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Find video protocols related to scientific articles indexed in Pubmed.
TGF? responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness.
Ann. Rheum. Dis.
PUBLISHED: 11-08-2014
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In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTP? (RPTP?), encoded by the transforming growth factor (TGF) ?-target gene, PTPRK, promotes RA FLS invasiveness.
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Evolution of Bluetongue Virus Serotype 1 in Northern Australia over 30 Years.
J. Virol.
PUBLISHED: 09-24-2014
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BTV 1 was first isolated in Australia from cattle blood collected in 1979 at Beatrice Hill Farm (BHF), Northern Territory (NT). From long term surveillance programs (1977 to 2011), 2487 isolations of ten BTV serotypes were made. The most frequently isolated serotype was BTV 1 (41%, 1019) followed by BTV 16 (17.5%, 436) and BTV 20 (14%, 348). In three years no BTVs were isolated and in twelve years no BTV 1 was isolated. Seventeen BTV 1 isolates were sequenced and analyzed in comparison with ten Australian prototype serotypes. BTV 1 showed an episodic pattern of evolutionary change characterized by four distinct periods. Each period consisted primarily of slow genetic drift which was punctuated from time to time by genetic shifts generated by segment reassortment and the introduction of new genome segments. Evidence was found for co-evolution of BTV genome segments. Evolutionary dynamics and selection pressures estimates showed strong temporal and clock-like molecular evolutionary dynamics of six Australian BTV genome segments. Bayesian coalescent estimates of mean substitution rates clustered in the range of 3.5 to 5.3 x 10(-4) substitutions per site per year. All BTV genome segments evolved under strong purifying (negative) selection with only three sites identified as under pervasive diversifying (positive) selection. The obligate replication in alternate hosts (insect vector and vertebrate host) imposed strong evolutionary constraints. The dominant mechanism generating genetic diversity of BTV 1 at BHF was through the introduction of new viruses and reassortment of genome segments with existing viruses.
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Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.
PLoS ONE
PUBLISHED: 08-13-2014
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Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.
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Stratified mucin producing intraepithelial lesion (smile): report of a case series with associated pathological findings.
Histopathology
PUBLISHED: 07-22-2014
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Stratified mucin producing intraepithelial lesion (SMILE) is an uncommon premalignant cervical lesion with morphological overlap between cervical intraepithelial neoplasia (CIN) and adenocarcinoma in situ (AIS)/ cervical glandular intraepithelial neoplasia. Given the limited literature on SMILE, we aimed to document its frequency and assess the associated pathology in a large series of cases.
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Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium.
Arthritis Res. Ther.
PUBLISHED: 05-20-2014
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Acid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).
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Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression.
Mod. Pathol.
PUBLISHED: 05-05-2014
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The oncogenic role of WNT is well characterized. Wntless (WLS) (also known as GPR177, or Evi), a key modulator of WNT protein secretion, was recently found to be highly overexpressed in malignant astrocytomas. We hypothesized that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signaling such as ovarian, gastric, and breast cancers. Immunohistochemical analysis using a TMA platform revealed WLS overexpression in a subset of ovarian, gastric, and breast tumors; this overexpression was associated with poorer clinical outcomes in gastric cancer (P=0.025). In addition, a strong correlation was observed between WLS expression and human epidermal growth factor receptor 2 (HER2) overexpression. Indeed, 100% of HER2-positive intestinal gastric carcinomas, 100% of HER2-positive serous ovarian carcinomas, and 64% of HER2-positive breast carcinomas coexpressed WLS protein. Although HER2 protein expression or gene amplification is an established predictive biomarker for trastuzumab response in breast and gastric cancers, a significant proportion of HER2-positive tumors display resistance to trastuzumab, which may be in part explainable by a possible mechanistic link between WLS and HER2.Modern Pathology advance online publication, 26 September 2014; doi:10.1038/modpathol.2014.114.
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TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.
Oncotarget
PUBLISHED: 04-19-2014
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TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.
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Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels.
Arthritis Res. Ther.
PUBLISHED: 04-11-2014
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We hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity.
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Progresses in DNA-based heterologous prime-boost immunization strategies.
Methods Mol. Biol.
PUBLISHED: 04-10-2014
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Although recombinant DNA and recombinant viral vectors expressing HIV antigens have yielded positive outcomes in animal models, these vaccines have not been effectively translated to humans. Despite this, there is still a high level of optimism that poxviral-based vaccine strategies could offer the best hope for developing an effective vaccine against not only HIV-1 but also other chronic diseases where good-quality T and B cell immunity is needed for protection. In this chapter we discuss step by step (1) how recombinant poxviral vectors co-expressing HIV antigens and promising mucosal/systemic adjuvants (e.g., IL-13R?2) are constructed, (2) how these vectors can be used in alternative heterologous prime-boost immunization strategies, (3) how systemic and mucosal samples are prepared for analysis, followed by (4) two immunological assays: multicolor intracellular cytokine staining and tetramer/homing maker analysis that are used to evaluate effective systemic and mucosal T cell immunity.
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Effects of metal nanoparticles on the lateral line system and behaviour in early life stages of zebrafish (Danio rerio).
Aquat. Toxicol.
PUBLISHED: 04-04-2014
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The unique physicochemistry and potential toxicity of manufactured nanoparticles (NPs) requires innovative approaches for the assessment of toxicity to aquatic organisms. Here, the toxicity of Cu-NPs, Ag-NPs and TiO2-NPs on the lateral line system of free-swimming zebrafish embryos was investigated and compared to appropriate metal salts or bulk material controls. Fish were exposed for 4h at 96-h post-fertilization. Metal salt (CuSO4 and AgNO3) controls reduced the number of functional lateral line neuromasts (LLN) to <5% of unexposed controls, but no effect on LLN was observed for TiO2-NPs or Ag-NPs. Exposure to Cu-NPs caused only a 15% reduction in LLN. Performance of positive rheotaxis was reduced by Cu-NPs, Ag-NPs, and the metal salt controls. The data show that some metal NPs can affect LLN and fish behaviour (rheotaxis) important for survival, and that effects were different from those of comparable metal ion controls. Capsule: We demonstrate that behaviour is a particularly sensitive indicator of metal NP exposure in fish and highlight the interaction between behaviour and external tissue surfaces.
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Differential distribution of PI3K isoforms in spinal cord and dorsal root ganglia: potential roles in acute inflammatory pain.
Pain
PUBLISHED: 03-03-2014
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PI3-kinases (PI3Ks) participate in nociception within spinal cord, dorsal root ganglion (DRG), and peripheral nerves. To extend our knowledge, we immunohistochemically stained for each of the 4 class I PI3K isoforms along with several cell-specific markers within the lumbar spinal cord, DRG, and sciatic nerve of naive rats. Intrathecal and intraplantar isoform specific antagonists were given as pretreatments before intraplantar carrageenan; pain behavior was then assessed over time. The ?-isoform was localized to central terminals of primary afferent fibers in spinal cord laminae IIi to IV as well as to neurons in ventral horn and DRG. The PI3K? isoform was the only class I isoform seen in dorsal horn neurons; it was also observed in DRG, Schwann cells, and axonal paranodes. The ?-isoform was found in spinal cord white matter oligodendrocytes and radial astrocytes, and the ?-isoform was seen in a subpopulation of IB4-positive DRG neurons. No isoform co-localized with microglial markers or satellite cells in naive tissue. Only the PI3K? antagonist, but none of the other antagonists, had anti-allodynic effects when administered intrathecally; coincident with reduced pain behavior, this agent completely blocked paw carrageenan-induced dorsal horn 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptor trafficking to plasma membranes. Intraplantar administration of the ?-antagonist prominently reduced pain behavior. These data suggest that each isoform displays specificity with regard to neuronal type as well as to specific tissues. Furthermore, each PI3K isoform has a unique role in development of nociception and tissue inflammation.
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The prognostic significance of the aberrant extremes of p53 immunophenotypes in breast cancer.
Histopathology
PUBLISHED: 02-19-2014
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The utility of p53 as a prognostic assay has been elusive. The aims of this study were to describe a novel, reproducible scoring system and assess the relationship between differential p53 immunohistochemistry (IHC) expression patterns, TP53 mutation status and patient outcomes in breast cancer.
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Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer.
J. Natl. Cancer Inst.
PUBLISHED: 01-10-2014
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There is no method routinely used to predict response to anthracycline and cyclophosphamide-based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection.
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Impaired behavioural response to alarm substance in rainbow trout exposed to copper nanoparticles.
Aquat. Toxicol.
PUBLISHED: 01-02-2014
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To date, studies of the toxicity of engineered nanoparticles (NPs) in fish have not fully considered effects on olfactory-mediated behaviours, despite their ecological importance. In this study the effects of copper NPs (Cu NPs) on the anti-predator behavioural responses of juvenile rainbow trout (Oncorhynchus mykiss) to trout alarm substance was investigated. Individual fish were exposed for 12h to a control (no added Cu), 50?gl(-1) of Cu as Cu NPs, or 50?gl(-1) Cu as CuSO4, after which fish behaviours were analyzed in 10min periods before and after the addition of the alarm substance stimulus. The response of control fish to deionised water (negative control, no alarm substance stimulus) was also analyzed. The alarm substance elicited a behavioural response in the control fish characterized by an immediate freeze response and the slower resumption of swimming activity compared to negative controls exposed to the sham deionised water stimuli. In fish exposed to Cu NPs, the behavioural response to alarm substance was eliminated, with no significant difference in behaviours compared to negative controls. In comparison, exposure to 50?gl(-1) Cu as CuSO4 decreased, but did not eliminate the response of fish to alarm substance, which indicated a significantly greater effect of Cu NPs on olfactory mediated behaviours than of the equivalent concentration of Cu as CuSO4. Measurement of total Cu concentrations in the tissues of fish demonstrated no significant accumulation of Cu from any treatment in gill, liver or brain, confirming the effects of Cu NPs, and to a lesser extent CuSO4, on behavioural responses were mostly associated with the interaction of the materials with the external surfaces of the fish. Scanning electron microscopy revealed that Cu as CuSO4 caused a pronounced depletion of ciliated sensory and non-sensory cells in the olfactory rosette surrounding the midline raphe, whereas Cu NPs had no impact on the structure of the rosette. However, exposure to Cu NPs caused a significant increase in the ratio of oxidized to reduced glutathione in brains of fish, indicating some systemic oxidative stress that was not observed in either controls or fish exposed to CuSO4. Overall, the study showed that the olfactory mediated behaviours of fish were potentially more sensitive to Cu NPs than CuSO4 and NPs elicited effects via a mechanism that is distinct from that of the metal salt.
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Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA.
PLoS ONE
PUBLISHED: 01-01-2014
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Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25-43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.
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Differential regulation of anti-inflammatory genes by p38 MAP kinase and MAP kinase kinase 6.
J Inflamm (Lond)
PUBLISHED: 01-01-2014
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Conventional p38? inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3-/-, MKK6-/-, and p38 inhibitor-treated wildtype cells.
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Differential roles of MAPK kinases MKK3 and MKK6 in osteoclastogenesis and bone loss.
PLoS ONE
PUBLISHED: 01-01-2014
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Bone mass is maintained by osteoclasts that resorb bone and osteoblasts that promote matrix deposition and mineralization. Bone homeostasis is altered in chronic inflammation as well as in post-menopausal loss of estrogen, which favors osteoclast activity that leads to osteoporosis. The MAPK p38? is a key regulator of bone loss and p38 inhibitors preserve bone mass by inhibiting osteoclastogenesis. p38 function is regulated by two upstream MAPK kinases, namely MKK3 and MKK6. The goal of this study was to assess the effect of MKK3- or MKK6-deficiency on osteoclastogenesis in vitro and on bone loss in ovariectomy-induced osteoporosis in mice. We demonstrated that MKK3 but not MKK6, regulates osteoclast differentiation from bone marrow cells in vitro. Expression of NFATc1, a master transcription factor in osteoclastogenesis, is decreased in cells lacking MKK3 but not MKK6. Expression of osteoclast-specific genes Cathepsin K, osteoclast-associated receptor and MMP9, was inhibited in MKK3-/- cells. The effect of MKK-deficiency on ovariectomy-induced bone loss was then evaluated in female WT, MKK3-/- and MKK6-/- mice by micro-CT analysis. Bone loss was partially inhibited in MKK3-/- as well as MKK6-/- mice, despite normal osteoclastogenesis in MKK6-/- cells. This correlated with the lower osteoclast numbers in the MKK-deficient ovariectomized mice. These studies suggest that MKK3 and MKK6 differentially regulate bone loss due to estrogen withdrawal. MKK3 directly mediates osteoclastogenesis while MKK6 likely contributes to pro-inflammatory cytokine production that promotes osteoclast formation.
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Novel Phosphoinositide 3-Kinase ?,? Inhibitor: Potent Anti-Inflammatory Effects and Joint Protection in Models of Rheumatoid Arthritis.
J. Pharmacol. Exp. Ther.
PUBLISHED: 11-15-2013
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Phosphoinositide 3-kinases ? and ? (PI3K? and PI3K?) are expressed in rheumatoid arthritis (RA) synovium and regulate innate and adaptive immune responses. We determined the effect of a potent PI3K?,? inhibitor, IPI-145, in two preclinical models of RA. IPI-145 was administered orally in rat adjuvant-induced arthritis (AA) and intraperitoneally in mouse collagen-induced arthritis (CIA). Efficacy was assessed by paw swelling, clinical scores, histopathology and radiography, and microcomputed tomography scanning. Gene expression and Akt phosphorylation in joint tissues were determined by quantitative real-time polymerase chain reaction and Western blot analysis. Serum concentrations of anti-type II collagen (CII) IgG and IgE were measured by immunoassay. T-cell responses to CII were assayed using thymidine incorporation and immunoassay. IPI-145 significantly reduced arthritis severity in both RA models using dosing regimens initiated before onset of clinical disease. Treatment of established arthritis with IPI-145 in AA, but not CIA, significantly decreased arthritis progression. In AA, histology scores, radiographic joint damage, and matrix metalloproteinase (MMP)-13 expression were reduced in IPI-145-treated rats. In CIA, joint histology scores and expression of MMP-3 and MMP-13 mRNA were lower in the IPI-145 early treatment group than in the vehicle group. The ratio of anti-CII IgG2a to total IgG in CIA was modestly reduced. Interleukin-17 production in response to CII was decreased in the IPI-145-treated group, suggesting an inhibitory effect on T-helper cell 17 differentiation. These data show that PI3K?,? inhibition suppresses inflammatory arthritis, as well as bone and cartilage damage, through effects on innate and adaptive immunity and that IPI-145 is a potential therapy for RA.
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Minimal effects of waterborne exposure to single-walled carbon nanotubes on behaviour and physiology of juvenile rainbow trout (Oncorhynchus mykiss).
Aquat. Toxicol.
PUBLISHED: 09-24-2013
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Fish behaviours are often considered to be sensitive endpoints of waterborne contaminants, but little attention has been given to engineered nanomaterials. The present study aimed to determine the locomotor and social behaviours of rainbow trout (Oncorhynchus mykiss) during waterborne exposure to single-walled carbon nanotubes (SWCNTs), and to ascertain the physiological basis for any observed effects. Dispersed stock suspensions of SWCNTs were prepared by stirring in sodium dodecyl sulphate (SDS), an anionic surfactant, on an equal w/w basis. Trout were exposed to control (no SWCNT or SDS), 0.25mgL(-1) SDS (dispersant control), or 0.25mgL(-1) of SWCNT for 10 days. Video tracking analysis of spontaneous locomotion of individual fish revealed no significant effects of SWCNT on mean velocity when active, total distance moved, or the distribution of swimming speeds. Hepatic glycogen levels were also unaffected. Fish exposed to SWCNTs retained competitive fitness when compelled to compete in energetically costly aggressive interactions with fish from both control groups. Assessment of the respiratory physiology of the fish revealed no significant changes in ventilation rate or gill injuries. Haematocrit and haemoglobin concentrations in the blood were unaffected by SWCNT exposure; and the absence of changes in the red and white pulp of the spleen excluded a compensatory haematopoietic response to protect the circulation. Despite some minor histological changes in the kidneys of fish exposed to SWCNT compared to controls, plasma ion concentrations and tissue electrolytes were largely unaffected. Direct neurotoxicity of SWCNT was unlikely with the brains showing mostly normal histology, and with no effects on acetylcholinesterase or Na(+)/K(+)-ATPase activities in whole brain homogenates. The minimal effects of waterborne exposure to SWCNT observed in this study are in contrast to our previous report of SWCNT toxicity in trout, suggesting that details of the dispersion method and co-exposure concentration of the dispersing agent may alter toxicity.
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Molecular and clinicopathological markers of prognosis in breast cancer.
Expert Rev. Mol. Diagn.
PUBLISHED: 06-21-2013
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A vast body of research in breast cancer prognostication has accumulated. Yet despite this, patients within current prognostic categories may have significantly different outcomes. There is a need to more accurately divide those cancer types associated with an excellent prognosis from those requiring more aggressive therapy. Gene expression array studies have revealed the numerous molecular breast cancer subtypes that are associated with differing outcomes. Furthermore, as next generation technologies evolve and further reveal the complexities of breast cancer, it is likely that existing prognostic approaches will become progressively refined. Future prognostication in breast cancer requires a morphomolecular, multifaceted approach involving the assessment of anatomical disease extent and levels of protein, DNA and RNA expression. One of the major challenges in prognostication will be the integration of potential assays into existing clinical systems and identification of appropriate patient subgroups for analysis.
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Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.
MBio
PUBLISHED: 04-04-2013
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Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.
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Critical comparison of intravenous injection of TiO2 nanoparticles with waterborne and dietary exposures concludes minimal environmentally-relevant toxicity in juvenile rainbow trout Oncorhynchus mykiss.
Environ. Pollut.
PUBLISHED: 03-21-2013
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A critical comparison of studies that have investigated tissue accumulation and toxicity of TiO2-NPs in fish is necessary to resolve inconsistencies. The present study used identical TiO2-NPs, toxicological endpoints, and fish (juvenile rainbow trout Oncorhynchus mykiss) as previous studies that investigated waterborne and dietary toxicity of TiO2-NPs, and conducted a critical comparison of results after intravenous caudal-vein injection of 50 ?g of TiO2-NPs and bulk TiO2. Injected TiO2-NPs accumulated only in kidney (94% of measured Ti) and to a lesser extent in spleen; and injected bulk TiO2 was found only in kidney. No toxicity of TiO2 was observed in kidney, spleen, or other tissues. Critical comparison of these data with previous studies indicates that dietary and waterborne exposures to TiO2-NPs do not lead to Ti accumulation in internal tissues, and previous reports of minor toxicity are inconsistent or attributable to respiratory distress resulting from gill occlusion during waterborne exposure.
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Molecular mapping the presence of druggable targets in preinvasive and precursor breast lesions: a comprehensive review of biomarkers related to therapeutic interventions.
Biochim. Biophys. Acta
PUBLISHED: 01-28-2013
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The analysis of clinical breast samples using biomarkers is integral to current breast cancer management. Currently, a limited number of targeted therapies are standard of care in breast cancer treatment. However, these targeted therapies are only suitable for a subset of patients and resistance may occur. Strategies to prevent the occurrence of invasive lesions are required to reduce the morbidity and mortality associated with the development of cancer. In theory, application of targeted therapies to pre-invasive lesions will prevent their progression to invasive lesions with full malignant potential. The diagnostic challenge for pathologists is to make interpretative decisions on early detected pre-invasive lesions. Overall, only a small proportion of these pre-invasive lesions will progress to invasive carcinoma and morphological assessment is an imprecise and subjective means to differentiate histologically identical lesions with varying malignant potential. Therefore differential biomarker analysis in pre-invasive lesions may prevent overtreatment with surgery and provide a predictive indicator of response to therapy. There follows a review of established and emerging potential druggable targets in pre-invasive lesions and correlation with lesion morphology.
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Protein tyrosine phosphatase expression profile of rheumatoid arthritis fibroblast-like synoviocytes: a novel role of SH2 domain-containing phosphatase 2 as a modulator of invasion and survival.
Arthritis Rheum.
PUBLISHED: 01-10-2013
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The fibroblast-like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage-degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS.
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Acid-sensing ion channel 3 deficiency increases inflammation but decreases pain behavior in murine arthritis.
Arthritis Rheum.
PUBLISHED: 01-03-2013
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Through its location on nociceptors, acid-sensing ion channel 3 (ASIC-3) is activated by decreases in pH and plays a significant role in musculoskeletal pain. We recently showed that decreases in pH activate ASIC-3 located on fibroblast-like synoviocytes (FLS), which are key cells in the inflammatory process. The purpose of this study was to test whether ASIC-3-deficient mice with arthritis have altered inflammation and pain relative to controls.
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Regulation of DNA methylation in rheumatoid arthritis synoviocytes.
J. Immunol.
PUBLISHED: 01-02-2013
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Rheumatoid arthritis (RA) is a chronic inflammatory disease in which fibroblast-like synoviocytes (FLS) exhibit an aggressive phenotype. Although the mechanisms responsible are not well defined, epigenetic determinants such as DNA methylation might contribute. DNA methyltransferases (DNMTs) are critical enzymes that establish and maintain DNA methylation. We evaluated whether proinflammatory cytokines might contribute to differential DNA methylation previously described in RA FLS through altered DNMT expression. FLS were obtained from RA and osteoarthritis (OA) synovium at the time of total joint replacement. Gene expression was determined by quantitative real-time PCR and protein expression by Western blot analysis. DNMT activity was measured with a functional assay, and global methylation was determined by an immunoassay that detects methylcytosine. Resting expression of DNMT1, -3a, and -3b mRNA were similar in RA and OA FLS. Western blot showed abundant DNMT1 and DNMT3a protein. Exposure to IL-1 decreased DNMT1 and DNMT3a mRNA expression in FLS. Dose responses demonstrated decreased DNMT expression at concentrations as low as 1 pg/ml of IL-1. DNMT mRNA levels decreased rapidly, with significant suppression after 2-8 h of IL-1 stimulation. IL-1 stimulation of OA FLS did not affect methylation of LINE1 sites but led to demethylation of a CHI3L1 locus that is hypomethylated in RA FLS. Chronic IL-1 stimulation also mimicked the effect of a DNMT inhibitor on FLS gene expression. Exposure to proinflammatory mediators reversibly alters DNA methylation in FLS by decreasing DNMT expression and function. These data suggest that IL-1 can potentially imprint cells in chronic inflammatory diseases.
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An imprinted rheumatoid arthritis methylome signature reflects pathogenic phenotype.
Genome Med
PUBLISHED: 01-01-2013
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BACKGROUND: A DNA methylation signature has been characterized that distinguishes rheumatoid arthritis (RA) fibroblast like synoviocytes (FLS) from osteoarthritis (OA) FLS. The presence of epigenetic changes in long-term cultured cells suggest that rheumatoid FLS imprinting might contribute to pathogenic behavior. To understand how differentially methylated genes (DMGs) might participate in the pathogenesis of RA, we evaluated the stability of the RA signature and whether DMGs are enriched in specific pathways and ontology categories. METHODS: To assess the RA methylation signatures the Illumina HumanMethylation450 chip was used to compare methylation levels in RA, OA, and normal (NL) FLS at passage 3, 5, and 7. Then methylation frequencies at CpGs within the signature were compared between passages. To assess the enrichment of DMGs in specific pathways, DMGs were identified as genes that possess significantly differential methylated loci within their promoter regions. These sets of DMGs were then compared to pathway and ontology databases to establish enrichment in specific categories. RESULTS: Initial studies compared passage 3, 5, and 7 FLS from RA, OA, and NL. The patterns of differential methylation of each individual FLS line were very similar regardless of passage number. Using the most robust analysis, 20 out of 272 KEGG pathways and 43 out of 34,400 GO pathways were significantly altered for RA compared with OA and NL FLS. Most interestingly, we found that the KEGG Rheumatoid Arthritis pathway was consistently the most significantly enriched with differentially methylated loci. Additional pathways involved with innate immunity (Complement and Coagulation, Toll-like Receptors, NOD-like Receptors, and Cytosolic DNA-sensing), cell adhesion (Focal Adhesion, Cell Adhesion Molecule), and cytokines (Cytokine-cytokine Receptor). Taken together, KEGG and GO pathway analysis demonstrates non-random epigenetic imprinting of RA FLS. CONCLUSIONS: The DNA methylation patterns include anomalies in key genes implicated in the pathogenesis of RA and are stable for multiple cell passages. Persistent epigenetic alterations could contribute to the aggressive phenotype of RA synoviocytes and identify potential therapeutic targets that could modulate the pathogenic behavior.
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Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a single multiplex PCR and DNA sequencing.
J. Clin. Microbiol.
PUBLISHED: 12-07-2011
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Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.
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The JAK inhibitor CP-690,550 (tofacitinib) inhibits TNF-induced chemokine expression in fibroblast-like synoviocytes: autocrine role of type I interferon.
Ann. Rheum. Dis.
PUBLISHED: 11-25-2011
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The objective of this study was to investigate the effect of the novel Janus kinase inhibitor CP-690,550 in fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA).
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Pseudoinvasion of benign squamous epithelium following cervical biopsy: a pseudoneoplastic phenomenon mimicking invasive squamous carcinoma.
J. Clin. Pathol.
PUBLISHED: 09-06-2011
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To describe an unusual and hitherto unreported pseudoneoplastic phenomenon that is characterised by the entrapment of benign squamous epithelium following cervical loop excision or punch biopsy and that may mimic invasive squamous carcinoma.
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Suppression of collagen-induced arthritis in growth arrest and DNA damage-inducible protein 45?-deficient mice.
Arthritis Rheum.
PUBLISHED: 06-25-2011
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Growth arrest and DNA damage-inducible protein 45? (GADD45?) is involved in stress responses, cell cycle regulation, and oncogenesis. Previous studies demonstrated that GADD45? deficiency exacerbates K/BxN serum-induced arthritis and experimental allergic encephalomyelitis (EAE) in mice, indicating that GADD45? plays a suppressive role in innate and adaptive immune responses. To further understand how GADD45? regulates autoimmunity, we evaluated collagen-induced arthritis in GADD45?-/- mice.
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Field-friendly techniques for assessment of biomarkers of nutrition for development.
Am. J. Clin. Nutr.
PUBLISHED: 06-15-2011
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Whereas cost-effective interventions exist for the control of micronutrient malnutrition (MN), in low-resource settings field-friendly tools to assess the effect of these interventions are underutilized or not readily available where they are most needed. Conventional approaches for MN measurement are expensive and require relatively sophisticated laboratory instrumentation, skilled technicians, good infrastructure, and reliable sources of clean water and electricity. Consequently, there is a need to develop and introduce innovative tools that are appropriate for MN assessment in low-resource settings. These diagnostics should be cost-effective, simple to perform, robust, accurate, and capable of being performed with basic laboratory equipment. Currently, such technologies either do not exist or have been applied to the assessment of a few micronutrients. In the Demographic and Health Surveys (DHS), a few such examples for which "biomarkers" of nutrition development have been assessed in low-resource settings using field-friendly approaches are hemoglobin (anemia), retinol-binding protein (vitamin A), and iron (transferrin receptor). In all of these examples, samples were collected mainly by nonmedical staff and analyses were conducted in the survey country by technicians from the local health or research facilities. This article provides information on how the DHS has been able to successfully adapt field-friendly techniques in challenging environments in population-based surveys for the assessment of micronutrient deficiencies. Special emphasis is placed on sample collection, processing, and testing in relation to the availability of local technology, resources, and capacity.
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Synoviocyte innate immune responses: TANK-binding kinase-1 as a potential therapeutic target in rheumatoid arthritis.
Rheumatology (Oxford)
PUBLISHED: 05-25-2011
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Innate immune responses in the rheumatoid synovium contribute to inflammation and joint destruction in RA. Two I?B kinase (IKK)-related kinases, TNF receptor associated factor (TRAF) family member-associated nuclear factor ?-light-chain enhancer of activated B cells (NF-?B) activator (TANK)-binding kinase 1 (TBK1) and IKK?, potentially regulate synovitis by activating IFN response genes. These kinases induce the expression of inflammatory mediators such as C-X-C motif ligand 10 (CXCL10)/IFN-?-induced protein 10?kDa (IP-10) in fibroblast-like synoviocytes (FLS). Since IP-10 is a promising therapeutic target in RA, we evaluated whether blocking TBK1 might be an effective way to modulate IP-10 expression.
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Tibrogargan and Coastal Plains rhabdoviruses: genomic characterization, evolution of novel genes and seroprevalence in Australian livestock.
J. Gen. Virol.
PUBLISHED: 05-18-2011
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Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle in Australia and TIBV has also been isolated from the biting midge Culicoides brevitarsis. Complete genomic sequencing revealed that the viruses share a novel genome structure within the family Rhabdoviridae, each virus containing two additional putative genes between the matrix protein (M) and glycoprotein (G) genes and one between the G and viral RNA polymerase (L) genes. The predicted novel protein products are highly diverged at the sequence level but demonstrate clear conservation of secondary structure elements, suggesting conservation of biological functions. Phylogenetic analyses showed that TIBV and CPV form an independent group within the dimarhabdovirus supergroup. Although no disease has been observed in association with these viruses, antibodies were detected at high prevalence in cattle and buffalo in northern Australia, indicating the need for disease monitoring and further study of this distinctive group of viruses.
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A simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings.
PLoS ONE
PUBLISHED: 04-14-2011
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Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation).
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Rapid molecular strategy for orbivirus detection and characterization.
J. Clin. Microbiol.
PUBLISHED: 03-30-2011
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Orbiviruses infect a wide range of hosts, including humans. The ability to detect them has been hampered by their diversity. Here we present a simple consensus reverse transcription (RT)-PCR method targeting the polymerase gene for orbivirus recognition and characterization. Phylogenetic assignment is achieved by automated Web-based sequence analysis of amplification products.
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Emerging technologies for point-of-care CD4 T-lymphocyte counting.
Trends Biotechnol.
PUBLISHED: 03-25-2011
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A CD4 T-lymphocyte count determines eligibility for antiretroviral therapy (ART) in patients recently diagnosed with HIV and also monitors the efficacy of ART treatment thereafter. ART slows the progression of HIV to AIDS. In the developing world, CD4 tests are often performed in centralized laboratories, typically in urban areas. The expansion of ART programs into rural areas has created a need for rapid CD4 counting because logistical barriers can delay the timely dissemination of test results and affect patient care through delay in intervention or loss of follow-up care. CD4 measurement at the point-of-care (POC) in rural areas could help the facilitation of ART and monitoring of treatment. This review highlights recent technology developments with applications towards determining CD4 counts at the POC.
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Physiological response to a metal-contaminated invertebrate diet in zebrafish: importance of metal speciation and regulation of metal transport pathways.
Aquat. Toxicol.
PUBLISHED: 02-23-2011
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Dietary metal uptake in fish is determined by metal bioavailability in prey and the metal requirements of the fish. In this study zebrafish were fed the intertidal polychaete worm Nereis diversicolor (3% wet weight day(-1)) collected from Ag, Cd and Cu-impacted Restronguet Creek (RC) or a reference site, Blackwater estuary (BW), for 21 days. On days 0, 7, 14 and 21 fish were fed a single meal of RC or BW N. diversicolor labeled with (110m)Ag or (109)Cd for measurements of metal assimilation efficiency (AE). Zebrafish intestines were also taken for mRNA expression analysis of copper transporter 1 (ctr1), divalent metal transporter 1 (dmt1) and metallothionein 2 (mt2). No significant difference was observed in the AE of (109)Cd in metal naïve fish at day 0 between RC and BW worms, 11.8±2.1 and 15.3±2.8%, respectively. However, AE of (110m)Ag was significantly greater in fish fed worms from BW compared to RC, 5±1.2% and 1.6±0.5%, respectively at day 0. Fractionation analysis of radiolabeled metal partitioned in N. diversicolor from RC revealed a greater proportion of Ag (40±1.1%) in a fraction containing protein and organelle bound metal, associated with high trophic availability, compared to BW polychaetes (24±2.5%). Lower AE of (110m)Ag from RC polychaetes is therefore unlikely due to speciation of (110m)Ag in N. diversicolor from RC, but to the high concentration of Cu, a potential Ag antagonist. Exposure to RC polychaetes significantly increased the AE of (110m)Ag (6.2±1%), but not (109)Cd, from RC worms, after 21 days. AE of (110m)Ag and (109)Cd was unaffected by pre-exposure to BW. Elevated concentration of intestinal Cu and increased expression of ctr1, dmt1 and mt2 after 14 days exposure in fish fed worms from RC suggest altered Cu handling strategy of these fish which may increase AE of Ag via shared Ag and Cu transport pathways. These data suggest metal exposure history of invertebrates may affect metal bioavailability to fish, and fish may alter intestinal uptake physiology during chronic dietary exposure with implications for the assimilation and toxicity of dietary metals.
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Preclinical efficacy studies of influenza A haemagglutinin precursor cleavage loop peptides as a potential vaccine.
J. Gen. Virol.
PUBLISHED: 02-02-2011
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A universal influenza vaccine that does not require annual reformulation would have clear advantages over the currently approved seasonal vaccine. In this study, we combined the mucosal adjuvant alpha-galactosylceramide (?GalCer) and peptides designed across the highly conserved influenza precursor haemagglutinin (HA(0)) cleavage loop as a vaccine. Peptides designed across the HA(0) of influenza A/H3N2 viruses, delivered to mice via the intranasal route with ?GalCer as an adjuvant, provided 100?% protection following H3N2 virus challenge. Similarly, intranasal inoculation of peptides across the HA(0) of influenza A/H5N1 with ?GalCer completely protected mice against heterotypic challenge with H3N2 virus. Our data suggest that these peptide vaccines effectively inhibited subsequent influenza A/H3N2 virus replication. In contrast, only 20?% of mice vaccinated with ?GalCer-adjuvanted peptides spanning the HA(0) of H5N1 survived homologous viral challenge, possibly because the HA(0) of this virus subtype is cleaved by intracellular furin-like enzymes. Results of these studies demonstrated that HA(0) peptides adjuvanted with ?GalCer have the potential to form the basis of a synthetic, intranasal influenza vaccine.
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A comparative analysis of HIV-specific mucosal/systemic T cell immunity and avidity following rDNA/rFPV and poxvirus-poxvirus prime boost immunisations.
Vaccine
PUBLISHED: 01-19-2011
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In this study we have firstly compared a range of recombinant DNA poxvirus prime-boost immunisation strategies and shown that combined intramuscular (i.m.) 2× DNA-HIV/intranasal (i.n.) 2× FPV-HIV prime-boost immunisation can generate high-level of HIV-specific systemic (spleen) and mucosal (genito-rectal nodes, vaginal tissues and lung tissues) T cell responses and HIV-1 p24 Gag-specific serum IgG1, IgG2a and mucosal IgG, SIgA responses in vaginal secretions in BALB/c mice. Data indicate that following rDNA priming, two rFPV booster immunisations were necessary to generate good antibody and mucosal T cell immunity. This data also revealed that mucosal uptake of recombinant fowl pox (rFPV) was far superior to plasmid DNA. To further evaluate CD8+ T cell immunity, i.m. 2× DNA-HIV/i.n. 1× FPV-HIV immunisation strategy was directly compared with single shot poxvirus/poxvirus, i.n. FPV-HIV/i.m. VV-HIV immunisation. Results indicate that the latter strategy was able to generate strong sustained HIV-specific CD8+ T cells with higher avidity, broader cytokine/chemokine profiles and better protection following influenza-K(d)Gag(197-205) challenge compared to rDNA poxvirus prime-boost strategy. Our findings further substantiate the importance of vector selection/combination, order and route of delivery when designing effective vaccines for HIV-1.
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Point-of-care nucleic acid testing for infectious diseases.
Trends Biotechnol.
PUBLISHED: 01-17-2011
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Nucleic acid testing for infectious diseases at the point of care is beginning to enter clinical practice in developed and developing countries; especially for applications requiring fast turnaround times, and in settings where a centralized laboratory approach faces limitations. Current systems for clinical diagnostic applications are mainly PCR-based, can only be used in hospitals, and are still relatively complex and expensive. Integrating sample preparation with nucleic acid amplification and detection in a cost-effective, robust, and user-friendly format remains challenging. This review describes recent technical advances that might be able to address these limitations, with a focus on isothermal nucleic acid amplification methods. It briefly discusses selected applications related to the diagnosis and management of tuberculosis, HIV, and perinatal and nosocomial infections.
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Interleukin 1 receptor antagonist mediates the beneficial effects of systemic interferon beta in mice: implications for rheumatoid arthritis.
Ann. Rheum. Dis.
PUBLISHED: 01-07-2011
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Interferon beta (IFN?) therapy is effective in multiple sclerosis and murine models of arthritis. Surprisingly, systemic IFN? treatment induces only minimal improvement in rheumatoid arthritis (RA). To explain this paradox, the authors evaluated the mechanism of IFN? benefit in passive K/BxN arthritis and the effect of IFN? treatment on RA synovium.
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CXCL13: a novel biomarker of B-cell return following rituximab treatment and synovitis in patients with rheumatoid arthritis.
Rheumatology (Oxford)
PUBLISHED: 11-23-2010
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The B-cell chemokine, CXCL13, is a proposed serum biomarker of synovitis in RA. Its behaviour in the context of B-cell depletion therapy and reconstitution was studied during treatment of RA with rituximab.
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Bioavailability of a natural lead-contaminated invertebrate diet to zebrafish.
Environ. Toxicol. Chem.
PUBLISHED: 09-08-2010
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Dietary metals are increasingly recognized as key determinants of total metal burdens in fish, yet their ecotoxicological significance remains unclear. In this study, a pairwise experimental design was used to assess reproductive performance of zebrafish (Danio rerio) fed diets supplemented with a natural Pb-enriched polychaete, Nereis diversicolor. Zebrafish were fed 1% flake food (dry wt diet/wet wt fish/d), 1% brine shrimp, and 1% N. diversicolor collected from either Gannel estuary, Cornwall, United Kingdom (UK), an estuary with legacy Pb contamination, or Blackwater estuary, Essex, UK, a reference site with low background metal concentrations, for 63 d. Mean daily dietary doses of Pb were 0.417 and 0.1 mg/kg/d (dry wt feed:wet wt fish) for fish fed N. diversicolor from Gannel and Blackwater estuaries, respectively. With the exception of Ag, which was higher for fish fed N. diversicolor from Gannel estuary, there were no differences in daily dietary exposures to other metals (As, Cd, Cu, Fe, and Zn) between treatment groups. Fish fed Pb-enriched Gannel N. diversicolor exhibited no significant impairment to incidence of spawning, numbers of eggs per breeding pair or hatch rate of embryos compared with pre-exposure levels, when N. diversicolor was omitted from the dietary regimen. Nevertheless, metal analysis revealed significant increases in whole-body Pb burdens of male fish fed polychaetes from Gannel estuary, Ag in female fish fed Gannel worms, and Ag and Cd in male fish fed the Blackwater worms. These data demonstrate that Pb naturally incorporated in N. diversicolor is bioavailable to fish, and fish exhibit sex-dependent dietary metal accumulation patterns, but after 63 d of the experimental feeding regimen, reproductive performance was unaffected.
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Role of interleukin 17 in arthritis chronicity through survival of synoviocytes via regulation of synoviolin expression.
PLoS ONE
PUBLISHED: 08-12-2010
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The use of TNF inhibitors has been a major progress in the treatment of chronic inflammation. However, not all patients respond. In addition, response will be often lost when treatment is stopped. These clinical aspects indicate that other cytokines might be involved and we focus here on the role of IL-17. In addition, the chronic nature of joint inflammation may contribute to reduced response and enhanced chronicity. Therefore we studied the capacity of IL-17 to regulate synoviolin, an E3 ubiquitin ligase implicated in synovial hyperplasia in human rheumatoid arthritis (RA) FLS and in chronic reactivated streptococcal cell wall (SCW)-induced arthritis.
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Molecular analysis of H7 avian influenza viruses from Australia and New Zealand: genetic diversity and relationships from 1976 to 2007.
J. Virol.
PUBLISHED: 07-28-2010
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Full-genome sequencing of 11 Australian and 1 New Zealand avian influenza A virus isolate (all subtype H7) has enabled comparison of the sequences of each of the genome segments to those of other subtype H7 avian influenza A viruses. The inference of phylogenetic relationships for each segment has been used to develop a model of the natural history of these viruses in Australia. Phylogenetic analysis of the hemagglutinin segment indicates that the Australian H7 isolates form a monophyletic clade. This pattern is consistent with the long-term, independent evolution that is, in this instance, associated with geographic regions. On the basis of the analysis of the other H7 hemagglutinin sequences, three other geographic regions for which similar monophyletic clades have been observed were confirmed. These regions are Eurasia plus Africa, North America, and South America. Analysis of the neuraminidase sequences from the H7N1, H7N3, and H7N7 genomes revealed the same region-based relationships. This pattern of independent evolution of Australian isolates is supported by the results of analysis of each of the six remaining genomic segments. These results, in conjunction with the occurrence of five different combinations of neuraminidase subtypes (H7N2, H7N3, H7N4, H7N6, H7N7) among the 11 Australian isolates, suggest that the maintenance host(s) is nearly exclusively associated with Australia. The single lineage of Australian H7 hemagglutinin sequences, despite the occurrence of multiple neuraminidase types, suggests the existence of a genetic pool from which a variety of reassortants arise rather than the presence of a small number of stable viral clones. This pattern of evolution is likely to occur in each of the regions mentioned above.
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Development of microarray and multiplex polymerase chain reaction assays for identification of serovars and virulence genes in Salmonella enterica of human or animal origin.
J. Vet. Diagn. Invest.
PUBLISHED: 07-13-2010
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Salmonella enterica is an important enteric pathogen consisting of many serovars that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiologic monitoring and controlling outbreaks of disease. Although immunologic and DNA-based serovar identification methods are available for rapid identification of isolates, they are time consuming or costly or both. In the current study, 2 molecular methods for identification of Salmonella serovars were developed and validated. A 70-mer oligonucleotide spotted microarray was developed that consisted of probes that detected genes responsible for genetic variation among isolates of Salmonella that can be used for serotyping. A multiplex polymerase chain reaction (PCR) assay was also developed, which is capable of identifying 42 serovars, thus providing a valuable prediction of the pathogenicity of the isolates by detecting the presence of virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. In a blind study, traditional serologic methods were correlated at 93.3% with the microarray-based method and 100% with the multiplex PCR-based serovar determination.
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The development of persistent duck hepatitis B virus infection can be prevented using antiviral therapy combined with DNA or recombinant fowlpoxvirus vaccines.
Vaccine
PUBLISHED: 04-09-2010
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We recently reported the development of a successful post-exposure combination antiviral and "prime-boost" vaccination strategy using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection. The current study aimed to simplify the vaccination strategy and to test the post-exposure efficacy of combination therapy with the Bristol-Myers Squibb antiviral drug, entecavir (ETV) and either a single dose of DHBV DNA vaccines on day 0 post-infection (p.i.) or a single dose of recombinant fowlpoxvirus (rFPV-DHBV) vaccines on day 7 p.i. Whilst untreated control ducks infected with an equal dose of DHBV all developed persistent and wide spread DHBV infection of the liver, ducks treated with ETV combined with either the DHBV DNA vaccines on day 0 p.i. or the rFPV-DHBV vaccines on day 7 p.i. had no detectable DHBV-infected hepatocytes by day 14 p.i. and were protected from the development of persistent DHBV infection.
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Oral treatment with HE3286 ameliorates disease in rodent models of rheumatoid arthritis.
Int. J. Mol. Med.
PUBLISHED: 03-04-2010
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HE3286 (17alpha-ethynyl-5-androstene-3beta, 7beta, 17beta-triol) is an orally bio-available synthetic derivative of naturally occurring androstene-3beta, 7beta, 17beta-triol. Our present data show that oral treatment with HE3286, favourably influenced the course of arthritis in the rat model of adjuvant-induced arthritis (reduced cumulative disease scores and paw edema), and in the mouse model of collagen antibody-induced arthritis (reduced clinical paw scores). Importantly, HE3286 was not immune suppressive in human mixed lymphocyte reaction or in animals challenged with Coxsackie B3 virus. HE3286 is currently in phase I/II clinical trials in rheumatoid arthritis and ulcerative colitis and these findings further strengthen the possibility that HE3286 may represent an effective anti-inflammatory agent useful for treating chronic inflammation with a more attractive safety profile than glucocorticoids or cyclooxygenase inhibitors.
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Platelet-derived growth factor and transforming growth factor beta synergistically potentiate inflammatory mediator synthesis by fibroblast-like synoviocytes.
Arthritis Res. Ther.
PUBLISHED: 02-19-2010
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The objective of this study was to model the effects of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF), both present in rheumatoid arthritis (RA) synovia, on the behavior of fibroblast-like synoviocytes (FLS) in response to pro-inflammatory cytokine (interleukin (IL)1beta, tumor necrosis factor-alpha (TNFalpha)) challenge.
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Ngaingan virus, a macropod-associated rhabdovirus, contains a second glycoprotein gene and seven novel open reading frames.
Virology
PUBLISHED: 01-20-2010
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Ngaingan virus (NGAV) was isolated from a pool of biting midges that were collected in the tropics of northern Australia. Reported here is the full-length sequence of the NGAV genome, which, at over 15.7 kb, is the largest in any rhabdovirus described to date and contains 13 genes, the highest number of genes observed in any (-) ssRNA virus. Seven of these putative genes show no significant homology to known proteins. Like viruses in the genus Ephemerovirus, NGAV possesses a second glycoprotein gene (G(NS)). Phylogenetic analyses, however, place NGAV within the yet to be classified "Hart Park" group containing Wongabel and Flanders viruses, which do not contain a second glycoprotein gene. Screening of various animal sera from northern Australia has indicated that NGAV is currently circulating in macropods (wallabies, wallaroos and kangaroos), highlighting the need for further studies to determine its potential to cause disease in these species.
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Inflammatory characteristics on ultrasound predict poorer longterm response to intraarticular corticosteroid injections in knee osteoarthritis.
J. Rheumatol.
PUBLISHED: 01-15-2010
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To assess whether inflammation on ultrasound is predictive of clinical response to intraarticular (IA) corticosteroid injections in patients with knee osteoarthritis (OA).
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Ethics education in neonatal-perinatal medicine in the United States.
Semin. Perinatol.
PUBLISHED: 11-17-2009
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Neonatology is one of the specialties that has immensely benefited from advances in medical technology in the last few decades. These advances have paralleled the rise of the civil rights movements and wider recognition of individual rights. As a result, ethical decision-making has become more complex, involving patients, parents, members of the health care team, and society in general. This has created a need for formal ethics education in neonatal-perinatal medicine fellowship programs. In this article, we briefly explore the current published data on ethics education in pediatric residency and neonatal-perinatal medicine fellowship programs. Then, we discuss the questions an academic educator may face while developing an ethics curriculum in his/her medical institution. Finally, we present the ethics curriculum that we developed in our neonatal-perinatal medicine fellowship program.
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Gadd45beta deficiency in rheumatoid arthritis: enhanced synovitis through JNK signaling.
Arthritis Rheum.
PUBLISHED: 10-31-2009
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JNK-mediated cell signaling plays a critical role in matrix metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45beta, which is an NF-kappaB-regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway, since it could block the upstream kinase MKK-7. This study was carried out to evaluate whether low Gadd45beta expression in RA enhances JNK activation and overproduction of MMPs in RA, and whether Gadd45beta deficiency increases arthritis severity in passive K/BxN murine arthritis.
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Regulation of dendritic cells and macrophages by an anti-apoptotic cell natural antibody that suppresses TLR responses and inhibits inflammatory arthritis.
J. Immunol.
PUBLISHED: 06-29-2009
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Although natural Abs (NAbs) are present from birth, little is known about what drives their selection and whether they have housekeeping functions. The prototypic T15-NAb, first identified because of its protective role in infection, is representative of a special type of NAb response that specifically recognizes and forms complexes with apoptotic cells and which promotes cell-corpse engulfment by phagocytes. We now show that this T15-NAb IgM-mediated clearance process is dependent on the recruitment of C1q and mannose-binding lectin, which have known immune modulatory activities that also provide "eat me" signals for enhancing phagocytosis. Further investigation revealed that the addition of T15-NAb significantly suppressed in vitro LPS-induced TNF-alpha and IL-6 secretion by the macrophage-like cell line, RAW264.7, as well as TLR3-, TLR4-, TLR7-, and TLR9-induced maturation and secretion of a range of proinflammatory cytokines and chemokines by bone marrow-derived conventional dendritic cells. Significantly, high doses of this B-1 cell produced NAb also suppressed in vivo TLR-induced proinflammatory responses. Although infusions of apoptotic cells also suppressed such in vivo inflammatory responses and this effect was associated with the induction of high levels of IgM antiapoptotic cell Abs, apoptotic cell treatment was not effective at suppressing such TLR responses in B cell-deficient mice. Moreover, infusions of T15-NAb also efficiently inhibited both collagen-induced arthritis and anti-collagen II Ab-mediated arthritis. These studies identify and characterize a previously unknown regulatory circuit by which a NAb product of innate-like B cells aids homeostasis by control of fundamental inflammatory pathways.
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Role of MAPK kinase 6 in arthritis: distinct mechanism of action in inflammation and cytokine expression.
J. Immunol.
PUBLISHED: 06-26-2009
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Development of p38alpha inhibitors for rheumatoid arthritis has been hindered by toxicity and limited efficacy. Therefore, we evaluated whether MKK6, an upstream kinase that regulates multiple p38 isoforms, might be an alternative therapeutic target in inflammatory arthritis. Wild-type (WT), MKK6(-/-), and MKK3(-/-) mice were administered K/BxN serum to induce arthritis. Articular expression of activated kinases and cytokines was determined by Western blot, qPCR, ELISA, and multiplex analysis. Immunoprecipitation and confocal microscopy experiments were performed to determine the subcellular location of MKK6, P-p38, and MAPKAPK2 (MK2). Arthritis scores were significantly lower in MKK6(-/-) mice compared with WT mice. Joint destruction and osteoclast differentiation were lower in MKK6(-/-), as were articular IL-6 and matrix metalloproteinase-3 expression. Phospho-p38 levels were modestly decreased in the joints of arthritic MKK6(-/-) mice compared with WT but were significantly higher than MKK3(-/-) mice. P-MK2 was low in MKK6(-/-) and MKK3(-/-) mice. Uncoupled p38 and MK2 activation was also observed in cultured, MKK6(-/-) FLS and confirmed using kinase assays. Immunoprecipitation assays and confocal microscopy showed that P-p38 and MK2 colocalized in activated WT but not MKK6(-/-) FLS. Distinct patterns of cytokine production were observed in MKK6(-/-) and MKK3(-/-) cells. MKK6 deficiency suppresses inflammatory arthritis and joint destruction, suggesting it might be a therapeutic target for inflammation. Although MKK3 and MKK6 activate the p38 pathway, they regulate distinct subsets of proinflammatory cytokines. MKK6 appears mainly to facilitate p38 and MK2 colocalization in the nucleus rather than to phosphorylate p38.
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Kinetic analysis of synovial signalling and gene expression in animal models of arthritis.
Ann. Rheum. Dis.
PUBLISHED: 05-26-2009
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Animal models of arthritis are frequently used to evaluate novel therapeutic agents. However, their ability to predict responses in humans is variable.
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Preliminary evaluation of a procedure for improved detection of Shiga toxin-producing Escherichia coli in fecal specimens.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 05-12-2009
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Culture confirmation of Shiga toxin-producing Escherichia coli (STEC) is very important for epidemiologic analysis. However, isolation of non-O157 STEC on conventional selective media such as sorbitol-MacConkey agar (SMAC) can be difficult because of heavy growth of competing bacteria and its phenotypical similarity to commensal nonpathogenic E. coli. An acid enrichment procedure was introduced in this study to facilitate detection of STEC from patients who were symptomatic. Forty-seven clinical fecal broths, which tested positive for Shiga toxin by commercial immunoassay, were processed for the isolation of STEC by both conventional and the acid enrichment methods. The acid enrichment method and conventional culture recovered STEC from 91% (43/47) and 70% (33/47) of the fecal broths, respectively. Neither method retrieved STEC in 3 specimens. Thirty-six STEC were successfully serogrouped, which included O26 (n = 11), O157 (n = 9), O103 (n = 7), O121 (n = 3), O111 (n = 2 each), O28AC, O146, O76, and O undetermined (n = 1 each). The analysis of STEC isolates by real-time PCR indicated that all 9 E. coli O157 contained stx2 gene alone or in combination with stx1. Non-O157 STEC more frequently contained stx1 only, and about one-third possessed stx2. The novel acid enrichment protocol greatly reduced the growth of competitor colonies on RTN and TCSMAC. The study demonstrated that incorporation of an acid enrichment procedure in clinical testing improved the isolation of STEC in fecal specimens.
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Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity.
Immunity
PUBLISHED: 04-24-2009
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NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.
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An orally bioavailable synthetic analog of an active dehydroepiandrosterone metabolite reduces established disease in rodent models of rheumatoid arthritis.
J. Pharmacol. Exp. Ther.
PUBLISHED: 03-18-2009
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Dehydroepiandrosterone (DHEA) treatment provides diverse anti-inflammatory benefits in rodent models of diseases, including rheumatoid arthritis (RA), but only limited benefits to patients. In rodents, DHEA is metabolized to (among others) androstene-3beta,7beta,17beta-triol (AET), which retains potent anti-inflammatory activity. 17Alpha-ethynyl-5-androstene-3beta,7beta,17beta-triol (HE3286) is a novel, metabolically stabilized, orally bioavailable derivative of AET. In the DBA mouse model of collagen-induced arthritis (CIA), once-daily oral treatments (gavage) with HE3286 (40 mg/kg), beginning at onset of disease, significantly decreased disease. Benefit was associated with reduction in joint inflammation, erosion, and synovial proliferation as measured by histological analysis and mRNA of proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin (IL)-6, IL-1beta, and IL-23. Significant benefit was also observed in the CIA model even when treatments were delayed until 7 days after the onset of arthritis. Furthermore, dose-dependent benefit was observed in the DBA mouse model of collagen antibody-induced arthritis, as well as reductions in IL-6 and matrix metalloproteinase-3 mRNA levels in joints at the peak of disease and at the end of the study. HE3286, in contrast to dexamethasone, was not immune-suppressive in several classic animal models of immune function. Instead, HE3286 treatment was associated with reduced nuclear factor-kappaB activation and in our previous studies, with increased regulatory T cells. We hypothesize that HE3286 may represent a novel, perhaps first-in-class, anti-inflammatory agent and may more fully translate the benefits of DHEA, heretofore largely limited to rodents, into treatments for human diseases, including autoimmune disorders such as RA.
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Genetic and epidemiological characterization of Stretch Lagoon orbivirus, a novel orbivirus isolated from Culex and Aedes mosquitoes in northern Australia.
J. Gen. Virol.
PUBLISHED: 03-12-2009
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Stretch Lagoon orbivirus (SLOV) was isolated in 2002 from pooled Culex annulirostris mosquitoes collected at Stretch Lagoon, near the Wolfe Creek national park in the Kimberley region of Western Australia. Conventional serological tests were unable to identify the isolate, and electron microscopy indicated a virus of the genus Orbivirus, family Reoviridae. Here, a cDNA subtraction method was used to obtain approximately one-third of the viral genome, and further sequencing was performed to complete the sequences of segment 1 (viral polymerase) and segment 2 (conserved inner-core protein). Phylogenetic analysis showed that SLOV should be considered a new species within the genus Orbivirus. A real-time RT-PCR test was designed to study the epidemiology of SLOV in the field. Six additional isolates of SLOV were identified, including isolates from four additional locations and two additional mosquito species. Horses, donkeys and goats were implicated as potential vertebrate hosts in a serological survey.
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High-throughput molecular determination of salmonella enterica serovars by use of multiplex PCR and capillary electrophoresis analysis.
J. Clin. Microbiol.
PUBLISHED: 03-04-2009
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Salmonella enterica is a leading cause of food-borne illness worldwide and is also a major cause of morbidity and mortality in domestic and wild animals. In the current study, a high-throughput molecular assay was developed to determine the most common clinical and nonhuman serovars of S. enterica in the United States. Sixteen genomic targets were identified based on their differential distribution among common serovars. Primers were designed to amplify regions of each of these targets in a single multiplex PCR while incorporating a 6-carboxyfluorescein-labeled universal primer to fluorescently label all amplicons. The fluorescently labeled PCR products were separated using capillary electrophoresis, and a Salmonella multiplex assay for rapid typing (SMART) code was generated for each isolate, based upon the presence or absence of PCR products generated from each target gene. Seven hundred fifty-one blind clinical isolates of Salmonella from Washington State, collected in 2007 and previously serotyped via antisera, were screened with the assay. A total of 89.6% of the isolates were correctly identified based on comparison to a panel of representative SMART codes previously determined for the top 50 most common serovars in the United States. Of the remaining isolates, 6.2% represented isolates that produced a new SMART code for a previously determined serotype, while the final 8.8% were from serotypes not screened in the original panel used to score isolates in the blinded study. This high-throughput multiplex PCR assay allowed simple and accurate typing of the most prevalent clinical serovars of Salmonella enterica at a level comparable to that of conventional serotyping, but at a fraction of both the cost and time required per test.
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Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals.
Vet. Microbiol.
PUBLISHED: 03-04-2009
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Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.
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Detection of antibodies specific for sheeppox and goatpox viruses using recombinant capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay.
J. Virol. Methods
PUBLISHED: 03-02-2009
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Viruses in the genus Capripoxvirus, family Poxviridae, cause sheeppox, goatpox and lumpy skin disease, which are the most serious poxvirus diseases of production animals. Despite the considerable threat that these viruses pose to livestock production and global trade in sheep, goats, cattle and their products, convenient and effective serodiagnostic tools are not readily available. To develop a more effective antibody detection capability, selected open reading frames from capripoxvirus DNA were amplified and expressed in Escherichia coli as His-tagged fusion proteins. By screening 42 candidate antigens, two sheeppox virus virion core proteins that were expressed efficiently, purified readily using affinity chromatography and reactive against capripoxvirus immune sera in an indirect enzyme-linked immunosorbent assay (ELISA) were identified. The ELISA performed favourably when sera from sheep and goats infected experimentally with virulent capripoxvirus isolates were tested, with sensitivity and diagnostic specificity ranging between 95 and 97%, but it was unable to detect antibodies reliably in vaccinated sheep or goats. Furthermore, no cross-reactivity with antibodies against orf virus was detected. This assay offers the prospect of a convenient and standardised ELISA-based serodiagnostic test, with no requirement for infectious reagents, that is well suited to high-throughput capripoxvirus surveillance on a flock or herd basis.
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Yemen and Vietnam capripoxviruses demonstrate a distinct host preference for goats compared with sheep.
J. Gen. Virol.
PUBLISHED: 02-13-2009
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Sheeppox and goatpox are caused by viruses that are members of the genus Capripoxvirus, and globally result in significant production losses. To improve the understanding of disease pathogenesis and evaluate host species preferences, sheep and goats were inoculated either with a capripoxvirus isolate from Yemen or from a recent outbreak in Vietnam. Blood, swabs and tissues were collected at various time points following experimental challenge and assessed for viral DNA content using real-time PCR and infectivity using virus isolation. The Yemen isolate was considerably more pathogenic in goats with 100 % mortality and morbidity compared with sheep with 0 % mortality and 100 % morbidity. The Vietnam isolate was also more pathogenic in goats with 100 % morbidity and an estimated 33 % mortality rate compared with mild morbidity and a 0 % mortality rate in sheep. Higher viral titres were observed in nasal, oral and conjunctival swabs from goats inoculated with either the Yemen or Vietnam isolate compared with those collected from sheep. Although the highest viral titres were detected in primary and secondary skin lesions in sheep and goats, the severity of clinical disease observed in each species varied according to the inoculum used. Whereas both the Yemen and Vietnam isolates clearly caused more severe disease in goats, the Yemen isolate was also moderately pathogenic in sheep. The Vietnam isolate, in contrast, caused only very mild disease in sheep. Limited DNA sequencing revealed ORF 074 of the Vietnam isolate to be identical to that of several goatpox virus isolates from China, suggesting a possible Chinese origin.
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Intranasal flu vaccine protective against seasonal and H5N1 avian influenza infections.
PLoS ONE
PUBLISHED: 02-11-2009
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Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that gamma-ray inactivated flu virus can induce cross-reactive Tc cell responses.
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Subtle alterations in swimming speed distributions of rainbow trout exposed to titanium dioxide nanoparticles are associated with gill rather than brain injury.
Aquat. Toxicol.
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The effects of engineered nanomaterials on fish behaviours are poorly understood. The present study aimed to determine the locomotor behaviours of trout during waterborne exposure to titanium dioxide nanoparticles (TiO(2) NPs) as well as inform on the underlying physiological mechanisms involved. Trout were exposed to either control (without TiO(2)), 1 mg l(-1) TiO(2) NPs or 1 mg l(-1) bulk TiO(2) for 14 days. Titanium dioxide exposure resulted in 31 (bulk) and 22 fold (nano) increases in the Ti concentrations of gill tissue compared to controls, but there were no measurable increases of Ti in the internal organs including the brain. Gill pathologies were observed in both TiO(2) treatments. Locomotor behaviours were quantified using video tracking software and the proportion of time spent swimming at high speed (>20 cms(-1)) was significantly decreased in fish exposed to TiO(2) NPs, compared to controls, but not fish exposed to bulk TiO(2). The shift in swimming speed distribution in the TiO(2) NP-exposed fish was associated with decreased area of red pulp in the spleen, increases in haematocrit and whole blood haemoglobin, all consistent with a compensation for respiratory hypoxia without the accumulation of plasma lactate. Fish exposed to TiO(2) NPs also retained competitive abilities when paired with controls in aggressive social encounters. The duration of competitive contests, the level of aggression and contest outcome were not affected by NP exposure. Neurological injury did not explain the changes in locomotor behaviour, although there was some apparent enlargement of the blood vessels on the brain. Whole brain homogenates showed a statistically significant increase in oxidative stress defences such as the total glutathione pool, but without loss of Na(+)K(+)-ATPase or acetylcholinesterase activities.
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Nucleic acid testing for tuberculosis at the point-of-care in high-burden countries.
Expert Rev. Mol. Diagn.
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Early diagnosis of tuberculosis (TB) facilitates appropriate treatment initiation and can limit the spread of this highly contagious disease. However, commonly used TB diagnostic methods are slow, often insensitive, cumbersome and inaccessible to most patients in TB endemic countries that lack necessary resources. This review discusses nucleic acid amplification technologies, which are being developed for rapid near patient TB diagnosis, that are in the market or undergoing clinical evaluation. They are based on PCR or isothermal methods and are implemented as manual assays or partially/fully integrated instrument systems, with associated tradeoffs between clinical performance, cost, robustness, quality assurance and usability in remote settings by minimally trained personnel. Unmet needs prevail for the identification of drug-resistant TB and for TB diagnosis in HIV-positive and pediatric patients.
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Serious and life-threatening pregnancy-related infections: opportunities to reduce the global burden.
PLoS Med.
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Michael Gravett and colleagues review the burden of pregnancy-related infections, especially in low- and middle-income countries, and offer suggestions for a more effective intervention strategy.
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