = ns). In conclusion, these results suggest that steady-state everolimus exposure over the wide range assessed in this study did not affect steady-state cyclosporine pharmacokinetics.

Three-year Efficacy and Safety Results from a Study of Everolimus Versus Mycophenolate Mofetil in De Novo Renal Transplant Patients

American Journal of Transplantation : Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. Oct, 2005  |  Pubmed ID: 16162203

Everolimus 1.5 or 3 mg/day was compared with mycophenolate mofetil (MMF) 2 g/day in a randomized, multicenter 36-month trial in de novo renal allograft recipients (n = 588) receiving cyclosporine microemulsion (CsA) and corticosteroids. The study was double-blind until all patients had completed 12 months, then open-label. By 36 months, graft loss occurred in 7.2, 16.7 and 10.7% of patients in the everolimus 1.5, 3 mg/day, and MMF groups, respectively (p = 0.0048 for everolimus 1.5 mg/day vs. 3 mg/day); efficacy failure (biopsy-proven acute rejection (BPAR), graft loss, death or lost to follow-up) occurred in 33.0, 38.9 and 37.2% of patients (p = 0.455 overall), respectively. Mortality and incidence of BPAR were comparable in all groups. Creatinine values were higher in everolimus groups, requiring a protocol amendment that recommended lower CsA exposure. Diarrhea, lymphocele, peripheral edema and hyperlipidemia were more common among everolimus-treated patients, whereas viral infections, particularly cytomegalovirus infection, increased in the MMF group. Overall safety and tolerability were better with MMF and everolimus 1.5 mg/day than with everolimus 3 mg/day. In conclusion, at 36 months, an immunosuppressive regimen containing everolimus 1.5 mg/day had equivalent patient, and graft survival and rejection rates compared with MMF in de novo renal transplant recipients, whereas everolimus 3 mg/day had inferior graft survival. Renal dysfunction in everolimus cohorts necessitates close monitoring.

1,2,3-Tridehydrobenzene

Angewandte Chemie (International Ed. in English). Oct, 2005  |  Pubmed ID: 16196083

RBP-Jkappa/SHARP Recruits CtIP/CtBP Corepressors to Silence Notch Target Genes

Molecular and Cellular Biology. Dec, 2005  |  Pubmed ID: 16287852

Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jkappa/CBF1. In the absence of Notch, RBP-Jkappa represses Notch target genes through the recruitment of a corepressor complex. We and others have identified SHARP as a component of this complex. Here, we functionally demonstrate that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. We identify the CtIP and CtBP corepressors as novel components of the human RBP-Jkappa/SHARP-corepressor complex and show that CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. We propose that a corepressor complex containing CtIP/CtBP facilitates RBP-Jkappa/SHARP-mediated repression of Notch target genes.

Large-scale Isolation of Human Hepatocytes for Therapeutic Application

Cell Transplantation. 2005  |  Pubmed ID: 16454359

During the last decade, hepatocyte transplantation has been suggested as a safe and potentially effective clinical option for the treatment of acute or decompensating chronic liver failure as well as for hereditary liver disease. Currently, one of the major limiting factors for clinical application is the insufficient access to suitable liver cell preparations. In cooperation with the German and Catalane organ procurement organizations, a routine procedure for the isolation of hepatocytes from donor organs rejected for transplantation (n = 117) has been established. The process is performed according to the current EC Guidelines for Good Manufacturing Practice (cGMP) and all corresponding national laws and regulations concerning donor organ and tissue procurement. In about 50% of the cases (n = 58) the three-step perfusion procedure has been completed with an average total cell yield of 5.9 x 10(9) cells per organ, the cell preparations displaying a mean viability of 64%. The mean specific yield was 3.6 x 10(6) total and 2.6 x 10(6) viable cells per gram liver tissue, respectively. Specific cell yields from three infantile donor livers were considerably higher. No correlation between isolation efficiency and cold ischemia time or donor age was found within the adult organ donors. In contrast, organs with a severe steatosis generally did not result in successful cell isolation. Results of sterility and endotoxin determination are also presented. In summary, a standardized and cGMP conform method of hepatocyte isolation from nontransplantable liver organs was established, which reproducibly yields large amounts of hepatocytes suitable for therapeutic application.

The Fulvenediyls and Related Biradicals: Molecular and Electronic Structure

The Journal of Physical Chemistry. A. Feb, 2005  |  Pubmed ID: 16833435

The structures, stabilities, and electronic properties of the nine fulvenediyls have been investigated and compared to the isomeric benzynes using density functional theory (DFT) and ab initio multireference configuration interaction methods (MRCI). Given the significant biradical character of several singlet fulvenediyls, the BLYP method reproduces the relative energies of these systems rather accurately. In contrast, some triplet states (3A'-12, 3A'-13, and 3B2-14) suffer from artifactual symmetry breaking towards a nonplanar geometry at the DFT level. The structures and properties of the title biradicals are readily rationalized within the framework of through-space and through-bond molecular orbital interactions. The degree of coupling between the formally unpaired electrons strongly depends on the number and arrangement of intervening sigma-bonds, and often parallels the trends observed for annellated arynes of similar topology. In some cases, novel structural patterns can be identified that are characteristic of five-membered-ring systems. These similarities and differences between five- and six-membered-ring arynes are discussed on the basis of molecular orbital arguments.

Generation and Reactivity of the Phenyl Cation in Cryogenic Argon Matrices: Monitoring the Reactions with Nitrogen and Carbon Monoxide Directly by IR Spectroscopy

The Journal of Organic Chemistry. Aug, 2006  |  Pubmed ID: 16901116

The phenyl cation 1 has been prepared by co-deposition of iodobenzene 6 or bromobenzene 7 with a microwave-induced argon plasma and characterized by IR spectroscopy in cryogenic argon matrices. The cation can clearly be identified by its strongest absorption at 3110 cm(-1) that is rapidly bleached upon visible light irradiation. This characteristic band is observed neither in the conventional photochemistry of 6 or 7 nor in discharge experiments with alkyl halides or chlorobenzene. The latter finding is in line with energetic considerations. According to density functional theory (DFT) computations, the strongest absorption of 1 is caused by a C-H stretching vibration that involves almost entirely the ortho-hydrogens. This is confirmed by isotopic labeling experiments. Co-deposition of halobenzene/N2 mixtures leads to a decrease of the 3110 cm(-1) absorption, whereas several new signals are detected in the 2200-2400 cm(-1) range of the IR spectrum. Annealing of a matrix that contains 1 and 1% N2 leads to an increase of a broad band at 2260 cm(-1) that is assigned to the benzenediazonium ion 2. A sharp signal at 2327 cm(-1) that had previously been assigned to the N-N stretching vibration of 2 is due to molecular nitrogen. The mechanism that triggers the IR activity of N2 is not yet understood. Annealing of a matrix that contains 1 and 0.5% CO leads to an increase of a broad band at 2217 cm(-1) that is considerably stronger than the 2260 cm(-1) absorption of 2. This signal is assigned to the C-O stretching vibration of the benzoyl cation 12, in excellent agreement with previous investigations of 12 in superacidic media. Some consequences of the measured frequencies with regard to bonding in 2 and 12 are discussed.

Human Cytomegalovirus-induced Reduction of Extracellular Matrix Proteins in Vascular Smooth Muscle Cell Cultures: a Pathomechanism in Vasculopathies?

The Journal of General Virology. Oct, 2006  |  Pubmed ID: 16963742

Human cytomegalovirus (HCMV) infection appears to be linked to the pathogenesis of atherosclerosis. An association between HCMV infection and an enhanced restenosis rate as well as the induction of vasculopathies after solid organ transplantation has been documented. Knowledge of the cellular and molecular basis of these findings is limited, however. By Northern blot and RT-PCR analysis of human foreskin fibroblasts (HFF) and human coronary artery smooth muscle cells (SMC), we identified extracellular matrix (ECM) genes that were downregulated after HCMV infection, including collagen type I and fibronectin. Quantitative immunoassays showed a significant reduction of soluble collagen type I and fibronectin proteins in supernatants of both cell types. This was shown to be a direct effect of HCMV infection and not due to a response to interferons released from infected cells, since neutralization of alpha and beta interferon activity could not block virus-induced downregulation of matrix proteins. As the amount of ECM depends on both synthesis and degradation, we also assessed the influence of HCMV on the activity of matrix metalloproteinases (MMP). Interestingly, a significant difference in virus-induced matrix degradation could be shown between the two cell types. HCMV upregulated MMP-2 protein and activity in SMC but not in HFF. Thus, HCMV infection of SMC reduces ECM dramatically by inducing two independent mechanisms that influence synthesis as well as degradation of ECM. These may represent molecular mechanisms for HCMV-induced pathogenesis of inflammatory vasculopathies and may facilitate dissemination of HCMV by promoting the detachment of infected cells in vivo.

Hyperacute Rejection in Ex Vivo-perfused Porcine Lungs Transgenic for Human Complement Regulatory Proteins

Transplant International : Official Journal of the European Society for Organ Transplantation. Mar, 2006  |  Pubmed ID: 16441772

Inhibition of complement activation via human membrane-associated complement regulators is known to prevent hyperacute rejection in heart and kidney pig-to-primate transplantation. The protective effect of such strategies in pulmonary xenografts, however, seems to be insufficient. In an ex vivo perfusion, model lungs from donor pigs transgenic for human CD55 (n = 6) or human CD59 (n = 5) were perfused with fresh human blood and compared with nontransgenic organs (n = 6). In addition, a soluble complement component 1 esterase inhibitor (C1-Inh) was applied in h-CD55 transgenic lungs (n = 3). In the h-CD55 transgenic group, survival was prolonged (P < 0.05), quality and maximal time of oxygenation significantly improved and pulmonary vascular resistance reduced compared with the control group. There was a decreased sequestration of platelets, less parenchymal injury and reduced deposition of C(5b-9) in the h-CD55 transgenic group. Additional soluble complement inhibition (C1-Inh) did not prolong survival of h-CD55 transgenic lungs. Survival and pulmonary function in lungs expressing h-CD59 was not significantly different from parameters observed in nontransgenic lungs. In this ex vivo model of pig-to-primate lung transplantation, membrane-based complement inhibition resulted in significantly improved pulmonary function. However, minor histopathological injuries observed in these transgenic xenografts suggested only partial protection from pulmonary dysfunction by complement inhibition alone.

Nitrogen-rich Oligoacenes: Candidates for N-channel Organic Semiconductors

Journal of the American Chemical Society. Feb, 2007  |  Pubmed ID: 17249669

The successive replacement of CH moieties by nitrogen atoms in oligoacenes (benzene to hexacene) has been studied computationally at the B3LYP/6-311+G(d,p)//6-31G(d) level of theory, and the effects of different heteroatomic substitution patterns on structures, electron affinities, excitation, ionization, and reorganization energies are discussed. The calculated tendencies are rationalized on the basis of molecular orbital arguments. To achieve electron affinities of 3 eV, a value required to allow for efficient electron injection from common metal electrodes, at least seven nitrogen atoms have to be incorporated into tetracenes or pentacenes. The latter require rather small reorganization energies for electron transfer (<0.20 eV) making these compounds promising candidates for n-channel semiconducting materials. Particularly interesting are heptaazapentacenes 5 and 6 in which the nitrogen atoms are arranged to form self-complementary systems with a maximum number of intermolecular CH-N contacts in planar oligomers. These interactions are expected to facilitate the formation of graphite-like sheet structures with cofacial arrangements of the pi systems and short interlayer distances due to attractive N-C(H) interlayer interactions. This should not only be ideal for charge transfer but also might contribute to improved air stability of these semiconductors. Self-complementarity is maintained in azaacenes containing two cyano groups in the terminal rings. These compounds require lower reorganization energies than the unsubstituted heterocycles (0.13-0.14 eV), show high electron affinities (3.3 eV), and are thus promising candidates for materials applications.

Dehydrophenylnitrenes: Matrix Isolation and Photochemical Rearrangements

The Journal of Organic Chemistry. Feb, 2007  |  Pubmed ID: 17253786

The photochemistry of 3-iodo-2,4,5,6-tetrafluorophenyl azide 8 and 3,5-diiodo-2,4,6-trifluorophenyl azide 9 was studied by IR and EPR spectroscopy in cryogenic argon and neon matrices. Both compounds form the corresponding nitrenes as primary photoproducts in photostationary equilibria with their azirine and ketenimine isomers. In contrast to fluorinated phenylnitrenes, ring-opened products are obtained upon short-wavelength irradiation of the iodine-containing systems, indicative of C-I bond cleavage in the nitrenes or didehydroazepines under these conditions. Neither 3-dehydrophenylnitrene 6 nor 3,5-didehydrophenylnitrene 7 could be detected directly. The structures of the acyclic photoproducts were identified by extensive comparison with DFT calculated spectra. Mechanistic aspects of the rearrangements leading to the observed products and the electronic properties of the title intermediates are discussed on the basis of DFT as well as high-level ab initio calculations. The computations indicate strong through-bond coupling of the exocyclic orbital in the meta position with the singly occupied in-plane nitrene orbital in the monoradical nitrenes. In contrast to the ortho or para isomers, this interaction results in low-spin ground states for meta nitrene radicals and a weakening of the C1-C2 bond causing the kinetic instability of these species even under low-temperature conditions. 3,5-Didehydrophenylnitrenes, on the other hand, in which a strong C3-C5 interaction reduces coupling of the radical sites with the nitrene unit, might be accessible synthetic targets if the intermediate formation of labile monoradicals could be circumvented.

Hepatic Gas Gangrene Following Liver Transplantation

Liver Transplantation : Official Publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. Mar, 2007  |  Pubmed ID: 17318863

The 1,2,3-tridehydrobenzene Triradical: 2B or Not 2B? The Answer is 2A!

The Journal of Physical Chemistry. A. Jun, 2007  |  Pubmed ID: 17518455

The molecular and electronic structure of 1,2,3-tridehydrobenzene was investigated by a variety of computational methods. The two lowest electronic states of the triradical are the (2)B(2) and (2)A(1) doublet states characterized by different interactions of the unpaired electrons. Vertically, the two states are well separated in energy-by 4.9 and 1.4 eV, respectively. However, due to different bonding patterns, their equilibrium structures are very different and, adiabatically, the two states are nearly degenerate. The adiabatic energy gap between the (2)B(2) and (2)A(1) states is estimated to be 0.7-2.1 kcal/mol, in favor of the (2)A(1) state. Harmonic vibrational frequencies and anharmonic corrections were calculated for both states. Comparison with the three experimentally observed IR transitions supports the assignment of the (2)A(1) ground state for the triradical with a weakly bonding distance of 1.67-1.69 A between the meta radical centers.

Trifluoro-1,3,5-tridehydrobenzene

Angewandte Chemie (International Ed. in English). 2007  |  Pubmed ID: 17568464

FTY720 Improves Survival After Transient Ischemia and Reperfusion of the Hind Limbs

The Journal of Trauma. Aug, 2007  |  Pubmed ID: 17693822

Ischemia and reperfusion (I/R) damage involves adhesion and transmigration of lymphocytes and neutrophils. FTY720 is an immunosuppressive agent that reduces the number of neutrophils and monocytes in peripheral blood as well as tissue lymphocyte infiltration. This study investigated the effect of FTY720 during hind limb I/R.

Duration of Development of Symptomatic In-stent Restenosis Correlates with the Stent-to-vessel-diameter Ratio: an Intravascular Ultrasound Study

Coronary Artery Disease. Nov, 2007  |  Pubmed ID: 17925602

Several predictors for in-stent restenosis (ISR) have been defined by intravascular ultrasound (IVUS) assessment; however, there is a lack of data correlating IVUS parameters with the speed of development of ISR. This study aims to investigate the relation between the duration of development of symptomatic ISR and the relative stent diameter.

Echocardiography in Leiomyomatosis of the Uterus: How to Guide Your Surgeon

Clinical Research in Cardiology : Official Journal of the German Cardiac Society. Feb, 2008  |  Pubmed ID: 18049830

Effects of Pharmacological Intervention on Coagulopathy and Organ Function in Xenoperfused Kidneys

Xenotransplantation. Feb, 2008  |  Pubmed ID: 18333913

Following pig to primate kidney transplantation, xenogenic activation of the coagulation (XAC) system of the recipient eventually leading to organ dysfunction and disseminated intravascular coagulation (DIC) can be observed.

Mechanistic Investigations of the Acid-catalyzed Cyclization of a Vinyl Ortho-quinone Methide

Chemistry (Weinheim an Der Bergstrasse, Germany). 2008  |  Pubmed ID: 18449871

Fluorescence-based Antiviral Assay for the Evaluation of Compounds Against Vaccinia Virus, Varicella Zoster Virus and Human Cytomegalovirus

Journal of Virological Methods. Jul, 2008  |  Pubmed ID: 18490063

Recombinant vaccinia virus (VACV), varicella zoster virus (VZV) and two human cytomegaloviruses (HCMV) expressing the green fluorescent protein (GFP) and the enhanced yellow fluorescent protein (EYFP) were used to develop a fluorescence-based assay for testing antiviral compounds. Infection of human embryonic lung fibroblasts (HEL) with the different recombinant viruses produced stable and detectable amount of GFP and EYFP signal as quantitated by automated fluorometry. The sensitivity of the recombinant viruses to a panel of antiviral drugs was measured and the fluorescence-based assay was compared to the cytopathic effect reduction assay (CPE-RA) in case of VACV and HCMV or to the plaque reduction assay (PRA) in case of VZV. The 50% inhibitory concentration (IC(50)) values for reference anti-pox and anti-herpesvirus compounds were comparable to those determined by CPE-RA or PRA assays. Furthermore the fluorimetric data could be confirmed by a flow cytometry assay. GFP- and EYFP-recombinant viruses proved to be a convenient tool for the evaluation of antiviral agents.

Singlet-triplet Energy Splitting and Excited States of Phenylnitrene

The Journal of Physical Chemistry. A. Sep, 2008  |  Pubmed ID: 18714972

The vertical and adiabatic singlet-triplet energy splittings (Delta E ST) of phenylnitrene were computed by a variety of multireference configuration interaction and perturbation theory methods employing basis sets of up to quadruple-xi quality and extrapolation to the complete basis set limit. The vertical and adiabatic energy gaps are 18.9 and 15.9 kcal mol (-1), respectively, the latter in reasonable agreement with the revised experimental value of 15.1 +/- 0.2 kcal mol (-1). The energy difference between both states at the geometry of the a (1)A 2 singlet state was also considered and amounts to 13.8 kcal mol (-1). In obtaining accurate state energy splittings, basis set completeness turns out to be a more important issue than the level of dynamical electron correlation treatment. Density functional theory that is frequently employed to investigate phenylnitrenes and their rearrangements yields varying results and, depending on the functional, gives adiabatic energy differences between 9 and 16 kcal mol (-1). The b (1)A 1 state has a similar geometry as the ground state of 1 and is 31 kcal mol (-1) higher in energy. According to best estimates, the next higher singlet states, c (1)A 1 and d (1)B 1, are 57 and 72 kcal mol (-1) above the ground state. In the triplet manifold, vertical excitation energies to the A (3)B 1 and B (3)A 2 states are 71 and 77 kcal mol (-1), respectively.

Human Cytomegalovirus: Host Immune Modulation by the Viral US3 Gene

The International Journal of Biochemistry & Cell Biology. Mar, 2009  |  Pubmed ID: 18992841

Human cytomegalovirus (HCMV) is a common infection, opportunistically causing disease in people with immune system deficits. HCMV expresses several proteins that contribute to avoidance of the host immune response. The US3 gene is one of the first immune evasion genes expressed following infection. Expression of the US3 gene is highly regulated, with the gene encoding autoregulatory proteins. The largest of the US3 proteins, a 22 kDa resident endoplasmic reticulum protein, binds to MHC class I heavy chain complexes and components of the peptide loading complex, delaying the maturation of the MHC class I complexes and presentation of viral antigen on the surface of infected cells. A smaller US3 protein, a 17 kDa US3 protein, competes with the 22 kDa for protein interactions, counteracting, in part, the effects of the larger protein. The US3 amino acid sequence is highly conserved among clinical isolates and laboratory strains, suggesting an important role for this gene in natural infections in the human host.

Pigs Transgenic for Human Thrombomodulin Have Elevated Production of Activated Protein C

Xenotransplantation. Nov-Dec, 2009  |  Pubmed ID: 20042048

The inability of porcine thrombomodulin (TM) to activate human anticoagulant protein C after pig-to-human xenotransplantation may lead to an aberrant activation of coagulation with microthrombosis and ultimately failure of the transplanted organ. Here, we describe the production of triple-transgenic pigs expressing hCD59/DAF and human thrombomodulin (hTM) and tested hTM-transgenic fibroblasts obtained from these pigs for their ability to activate human protein C in a new in vitro assay.

The Nucleotide-binding Oligomerization Domain-like Receptor NLRC5 is Involved in IFN-dependent Antiviral Immune Responses

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2010  |  Pubmed ID: 20061403

Nucleotide-binding oligomerization domain-like receptors (NLRs) are a group of intracellular proteins that mediate recognition of pathogen-associated molecular patterns or other cytosolic danger signals. Mutations in NLR genes have been linked to a variety of inflammatory diseases, underscoring their pivotal role in host defense and immunity. This report describes the genomic organization and regulation of the human NLR family member NLRC5 and aspects of cellular function of the encoded protein. We have analyzed the tissue-specific expression of NLRC5 and have characterized regulatory elements in the NLRC5 promoter region that are responsive to IFN-gamma. We show that NLRC5 is upregulated in human fibroblasts postinfection with CMV and demonstrate the role of a JAK/STAT-mediated autocrine signaling loop involving IFN-gamma. We demonstrate that overexpression and enforced oligomerization of NLRC5 protein results in activation of the IFN-responsive regulatory promoter elements IFN-gamma activation sequence and IFN-specific response element and upregulation of antiviral target genes (e.g., IFN-alpha, OAS1, and PRKRIR). Finally, we demonstrate the effect of small interfering RNA-mediated knockdown of NLRC5 on a target gene level in the context of viral infection. We conclude that NLRC5 may represent a molecular switch of IFN-gamma activation sequence/IFN-specific response element signaling pathways contributing to antiviral defense mechanisms.

Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins--characterization and Use in Antiviral Screening

PloS One. 2010  |  Pubmed ID: 20161802

Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus-infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.

Interaction of the Papillomavirus E8--E2C Protein with the Cellular CHD6 Protein Contributes to Transcriptional Repression

Journal of Virology. Sep, 2010  |  Pubmed ID: 20631145

Expression of the E6 and E7 oncogenes of high-risk human papillomaviruses (HPV) is controlled by cellular transcription factors and by viral E2 and E8--E2C proteins, which are both derived from the HPV E2 gene. Both proteins bind to and repress the HPV E6/E7 promoter. Promoter inhibition has been suggested to be due to binding site competition with cellular transcription factors and to interactions of different cellular transcription modulators with the different amino termini of E2 and E8--E2C. We have now identified the cellular chromodomain helicase DNA binding domain 6 protein (CHD6) as a novel interactor with HPV31 E8--E2C by using yeast two-hybrid screening. Pull-down and coimmunoprecipitation assays indicate that CHD6 interacts with the HPV31 E8--E2C protein via the E2C domain. This interaction is conserved, as it occurs also with the E8--E2C proteins expressed by HPV16 and -18 and with the HPV31 E2 protein. Both RNA knockdown experiments and mutational analyses of the E2C domain suggest that binding of CHD6 to E8--E2C contributes to the transcriptional repression of the HPV E6/E7 oncogene promoter. We provide evidence that CHD6 is also involved in transcriptional repression but not activation by E2. Taken together our results indicate that the E2C domain not only mediates specific DNA binding but also has an additional role in transcriptional repression by recruitment of the CHD6 protein. This suggests that repression of the E6/E7 promoter by E2 and E8--E2C involves multiple interactions with host cell proteins through different protein domains.

Management of Major Postsurgical Gastroesophageal Intrathoracic Leaks with an Endoscopic Vacuum-assisted Closure System

Gastrointestinal Endoscopy. Feb, 2010  |  Pubmed ID: 19879566

Endoscopic treatment options for postsurgical intrathoracic leaks include injection of fibrin glue, clip application, and stent placement. Endoscopic vacuum-assisted closure (E-VAC) may be an effective treatment option.

Training, Competency, and Certification in Cardiac CT: a Summary Statement from the Society of Cardiovascular Computed Tomography

Journal of Cardiovascular Computed Tomography. Sep-Oct, 2011  |  Pubmed ID: 21875825

Training and competency criteria in cardiac CT were developed to guide practitioners in the process of achieving and maintaining skills in performing and interpreting cardiac CT studies. Appropriate training and eventual certification in cardiac CT angiography may be obtained by adhering to the recommendations for competency as set forth by either the American College of Cardiology Foundation (ACCF) or the American College of Radiology (ACR). Competency under either pathway requires both knowledge and experience-based components, with benchmarks set for level of experience on the basis of the extent of training experience. Although these recommended parameters are substantial, meeting these training criteria does not guarantee competence or expertise, which is the responsibility of the individual practitioner and may require further training and experience. Separate from satisfying initial training for the achievement of competency, certification in cardiac CT may be achieved through formal certification under the Certification Board of Cardiovascular Computed Tomography. Eligibility for certification generally follows the ACCF/American Heart Association Level 2 or ACR competency pathways. The ACR also conducts a certificate program related to advanced proficiency in cardiac CT. This official document of the Society of Cardiovascular Computed Tomography summarizes the present criteria for competency and certification in the field of cardiac CT.

Preprocedural Planning with Prospectively Triggered Multidetector Row CT Angiography Prior to Bronchial Artery Embolization in Cystic Fibrosis Patients with Massive Hemoptysis

Lung. Oct, 2011  |  Pubmed ID: 22037830

STUDY OBJECTIVES: The aim of this study was to determine if electrocardiographically synchronized, prospectively triggered multidetector row computed tomography (ECG-MDR-CT) angiography of the aorta can accurately predict the location of ectopic bronchial arteries in patients with cystic fibrosis (CF) with massive hemoptysis prior to bronchial artery embolization (BAE). DESIGN AND SETTING: The study was a prospective, observational study from September 1, 2009 to June 30, 2011, conducted at a university hospital with an adult CF center. PATIENTS: The study included adult CF patients with massive hemoptysis. RESULTS: A total of four adult patients (mean [± SD] age = 31.5 ± 7.9 years) with CF and massive hemoptysis underwent ECG-MDR-CT angiography. The location of the bleeding source was predicted in each case based on lung pathology observed on ECG-MDR-CT angiography. All four patients eventually required BAE without the need for conventional aortograms since the locations of the bronchial arteries were determined prior to the procedure. Review of lung pathology and arterial networks from the ECG-MDR-CT angiography data limited the number of selective catheterizations necessary to complete the procedures. BAE resulted in complete resolution of hemoptysis in three patients and successful mitigation of the bleeding in the fourth patient until lung transplantation was performed 1 week later. CONCLUSIONS: ECG-MDR-CT angiography accurately depicted bronchial artery anatomy in CF patients with massive hemoptysis and provided excellent preprocedural planning for BAE. The information provided by ECG-MDR-CT angiography of the aorta prior to conventional angiography decreased the BAE radiation dose and contrast volume and likely reduced table time.

Boerhaave Syndrome As a Complication of Colonoscopy Preparation: a Case Report

Journal of Medical Case Reports. 2011  |  Pubmed ID: 22054124

ABSTRACT:

Transgenic Expression of Human Heme Oxygenase-1 in Pigs Confers Resistance Against Xenograft Rejection During Ex Vivo Perfusion of Porcine Kidneys

Xenotransplantation. Nov-Dec, 2011  |  Pubmed ID: 22168142

The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay.

The Influence of Space Dimension on the Large-time Behavior in a Reaction-diffusion System Modeling Diallelic Selection

Journal of Mathematical Biology. Mar, 2011  |  Pubmed ID: 20383710

We study a mathematical model from population genetics, describing a single-locus diallelic (A/a) selection-migration process. The model consists of a coupled system of three reaction-diffusion equations, one for the density of each genotype, posed in the whole space [Formula: see text]. The genotype AA is advantageous, due to a smaller death rate, and we consider the fully recessive case where the other two genotypes aa and Aa have the same (higher) death rate. In the nondiffusive (spatially homogeneous) case, the disadvantageous gene a is always eliminated in the large time limit. In the presence of diffusion, when the birth rate exceeds a certain threshold value, we prove that this conclusion is still true for dimensions n ≤ 2, whereas for n ≥ 3 there exist initial distributions for which the advantageous gene A ultimately disappears. This is the first rigorous result of this type for the full system, and it solves a problem which seems to have been open since the celebrated work of Aronson and Weinberger (Lecture notes in mathematics, vol 446, Springer, New York, pp 5-49, 1975; Adv Math 30, 33-76, 1977), where similar results had been obtained for a simplified scalar model, that they derived as an approximation of the full system. Interestingly, we moreover show that, at the threshold value of the birth rate, the cut-off dimension shifts from n = 2 to n = 6.