Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer.

The cancer­stromal interaction has been demonstrated to promote tumor progression, and cancer-associated fibroblasts (CAFs), which are the main components of stromal cells, have attracted attention as novel treatment targets. Chitinase 3-like 1 (CHI3L1) is a chitinase-like protein, which affects cell proliferation and angiogenesis. However, the mechanisms through which cells secrete CHI3L1 and through which CHI3L1 mediates tumor progression in the cancer microenvironment are still unclear. Accordingly, the present study assessed the secretion of CHI3L1 in the microenvironment of colorectal cancer and evaluated how CHI3L1 affects tumor angiogenesis. CAFs and normal fibroblasts (NFs) established from colorectal cancer tissue, and human colon cancer cell lines were evaluated using immunostaining, cytokine antibody array, RNA interference, reverse transcription-quantitative PCR (RT-qPCR), ELISA, western blotting and angiogenesis assays. The expression and secretion of CHI3L1 in CAFs were stronger than those in NFs and colorectal cancer cell lines. In addition, interleukin-13 receptor α2 (IL-13Rα2), a receptor for CHI3L1, was not expressed in colorectal cancer cell lines, but was expressed in fibroblasts, particularly CAFs. Furthermore, the expression and secretion of IL-8 in CAFs was stronger than that in NFs and cancer cell lines, and recombinant CHI3L1 addition increased IL-8 expression in CAFs, whereas knockdown of CHI3L1 suppressed IL-8 expression. Furthermore, IL-13Rα2 knockdown suppressed the enhancement of IL-8 expression induced by CHI3L1 treatment in CAFs. For vascular endothelial growth factor-A (VEGFA), similar results to IL-8 were observed in an ELISA for comparison of secretion between CAFs and NFs and for changes in secretion after CHI3L1 treatment in CAFs; however, no significant differences were observed for changes in expression after CHI3L1 treatment or IL-13Rα2 knockdown in CAFs assessed using RT-qPCR assays. Angiogenesis assays revealed that tube formation in vascular endothelial cells was suppressed by conditioned medium from CAFs with the addition of human CHI3L1 neutralizing antibodies compared with control IgG, and also suppressed by conditioned medium from CAFs transfected with CHI3L1, IL-8 or VEGFA small interfering RNA compared with negative control small interfering RNA. Overall, the present findings indicated that CHI3L1 secreted from CAFs acted on CAFs to increase the secretion of IL-8, thereby affecting tumor angiogenesis in colorectal cancer.

Interleukin-2-inducible T-cell kinase (Itk) signaling regulates potent noncanonical regulatory T cells.

Regulatory T cells (Tregs) play an important role in controlling autoimmunity and limiting tissue damage and inflammation. IL2-inducible T cell kinase (Itk) is part of the Tec family of tyrosine kinases and is a critical component of T cell receptor mediated signaling. Here, we showed that either genetic ablation of Itk signaling or inhibition of Itk signaling pathways resulted in increased frequency of "noncanonical" CD4+ CD25- FOXP3+ Tregs (ncTregs), as well as of "canonical" CD4+ CD25+ FOXP3+ Tregs (canTregs). Using in vivo models, we showed that ncTregs can avert the formation of acute graft-versus-host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft-versus-leukaemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in Itk-/- ncTregs. Furthermore, disrupting the SLP76: ITK interaction using a specific peptide inhibitor led to enhanced Treg development in both mouse and primary human cells. This peptide inhibitor also significantly reduced inflammatory cytokine production in primary GVHD patient samples and mouse T cells without causing cell death or apoptosis. We provide evidence that specifically targeting Itk signaling could be a therapeutic strategy to treat autoimmune disorders.

Comparison of ADAPT, FIB-4 and APRI as non-invasive predictors of liver fibrosis and NASH within the CENTAUR screening population.

BACKGROUND & AIMS: The development of accurate non-invasive tests to detect and measure the extent of fibrosis and disease activity in patients with non-alcoholic steatohepatitis (NASH) - the progressive phenotype of non-alcoholic fatty liver disease (NAFLD) - is of great clinical importance. Herein, we aimed to validate the performance of PRO-C3 and ADAPT for the detection of moderate/severe fibrosis within the CENTAUR screening population. METHODS: PRO-C3 was assessed in plasma from the screening population of the phase IIb CENTAUR study (NCT02217475) in adults with NASH and liver fibrosis. The relation between PRO-C3 and histologic features of NASH was evaluated, as well as the demographics of patients with high and low levels of PRO-C3. The diagnostic ability of PRO-C3, as a standalone marker or incorporated into ADAPT, to identify patients with F≥2 and NASH was estimated using receiver-operating characteristic analysis and logistic regression models. RESULTS: A total of 517 individuals with matched biopsy and PRO-C3 measurements were included. Patients with PRO-C3 levels ≥20.2 ng/ml showed increased levels of insulin, HOMA-IR, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and platelet count compared to patients with low PRO-C3 (p <0.05). PRO-C3 increased stepwise with increasing liver fibrosis, lobular inflammation, hepatocyte ballooning, steatosis, and NAFLD activity score (p <0.05), and could distinguish between NAFL and NASH (p <0.0001). PRO-C3 was independently associated with fibrosis and NASH when adjusted for clinical confounders. ADAPT outperformed Fibrosis-4, AST-to-platelet ratio index, and AST/ALT ratio as a predictor of advanced fibrosis and NASH (p <0.001). CONCLUSION: PRO-C3 was associated with NAFLD activity score and fibrosis. ADAPT outperformed other non-invasive scores for detecting NASH. These data support the use of PRO-C3 and ADAPT as diagnostic tools to identify patients with NASH eligible for inclusion in clinical trials. CLINICAL TRIAL NUMBER: NCT02217475 LAY SUMMARY: PRO-C3 is a serological biomarker associated with liver disease activity and fibrosis. Its performance for the detection of disease activity and fibrosis is improved when it is incorporated into the ADAPT score. Herein, we showed that ADAPT was better at selecting patients with non-alcoholic steatohepatitis for inclusion in clinical trials than other non-invasive scores.

Comparison of four commercial SARS-CoV-2 IgG immuno-assays in RT-PCR negative patients with suspect CT findings.

A subset of patients with Covid-19 presents with negative RT-PCR screening but suspect CT findings. Using four commercially available anti-SARS-CoV-2 IgG immuno-assays, we found this subset constituted 9.2% of all consecutively admitted outpatients with Covid-19 in our hospital. Clinical specificity for Covid-19 of some N protein-based immuno-assays was suboptimal, as positive results were observed in control patients with recent common human coronavirus, influenza B and adenovirus infections.

Intervalo de tempo decorrido entre o início dos sintomas e a realização do exame para COVID-19 nas capitais brasileiras, agosto de 2020* / Intervalo de tiempo entre el inicio de los síntomas y el examen de COVID-19 en las capitales brasileñas, agosto de 2020 / Time interval between onset of symptoms and COVID-19 testing in Brazilian state capitals, August 2020

Objetivo: Analisar as notificações de síndrome gripal segundo o intervalo de tempo decorrido entre início dos sintomas e realização do exame para COVID-19. Métodos: Estudo transversal, utilizando registros de casos de síndrome gripal contendo resultados de testes diagnósticos da COVID-19 nas capitais brasileiras e no Distrito Federal, no sistema e-SUS Notifica, entre 1º/março/2020 e 18/agosto/2020. Comparou-se o intervalo de tempo entre início dos sintomas e realização do exame (teste ANOVA), classificando-o segundo a adequação/oportunidade do exame. Resultados: Entre 1.942.514 notificações, o tempo médio entre início dos sintomas e execução dos testes foi de 10,2 dias (±17,1). Entre testados, predominou o sexo feminino (55,1%), idade de 20-39 anos (43,8%) e região Sudeste (43,0%). O teste ELISA IgM foi realizado em tempo adequado para 58,8%; e o teste rápido-antígeno, em tempo inadequado para 68,0%. Conclusão: Observou-se inadequação entre início dos sintomas e realização dos testes para COVID-19 nas regiões brasileiras.

Objetivo: Analizar las notificaciones de síndrome gripal según el intervalo de tiempo entre el inicio de los síntomas y el examen de COVID-19. Métodos: Estudio transversal utilizando registros de casos de síndrome gripal que contienen resultados de pruebas diagnósticas de COVID-19 en las capitales brasileñas y el Distrito Federal del sistema e-SUS Notifica, entre 1/marzo/2020 y 18/agosto/2020. El intervalo de tiempo se comparó entre el inicio de los síntomas y la realización del examen mediante la prueba ANOVA, clasificándolo según la adecuación/ oportunidad del examen. Resultados: Entre 1.942.514 notificaciones, el tiempo promedio entre el inicio de los síntomas y la ejecución del examen fue de 10,2 días (±17,1). Entre los evaluados, predominaron las mujeres (55,1%), 20-39 años (43,8%) y la región Sudeste (43,0%). El ELISA IgM se realizó en momento adecuado para 58,8% y la prueba de Antígeno Rápido en momento inadecuado para 68,0%. Conclusión: Se constata inadecuación de tiempo entre el inicio de los síntomas y las pruebas para COVID-19 en las regiones brasileñas.

Objective: To analyze notifications of flu-like syndrome according to the time interval between onset of symptoms and testing for COVID-19. Methods: This was a cross-sectional study using records of flu-like syndrome cases containing results of COVID-19 diagnostic tests in the Brazilian state capitals and Federal District, held on the e-SUS Notifica system, from March 1st, 2020 to August 18th, 2020. The time interval between symptom onset and testing was compared using the ANOVA test, classifying it according to test adequacy/timeliness. Results: Taking 1,942,514 notifications, average time between symptom onset and testing was 10.2 days (±17.1). Among those tested, females (55.1%), people aged 20-39 years (43.8%), and the Southeast region of Brazil (43.0%) predominated. 58.8% of IgM ELISA tests were performed at an adequate time while 68.0% of rapid antigen tests were not performed at an adequate time. Conclusion: Inadequacy was found between symptom onset and time taken to test for COVID-19 in the Brazilian regions.

Evaluation of the diagnostic accuracy of laboratory-based screening for hepatitis C in dried blood spot samples: A systematic review and meta-analysis.

The dried blood spot (DBS) is increasingly used for the hepatitis C virus (HCV) screening. Our objective was to perform a meta-analysis of the methodology for HCV screening in DBS samples, particularly in the type of diagnostic assay used. We performed a meta-analysis of all eligible studies published to date (March 2018). The literature search revealed 26 studies: 21 for detection of anti-HCV antibodies and 10 for detection of HCV-RNA. Statistical analyses were performed using Meta-DiSc and STATA (MIDAS module). For detection of HCV antibodies, pooled diagnostic accuracy measures were as follows: sensitivity 96.1%, specificity 99.2%, positive likelihood ratio (PLR) 105, negative likelihood ratio (NLR) 0.04, diagnostic odds ratio (DOR) 2692.9, and summary receiver operating characteristic (SROC) 0.997 ± 0.001. For detection of HCV-RNA, the pooled diagnostic accuracy measures were as follows: sensitivity 97.8%, specificity 99.2%, PLR 44.8, NLR 0.04, DOR 1966.9, and SROC 0.996 ± 0.013. Similar values of pooled diagnostic accuracy measures were found according to the type of anti-HCV antibody detection assay (enzyme-linked immunosorbent assay, rapid diagnostic test, and chemiluminescence assays) and HCV-RNA detection assay (real-time polymerase chain reaction and transcription-mediated amplification). The analysis of external validity showed a high negative predicted value (NPV) for both approaches, but a low positive predicted value (PPV) when prevalence was < 10%, particularly in HCV-RNA tests. Finally, this meta-analysis is subject to limitations, especially publication bias and significant heterogeneity between studies. In conclusion, HCV screening in DBS samples has an outstanding diagnostic performance, with no relevant differences between the techniques used. However, external validity may be limited when the HCV prevalence is low.

Seroprevalence of Visna-Maedi Virus (VMV) and Border Disease Virus (BDV) in Van province and around / Seroprevalência de Visna-Maedi Virus (VMV) e Border Disease Virus (BDV) na Província de Van e Proximidades

The present study investigated the seroprevalance of Visna Maedi Virus (VMV) and Border Disease Virus (BDV) infections in sheeps in regions in and around Van province, Turkey. Sample materials were taken from 360 sheep sent to slaughterhouses around Van. All serum samples were examined using ELISA for antibodies for Visna Maedi (VMV) and Border Disease (BDV) viruses. Of these, 38 (10.5%) tested positive for Visna Maedi virus antibodies and 163 (45.2%) for Border Disease virus antibodies. Varying numbers of samples were positive for both virus antibodies across the towns of Ercis, Çaldiran, Erçek and Baskale in Van, Agri and Hakkari provinces. Both infections should be eliminated by informing veterinarians and animal owners, identifying and eliminating persistently infected animals from flocks, and conducting appropriate eradication measures. Economic support should be provided for this.(AU)

O presente estudo investigou a seroprevalência de infecções por Visna Maedi Virus (VMV) e Border Disease Virus (BDV) em ovelhas nas redondezas da província de Van, na Turquia. Amostras foram retiradas de 360 ovelhas enviadas a um matadouro próximo de Van. Todas as amostras foram examinadas usando ELISA para anticorpos de visna Maedi (VMW) e Border Disease (BDV). Destes, 38 (10.5%) foram positivos para anticorpos virais de Visna Maedi e 163 (45.2%) para anticorpos virais de Border Disease. Números variados de amostras foram positivos para ambos os anticorpos nos municípios de Ercis, Çaldiran, Erçek e Baskale, nas províncias Van, Agri e Hakkari. Ambas as infecções devem ser eliminadas informando veterinários e proprietários, identificando e eliminando animais persistentemente infectados de rebanhos, e conduzindo medidas apropriadas de erradicação. Suporte financeiro deve ser providenciado para tal.(AU)

Serum relaxin as a diagnostic and prognostic marker in patients with epithelial ovarian cancer.

BACKGROUND AND AIM: Relaxin is a short circulating peptide hormone. The aim of this study was to understand the role of relaxin in progression of epithelial ovarian cancer (EOC) and to assess its diagnostic and prognostic significance. METHODS: A total of 124 patients with EOC, 46 patients with benign ovarian diseases, and 50 healthy controls were recruited. Serum levels of relaxin were determined by ELISA method. The relationship between serum relaxin level and each of the clinicopathological parameters was analyzed using the χ2 test. Survival curves were plotted using the Kaplan-Meier method. The statistical difference in survival between the different groups was compared using the log-rank test. Survival correlation with the prognostic factors was further investigated by multivariate analysis using the Cox proportional hazards model with backward stepwise likelihood ratio. RESULTS: The results showed that serum relaxin level was significantly higher in patients with EOC than those with benign ovarian diseases and healthy controls (p< 0.01). Serum relaxin level was associated with FIGO stage, lymph node metastasis, tumor resectability, survival of the patients, chemotherapy and tumor recurrence (p< 0.05). Analysis using the Kaplan-meier method indicated that patients with high serum relaxin had significantly shorter overall survival time than those with low relaxin (p< 0.01). In a multivariate analysis along with clinical prognostic parameters, serum relaxin was identified as an independent adverse prognostic variable for survival. CONCLUSIONS: These results indicated that serum relaxin may be a clinically useful indicator for diagnostic and prognostic evaluation in EOC patients.

Aspergillus antibody detection: diagnostic strategy and technical considerations from the Société Française de Mycologie Médicale (French Society for Medical Mycology) expert committee.

Until now, there has been no consensus on the best method for the detection of anti-Aspergillus antibodies, a key diagnostic tool for chronic aspergilloses. To better appreciate the usage of and confidence in these techniques, the Société Française de Mycologie Médicale (French Society for Medical Mycology; SFMM) performed a two-step survey of French experts. First, we administered an initial survey to French labs performing Aspergillus serology to depict usage of the different techniques available for Aspergillus serology. Second, an opinion poll was conducted of 40 experts via an online questionnaire. Each item was rated from 1 to 9 according to the level of agreement. The initial survey revealed that enzyme-linked immunosorbent assay (ELISA) (81%) and immunoelectrophoresis (IEP) (67%) were the most commonly used techniques for screening and confirmation, respectively. The distinction between screening and confirmation techniques was confirmed by the experts (median = 7) with a 44.2% variation coefficient. Only ELISA for screening and IEP and Western blot (WB) for confirmation were clearly considered valuable methods (median ≥8 with variation coefficients less than 30%). The use of a confirmation technique was recommended in the case of a positive result in a compatible clinical context (cystic fibrosis, for example) or during the patient's follow-up. In the case of discordant results between the screening and confirmation techniques, the experts recommended greater confidence in the results obtained with the confirmation technique. All experts emphasized the need to standardize Aspergillus serology techniques and to better define the place of each of them in the diagnosis of aspergillosis.

Preoperative serum levels of insulin-like growth factor-binding protein 2 predict prognosis of gastric cancer patients.

It has been reported that serum insulin-like growth factor-binding protein 2 (IGFBP2) levels are elevated in various types of cancers. However, the clinicopathologic and prognostic implications of circulating IGFBP2 have never been investigated in gastric cancer. We tested IGFBP2 levels in the sera of 118 gastric cancer patients and 34 healthy controls using enzyme-linked immunosorbent assay (ELISA). The mean serum IGFBP2 level was significantly elevated in the gastric cancer patients compared to controls (805.23 ± 590.56 ng/ml vs. 459.61 ± 277.01 ng/ml; P < 0.001). Serum IGFBP2 levels were significantly higher in larger (> 6 cm) tumors (956.8 ± 734.0 ng/ml vs. 548.6 ± 364.0 ng/ml; P = 0.007) and in higher (T3/4) T stages (854.8 ± 621.4 ng/ml vs. 546.5 ± 315.1 ng/ml; P = 0.037). Multivariate Cox analysis showed that higher serum IGFBP2 level (> 400.01 ng/ml) was an independent prognostic factor predicting worse overall survival in patients with gastric cancer (hazard ratio (HR): 3.749, P = 0.034). When we divided patients into four groups based on blood IGFBP2 levels, survival was stratified. The HRs for death in the 3rd and 4th quartiles of serum IGFBP2 levels in comparison to that in the 1st quartile were 2.527 (P = 0.043) and 3.092 (P = 0.012). In conclusion, circulating IGFBP2 has potential as a biomarker predicting prognosis for gastric cancer patients.

Influência do ressecamento da superfície radicular, do tratamento com EDTA e ácido Hialurônico na viabilidade e expressão de citocinas inflamatórias de fibroblastos do ligamento periodontal / Influence of root surface dryness and treatment with EDTA and Hyaluronic acid on the viability and expression of inflammatory cytokines of periodontal ligament fibroblasts

O momento ideal para o reimplante de um dente avulsionado é imediatamente após a avulsão, no entanto, isso nem sempre é possível. Uma série de fatores influenciam na viabilidade das células do ligamento periodontal (PDL) contribuindo para acelerar ou minimizar a ocorrência da reabsorção radicular ou anquilose, consequências mais frequentes dos reimplantes. Um destes fatores é o período extra-oral e o tratamento da superfície radicular. O EDTA (ácido etilenodiamino tetracético) a 17% e o ácido hialurônico (AH) são utilizados para tratar a superfície radicular, visando menor ocorrência de reabsorção inflamatória e por substituição. Sendo assim, os objetivos deste estudo foram: a) avaliar a viabilidade de fibroblastos do ligamento periodontal (PDLF) em contato com discos radiculares submetidos ou não ao ressecamento de superfície por diferentes tempos e tratados com EDTA e/ou AH; b) quantificar as citocinas inflamatórias IL-6, IL-8, IL-1ß e TNF-α expressas por PDLF; c) observar a adesão de PDLF na superfície do discos radiculares tratados. Foram obtidos 108 discos de dentina e cemento da superfície radicular de dentes bovinos, com 4,5 mm de diâmetro, que foram previamente submetidos a dissolução do ligamento periodontal em solução de hipoclorito de sódio 1% por 15 min. Em seguida os discos foram esterilizados em concentrações decrescentes de álcool (100%,90%,80% e 70%). Os espécimes foram submetidos ou não ao ressecamento radicular por 1h ou 24h e tratados com EDTA associado ou não ao ácido hialurônico, colocados em placas de 96 poços onde foram semeadas células de culturas primárias de PDLF, que ficaram em contato com os discos por 48 h. A viabilidade celular na superfície dos discos foi avaliada através do ensaio de XTT. A microscopia eletrônica de varredura foi utilizada para verificar a adesão de PDLF à superfície dos discos. A detecção e quantificação das citocinas foi realizada pelo teste ELISA. Os dados foram submetidos à análise estatística pelo teste ANOVA e Tukey (p < 0,05). A maior média foi apresentada no grupo sem ressecamento para o tratamento EDTA+AH (148,39), que diferiu significativamente dos grupos controle e EDTA. No grupo ressecamento 1 h EDTA+AH (144,91) foi diferente dos demais. Para ressecamento 24h, verificou-se que o grupo EDTA+AH diferiu do grupo controle. Não houve modificações na expressão das citocinas IL-6, IL-8, IL-1ß e TNF-α quando foi acrescentado os tratamentos propostos. A IL-6 mostrou uma diminuição quando em contato com AH no periodo de 24h. Foi observada pelo MEV adesão de PDLF na superfície de todos os discos tratados e em todos os períodos analisados. Conclui-se que o ácido hialurônico é uma alternativa de tratamento para casos de dentes avulsionados já que mostrou seu papel promovendo adesão e aumento da viabilidade(AU)

The ideal time for reimplantation of an avulsed tooth is immediately after avulsion, however, this is not always possible. A number of factors influence the viability of the cells of the periodontal ligament (PDL) contributing to accelerate or minimize the occurrence of root resorption or ankylosis, more frequent consequences of reimplantation. One of these factors is the extra-oral period and root surface treatment. EDTA (17% ethylenediaminetetraacetic acid) and hyaluronic acid (HA) are used to treat the root surface, aiming for a lower occurrence of inflammatory resorption and replacement. Thus, the objectives of this study were: a) to evaluate the viability of periodontal ligament fibroblasts (PDLF) in contact with root disks submitted to surface dryness at different times and treated with EDTA and / or AH; b) quantify the inflammatory cytokines IL-6, IL-8, IL-1ß and TNF-α expressed by PDLF; c) observe the adhesion of PDLF on the surface of the treated root discs. 108 dentin and cementum disks were obtained from the root surface of bovine teeth, 4.5 mm in diameter, which were previously submitted to dissolution of the periodontal ligament in 1% sodium hypochlorite solution for 15 min. Then the disks were sterilized in decreasing concentrations of alcohol (100%, 90%, 80% and 70%). The specimens were submitted to root resection for 1 h or 24 h and treated with EDTA, whether or not associated with hyaluronic acid, placed in 96-well plates where cells from PDLF primary cultures were seeded and left in contact with the discs for 48 h. Cell viability at disc surfaces was assessed by the XTT assay. Scanning electron microscopy was used to verify the adhesion of PDLF to the disc surface. Detection and quantification of cytokines was performed by ELISA. Data were submitted to statistical analysis by the ANOVA and Tukey test (p <0.05). The highest mean was presented in the non-dry group for EDTA + AH treatment (148,39), which differed significantly from the control and EDTA groups. In the dryness group 1 h EDTA + AH (144.91) was different from the others. For dryness 24 h, it was found that the EDTA + AH group differed from the control group. There were no changes in the expression of cytokines IL-6, IL-8, IL-1ß and TNFα when the proposed treatments were added. IL-6 showed a decrease when in contact with HA in the 24-hour period. It was observed by the MEV adhesion of PDLF on the surface of all treated discs and in all periods analyzed. It is concluded that hyaluronic acid is an alternative treatment for cases of avulsed teeth since it showed its role promoting adhesion and increased viability(AU)

Reliability and clinical utility of Enzyme-linked immunosorbent assay for detection of anti-aminoacyl-tRNA synthetase antibody.

Anti-aminoacyl-tRNA synthetase (ARS) antibody is one of the myositis-specific autoantibodies to make a diagnosis of polymyositis (PM) and dermatomyositis (DM). Recently a new enzyme-linked immunosorbent assay (ELISA) kit of concurrently detected anti-ARS antibodies (anti-Jo-1, anti-PL-7, anti-PL-12, anti-EJ and anti-KS) have become to measure in the clinical setting. To evaluate the reliability of this ELISA kit, we measured anti-ARS antibodies in 75 PM and DM patients using by this ELISA assay and compared them with the results by RNA immunoprecipitation assay. Between the measurements of anti-PL-7, anti-PL-12, anti-EJ and anti-KS autoantibodies by ELISA assay and RNA-IP assay, the concordance rate of reproducibility is 95.1% and the positive agreement rate is 90.9% and negative agreement rate is 96.0% and kappa statistic is 0.841. Between the measurements of existing anti-Jo-1 antibody ELISA kit and anti-ARS antibody ELISA kit, the concordance rate of reproducibility is 96.9%, the positive agreement rate is 100%, negative agreement rate is 96.1% and kappa statistic is 0.909. The lung involvement in patients with PM and DM patients are positive of anti-ARS antibodies and anti-melanoma differentiation associated gene5 (MDA5) antibody at a rate around 70%. Then most life-threatening ILD with anti-MDA5 positive clinically amyopathic dermatomyositis patients could be highly guessed when anti-ARS antibodies are negative.

HIV testing patterns for United States Air Force personnel, 2008-2012.

OBJECTIVE: This study evaluated 3rd generation human immunodeficiency virus (HIV) test patterns and HIV infection rates in the United States Air Force (USAF). STUDY DESIGN: Retrospective database study. METHODS: HIV enzyme-linked immunoassay (ELISA) and Western blot tests were analysed for all USAF personnel from 2008 to 2012. For new HIV cases, unadjusted and adjusted annual rates were calculated per 100,000 persons. RESULTS: In total, 1,608,665 tests were performed in 626,298 individuals, with a reactive ELISA observed in 809 (0.001%) persons. Western blot (n = 1949) results included 378 (19.4%) positive, 1283 (65.8%) negative, and 288 (15.0%) indeterminate (WBi). Unadjusted annual HIV rates were between 16.7 and 20.6 per 100,000 persons during the study period. The overall age-adjusted rate was 14.8 cases per 100,000 persons tested. Blacks/African Americans had the highest risk of HIV (risk ratio 7.9 [95% confidence interval 5.78, 9.95] compared to Whites). CONCLUSIONS: WBi results, which can cause delays in determining HIV status, were relatively common with the 3rd generation assay. However, this will be mitigated by a planned transition to a 4th generation assay. Although the overall rate of HIV in the USAF is lower than US civilian adults, HIV prevention efforts targeting young Blacks/African Americans may help to reduce HIV incidence in the USAF.

The clinical significance of vascular endothelial growth factor in malignant ascites.

Ascites can be caused by many kinds of diseases. Patients with undetermined ascites represent a diagnostic challenge. The aims of this study were to determine the diagnostic value of vascular endothelial growth factor (VEGF) in differentiation of malignant ascites from benign ascites and to investigate the clinical value of ascitic VEGF as an independent prognostic parameter. The study included 462 consecutive patients with malignant ascites and 550 patients with benign ascites, VEGF level in ascites were determined by a sandwich enzyme-linked immunosorbent assay. The survival rate was calculated by the Kaplan-Meier method and the log-rank test. Multivariate survival analysis was performed using the Cox hazards model. In our study, we found VEGF levels in malignant ascites (676.59 ± 303.86 pg/ml) were significantly higher than those in benign ascites (218.37 ± 98.15 pg/ml) (P < 0.001). Meanwhile, we also found that VEGF levels in malignant ascites from patients with ovarian cancer were higher than those with other cancers. Areas under the receiver operating characteristic (ROC) curves of ascitic VEGF was 0.940. At a cutoff value of 319.5 pg/ml, VEGF yielded a sensitivity of 89.2 % and a specificity of 88.4 %. Patients associated with the high-level VEGF value (≥613.38 pg/ml) in malignant ascites exhibited poor mean survival rates (8.3 ± 0.52 vs 15.11 ± 0.66 months, P < 0.001). In a multivariate Cox regression model, higher ascitic VEGF was an independent prognostic factor for overall survival. Planned subgroup analysis was performed for patients with tumor node metastasis (TNM) stage I. In the univariate analysis, only ascitic VEGF was associated with overall survival. VEGF was found to have a highly accurate sensitivity and specificity, suggesting that it could be considered as a new biomarker to differentiate malignant ascites from the benign one. The high level of VEGF value in malignant ascites may be used as an independent prognostic factor in patients with all stages of cancer.

Appropriate use of D-dimer testing can minimize over-utilization of venous duplex ultrasound in a contemporary high-volume hospital.

BACKGROUND: The sensitivity of d-dimer (DD) in detecting deep venous thrombosis (DVT) is remarkably high; however, many institutions send patients immediately for a venous duplex ultrasound (VDU). This study was designed to examine the appropriate utilization of DD and VDU in a high-volume hospital. METHODS: A retrospective study was conducted on consecutive patients who presented to a high-volume emergency department (ED) with lower extremity limb swelling/pain over a 30-day period, who were sent for VDU during an evaluation for DVT. VDU data were merged with electronic DD laboratory results. The enzyme-linked immunosorbent assay method was used to provide DD values and thresholds. Values above 0.60 mg/fibrinogen equivalent unit (FEU) were considered abnormal. RESULTS: We reviewed the medical records of 517 ED patients in the month of June 2013. After applying the Wells criteria, 157 patients (30.4%) were excluded because of a history of DVT or pulmonary embolism, having been screened for shortness of breath, or sent for surveillance-leaving 360 for analysis. The average age was 59.3 ± 16.5 years with more women (210, 58.3%) and the majority reported limb pain or swelling (73.9%). DD was performed on 51 patients with an average value of 3.6 ± 5.4 mg/FEU, of which 43 (84.3%) were positive. DD identified all positive and negative DVT patients (100% sensitivity and negative predictive value), but also included 40 false positives (16.7% specificity). On the other hand, 309 patients were sent directly to VDU without DD; of those, 43 (13.9%) were positive for DVT. However, 266 (86.1%) patients were negative for DVT by VDU without DD and these were deemed improper by our current study protocol. Potential charge savings were calculated as VDU for all (360 × $1000 = $360,000), DD for all (360 × $145 = $52,200), and VDU for both true and false positives (estimated to be about 25% of the cases; 90 × $1000 = $90,000); this equals a charge savings of $217,800 and would avoid unnecessary VDUs. CONCLUSIONS: Based on the results of our study, we suggest that the DD test be utilized during the initial work-up for patients with limb swelling/pain in the emergency room. Appropriate utilization of DD, as well as other clinical criteria, may limit the over-utilization and added cost of VDU, without a negative impact on patient care. The results of DD tests should be utilized to limit the number of patients sent for VDU to only those patients with a positive DD or other significant underlying concerns.

Heparin-induced thrombocytopenia testing is over-utilized in cirrhosis & correlates with poor clinical outcomes.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a serious complication seen in hospitalized, medically-ill patients. Evaluation for HIT using a commercially-available ELISA-based test has become increasingly common; however, it does have a high false positive rate. Implications of HIT testing in patients with cirrhosis have not yet been reported. MATERIAL AND METHODS: We conducted a single-institution, retrospective review of all patients with cirrhosis admitted over a 29-month period. The student's t-test and the χ2 test were used for comparisons. We performed a stratified survival analysis using Kaplan-Meier and log rank testing. RESULTS: A total of 1,305 patients had a HIT Ab sent during the study period. Of these patients, 106 had cirrhosis and were included in the study. Eighteen (17%) of the patients with cirrhosis were HIT Ab positive and four of the eighteen had a positive Serotonin Release Assay (SRA) confirmatory test. No difference was found in platelet nadir, thrombotic rate, length of stay, and patient survival between patients with positive HIT Ab and negative HIT Ab testing. No consistent treatment was used among patients who were HIT Ab positive, despite hematology service consultation. Patients who were HIT Ab negative were more likely to have undergone liver transplantation compared to those who were positive (27 vs. 5.5%, respectively; p = 0.048). CONCLUSION: Our data suggest that HIT Ab testing is over-used in patients with cirrhosis and is poorly predictive of outcomes. With a poor positive predictive value, HIT testing may add unnecessary complexity to an already complicated patient population.

Comparison of protein immunoprecipitation-multiple reaction monitoring with ELISA for assay of biomarker candidates in plasma.

Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978-0.995) between 10 and 640 ng/mL for the target proteins spiked into a "mock plasma" matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67-0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.

Development of an ELISA for the quantification of the C-terminal decapeptide prothymosin α(100-109) in sera of mice infected with bacteria.

Apoptosis is characterized by a series of discrete biochemical events, among which is the truncation of the nuclear polypeptide prothymosin alpha (proTα) by activated caspase-3. This early apoptotic event results in the generation of a carboxy-terminal fragment of proTα, the immunoactive decapeptide proTα(100-109). We hypothesized that the detection of increased levels of proTα(100-109) in serum can be directly correlated with the induction of massive cell apoptosis, resulting from a severe bacterial infection. Thus, using high-affinity-purified polyclonal antibodies (Abs), raised in rabbits and a prototype antibody-capture system, we developed a highly sensitive and specific competitive ELISA for proTα(100-109). The sensitivity of the ELISA (0.1ng/mL to 10µg/mL) is acceptable for the quantification of the decapeptide in serum samples. To assess our initial hypothesis, we determined the concentration of proTα(100-109) in the sera of mice infected with the bacterium Streptococcus pyogenes over the course of the infection. We show that serum concentration of proTα(100-109) was marginal to undetectable before infection, increased over time and peaked at 72h postinfection. In silico analysis suggests that the Abs generated are unlikely to cross-react with any other unrelated mouse or bacterial protein. Further validation of our ELISA using serum samples from humans, infected with bacteria, may provide a useful tool to differentiate the causative agent of a potentially lethal septic infection.

ELISA rescue protocol: recovery of sample concentrations from an assay with an unsuccessful standard curve.

The Enzyme-linked Immunosorbant Assay (ELISA) is a method commonly used to measure proteins in various biological matrices, due to its ease of performance and relatively low cost. In order for quantitative data to be generated, a reference standard curve must be prepared for each assay; however, due to investigator error or standard protein degradation, otherwise representative experimental sample data are rendered useless. Herein, we describe a protocol by which sample concentrations can be recovered from assays in which the standard curve fails. The ΔOD values of the experimental samples are used to generate a new standard curve, which is applied back to the original plate. For validation of this method, experimental sample concentrations obtained using acceptable standard curves were potted against those calculated using this new method. Using linear regression analysis, we show a near 1:1 correlation between sample concentrations, with r(2) values between 0.98 and 0.99 and slopes between 0.97 and 1.10. This method demonstrates that assays resulting in unusable standard curves do not require re-assay of all samples. Instead, the experimental sample concentrations can be retrieved saving the investigator the time and resources required to rerun samples or repeat entire experiments.

Comparing and combining data across multiple sources via integration of paired-sample data to correct for measurement error.

In biomedical research such as the development of vaccines for infectious diseases or cancer, study outcomes measured by an assay or device are often collected from multiple sources or laboratories. Measurement error that may vary between laboratories needs to be adjusted for when combining samples across data sources. We incorporate such adjustment in the main study by comparing and combining independent samples from different laboratories via integration of external data, collected on paired samples from the same two laboratories. We propose the following: (i) normalization of individual-level data from two laboratories to the same scale via the expectation of true measurements conditioning on the observed; (ii) comparison of mean assay values between two independent samples in the main study accounting for inter-source measurement error; and (iii) sample size calculations of the paired-sample study so that hypothesis testing error rates are appropriately controlled in the main study comparison. Because the goal is not to estimate the true underlying measurements but to combine data on the same scale, our proposed methods do not require that the true values for the error-prone measurements are known in the external data. Simulation results under a variety of scenarios demonstrate satisfactory finite sample performance of our proposed methods when measurement errors vary. We illustrate our methods using real enzyme-linked immunosorbent spot assay data generated by two HIV vaccine laboratories.