ELISA Ratio Research Articles

Genomic hybrid capture assay to detect Borrelia burgdorferi: an application to diagnose neuroborreliosis in horses.

Antemortem diagnosis of neuroborreliosis in horses has been hindered by both the low sensitivity of PCR testing for Borrelia burgdorferi in CSF and the low specificity of serum:CSF ELISA ratios used to determine intrathecal antibody production against the bacterium. PCR testing of the CSF of an adult horse with acute neurologic disease for the B. burgdorferi flagellin gene was negative. However, we enriched B. burgdorferi DNA through nucleic acid hybrid capture, followed by next-generation sequencing, and identified B. burgdorferi in the CSF of the horse, confirming a diagnosis of neuroborreliosis.

Preservation of anti-SARS-CoV-2 neutralising antibodies in convalescent plasma after pathogen reduction with methylene blue and visible light.

COVID-19 convalescent plasma (CCP) is an experimental treatment against SARS-CoV-2. Although there has so far been no evidence of transmission through transfusion, pathogen reduction technologies (PRT) have been applied to CCP to mitigate risk of infectious disease. This study aims to assess the impact of methylene blue (MB) plus visible light PRT on the virus-neutralising activity of the specific antibodies against SARS-CoV-2. Thirty-five plasma doses collected by plasmapheresis from COVID-19 convalescent donors were subjected to MB plus visible light PRT. Anti-SARS-CoV-2 RBD S1 epitope IgGs antibodies were quantified by ELISA. Titres of SARS-CoV-2 neutralising antibodies (NtAbs) were measured before and after the PRT process. A Spearman's correlation was run to determine the relationship between antibody neutralisation ability and SARS-CoV-2 IgG ELISA ratio. Pre- and post-inactivation neutralising antibody titres were evaluated using a Wilcoxon test. The plasma pathogen reduction procedure did not diminish NtAbS titres and so did not cause a change in the viral neutralisation capacity of CCP. There was a strong correlation between pre-and post-PRT NtAbs and anti-SARS-CoV-2 IgGs titres. Our results showed PRT with MB did not impair the CCP passive immunity preserving its potential therapeutic potency. Therefore, PRT of CCP should be recommended to mitigate the risk for transmission of transfusion-associated infectious disease. There is a good correlation between SARS-CoV-2 IgG titres determined by ELISA and the neutralising capacity. This allows blood centres to select CCP donors based on IgG ELISA titres avoiding the much more labour-intensive laboratory processes for determining neutralising antibodies.

Impact of Anti-Endothelial Cell Antibodies (AECAs) in Patients with Polycythemia Vera and Thrombosis.

Polycythemia vera (PV) causes thrombosis. Erythrocytosis and cell adhesiveness are responsible for thrombosis. JAK2V617F causes inflammation and autoimmunity; however, whether or not autoimmunity or inflammation causes thrombosis has yet to be proven. In 60 PV patients, we analyzed JAK2V671F and its allele burden, autoimmune Th17 cells, interleukin-17 (IL-17), anti-endothelial cell antibodies (AECAs), endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and von Willebrand factor antigen (VWF:Ag). Fifty blood donors were used as the controls. All patients were on phlebotomy-maintaining hematocrit <45% and aspirin. Of the 60 patients, 40 had thrombosis. Those patients with thrombosis had a higher JAK2V617F allele burden than those without thrombosis, andTh17 cells and IL-17 were also higher in patients with thrombosis. Interestingly, we observed a high AECA IgG ELISA ratio (ER) in patients with thrombosis, which was normal in patients without thrombosis. We found high ELAM-1 and ICAM-1 as well as high VWF:Ag in patients with thrombosis compared to patients without thrombosis. AECA-positive sera from patients with thrombosis showed enhanced binding to cytokine-treated HUVEC and a positive antibody-dependent cellular cytotoxicity, suggesting that AECA may contribute to vascular injury. A positive correlation between AECAs, allele burden, and thrombosis was found. These results suggest that autoimmunity may be an additional mechanism in PV thrombogenesis.

Early functional and metabolic disorders in children with type I diabetes mellitus and diabetic nephropathy.

Type 1 diabetes (T1D) is mainly adisease of children and young adults. The onset of disease is associated with 3 classical symptoms: polydipsia, polyphagia, and polyuria. Hyperglycaemia is the main and primary metabolic disorder in T1D. One of the complications developing as aresult of microangiopathy is diabetic nephropathy (DN). Additionally, diabetes remains the most common reason for progressing to end-stage renal disease (ESRD). The aim of the study was to evaluate the most initial metabolic and functional disorders in diabetic children and children with DN. The study involved 76 children with T1D and diabetic nephropathy (aged 3 to 17 years). The levels of ET-1 measured using ELISA assay and ratio of lipid oxidation measured spectrophotometrically. Main clinical parameters (blood pressure, glycaemia, albuminuria, creatininaemia, HbA1C, cholesterol levels) were measured using conventional methods available in Clinical Paediatric Hospital No. 6 (Kyiv, Ukraine). The glomerular filtration rate (GFR) was calculated using the Schwartz formula. Patterns of the BP changes, kidney function impairment, ET-level, and metabolic and functional disorders in children with T1D and DN were found.

Alcohol Use Is Associated With Intestinal Dysbiosis and Dysfunctional CD8+ T-Cell Phenotypes in Persons With Human Immunodeficiency Virus.

Inflammation persists among persons with human immunodeficiency virus (PWH) despite effective antiretroviral therapy and may contribute to T-cell dysfunction. Alcohol use is prevalent among PWH and promotes intestinal leak, dysbiosis, and a proinflammatory milieu. Whether alcohol use is associated with T-cell late differentiation remains to be investigated. Data and samples from PWH (N = 359 of 365) enrolled in the New Orleans Alcohol Use in HIV Study were used. Alcohol use was assessed by self-report (Alcohol Use Disorders Identification Test; lifetime alcohol exposure; 30-day Alcohol Timeline Followback) and phosphatidylethanol (PEth) quantitation. In a subset of participants, fecal bacterial content was assessed by ribosomal 16S marker gene deep sequencing and quantitative polymerase chain reaction. Intestinal leak was assessed by fecal-to-plasma α-1-antitrypsin (A1AT) enzyme-linked immunosorbent assay ratio. Peripheral T-cell populations were quantified by flow cytometry. Alcohol Use Disorder Identification Test scores were positively associated with activated-senescent, exhausted, and terminal effector memory CD45RA+CD8+ but not CD4+ T cells (cells/μL) after confounder adjustment (P < .050). Phosphatidylethanol was positively associated with A1AT (P < .050). The PEth and activated-senescent CD8+ were associated with bacterial β-diversity (P < .050) and positively associated with the relative abundance of coabundant Prevotellaceae members (q < .100). Alcohol use among PWH is associated with CD8+ T-cell late differentiation, intestinal leak, and dysbiosis. Alcohol-associated dysbiosis is implicated in CD8+ T-cell senescence.

Interest of Confirmation Tests in the Diagnosis of Viral Hepatitis C to Blood Donors in Abidjan-Côte d'Ivoire

Introduction The anti-HCV RIBA test verifies the presence of anti-HCV serum antibodies detected by the Elisa test. In Côte d'Ivoire, screening for hepatitis C is done exclusively by enzyme immunoassays. In order to reduce the number of HCV positive blood donor exclusions on ELISA, we conducted this study which aimed to demonstrate the value of the RIBA test in confirming diagnosis of viral hepatitis C to blood donors. Methods Our study, which took place from 02 to 23 February 2008 in the laboratory of Abidjan NBTC, focused on 200 sera of blood donors anti-HCV positive (Elisa test) selected according to the ratio. The DECISCAN HCV PLUS confirmation test of BIORAD was used. Results Among the 200 HCV samples positive by EIA, 49% (98/200) were confirmed positive. RIBA gave an indeterminate result in 40% of cases (80/200); and negative in 11% of cases (22/200) corresponding to false ELISA devices. In RIBA 96 samples had a low ELISA ratio of which 21% (20/96) were RIBA negative, and 79% (76/96) were indeterminate. RIBA positive samples (98/200) had a high ratio in 82% of cases (80/98). The presence of NS3 (C33) and NS4 (C100) was noted in 100% of cases (98/98, C2 in 37% (36/98) of cases and C1 in 18% of cases (18/98). RIBA indeterminate noted the presence of NS3 in 98% of cases (78/80) and NS4 in 30% of cases (24/80). Proteins C1, C2 and NS4 are essential for the diagnosis of confirmation of viral hepatitis C by RIBA. Conclusion These results attest to the lower specificity of enzyme immunoassays (ELISAs); hence the benefit of using RIBA confirmatory tests. A significant number of donors are excluded from blood donation in Côte d'Ivoire on the basis of false positive results obtained by the ELISA technique.

Neurocysticercosis among People Living Near Pigs Heavily Infected with Cysticercosis in Rural Endemic Peru.

Neurocysticercosis causes substantial neurologic morbidity in endemic regions around the world. In this cross-sectional study, we describe the frequency of neurocysticercosis among a presumed high-risk group of people in an endemic community in northern Peru. Participants who screened positive on a nine-question seizure survey were evaluated clinically to diagnose epilepsy using International League Against Epilepsy criteria. Those with epilepsy were offered a noncontrast computerized tomography (CT) of the head. We also tested sera from all participants using the lentil lectin-bound glycoprotein enzyme-linked immunoelectrotransfer blot (EITB) to detect anti-cysticercus antibodies and enzyme-linked immunosorbent assay (ELISA) B60/B158 to detect cysticercosis antigens. Participants with strongly positive ELISA (ratio ≥ 3) were offered a noncontrast magnetic resonance imaging (MRI) of the brain. We diagnosed 16 cases of epilepsy among 527 people screened (lifetime prevalence 30 per 1,000). Twelve with epilepsy accepted CT scan and five (41.7%) had parenchymal calcifications. None had viable cysts. Of the 514 who provided a blood sample, 241 (46.9%) were seropositive by EITB and 12 (2.9%) were strongly positive by ELISA (ratio ≥ 3). Eleven accepted MRI and eight (72.3%) had neurocysticercosis, including five with extraparenchymal cysts, five with parenchymal vesicular cysts, and two with parenchymal granulomas. These findings show that clinically relevant forms of neurocysticercosis and epilepsy can be found by applying screening interventions in communities endemic to Taenia solium. Longitudinal controlled studies are needed to better understand which subgroups are at highest risk and which are most likely to have improved prognosis as a result of screening.

Transplacental transmission of Theileria orientalis occurs at a low rate in field-affected cattle: infection in utero does not appear to be a major cause of abortion

BackgroundBovine theileriosis, caused by the haemoprotozoan Theileria orientalis, is an emerging disease in East Asia and Australasia. Previous studies have demonstrated transplacental transmission of various Theileria spp. but molecular confirmation of transplacental transmission of T. orientalis has never been confirmed in the field. In this study, cow-calf (< 48 h old) pairs were sampled across 3 herds; opportunistic samples from aborted foetuses or stillborn calves were also examined. Molecular (multiplex qPCR) and serological (ELISA) methods were used to determine infection prevalence and the presence of anti-Theileria antibodies in each herd. In addition, pregnant heifers and foetal calves were sampled at abattoir and tested for the presence of T. orientalis by qPCR.ResultsThe qPCR results indicated that, even though there was a high prevalence of T. orientalis infection in cows, the rate of transplacental transmission to their calves was low, with only one newborn calf from one herd and one foetus from the abattoir testing positive for T. orientalis DNA. Five aborted foetuses and stillborn calves, 3 of which were derived from a herd experiencing a high number of clinical theileriosis cases at the time of sampling, all tested negative for T. orientalis by qPCR. This suggests that in utero infection of calves with T. orientalis may not be a major driver of abortions during theileriosis outbreaks. Temporal monitoring of 20 calves born to T. orientalis-positive mothers indicated that T. orientalis was detectable in most calves between 10 and 27 days post-partum, consistent with prior field studies on adult cattle introduced to Theileria-affected herds. There was a positive correlation between the ELISA ratio of newborn calves and their mothers within 48 h of calving; however, maternal antibodies were only detectable in some calves and only for 4–4.5 weeks post-partum. All calves displayed high parasite loads peaking at 4–8 weeks post-partum, with only some calves subsequently mounting a detectable adaptive antibody response.ConclusionsThese findings indicate transplacental transmission of T. orientalis appears to play only a minor role in persistence of T. orientalis infection in the field; however calves are highly susceptible to developing high level T. orientalis infections at 4–8 weeks of age regardless of whether maternal antibodies are present post-partum.

Factors associated with seroconversion to the major piroplasm surface protein of the bovine haemoparasite Theileria orientalis.

BackgroundBovine theileriosis caused by Theileria orientalis is an emerging disease of cattle in the Asia-Pacific region where it causes a significant economic burden to meat and milk production. While host immunological responses to the lymphocyte-transforming species of Theileria, T. parva and T. annulata, have been well studied, little is known about the immune response to this non-transforming species.MethodsWe developed a recombinant antigen ELISA based on the major piroplasm surface protein (MPSP) of T. orientalis and investigated whether seroconversion to the MPSP was associated with clinical factors (anaemia), parasite burden and parasite genotype. We also examined the dynamics of seroconversion in animals acutely infected with T. orientalis.ResultsIn cattle testing qPCR positive for T. orientalis, seroconversion was more frequent in anaemic compared to normal cattle (P < 0.0001). The ELISA ratio (ER) was highly correlated with total parasite burden as measured by qPCR (r = 0.69; P < 0.0001); however when loads of individual genotypes of the parasite were examined, only the pathogenic Ikeda genotype was highly correlated with ER. Conversely, seroconversion was less frequently detected in the presence of benign T. orientalis genotypes. Temporal measurement of the serological response, parasite burden and packed cell volume (PCV) in acutely infected animals revealed that seroconversion to the MPSP occurs within 2-3 weeks of the initial qPCR detection of the parasite and coincides with a peak in infection intensity and a declining PCV.ConclusionWhether the serological response to the MPSP is immunoprotective against re-infection or recrudescence requires further investigation; however the MPSP represents a promising target for a subunit vaccine given that genetic variability within the MPSP results in differential pathogenicity of T. orientalis.

A novel breast cancer screening platform: An epitope-based biomarker coupled to electrochemical sensor.

9 Background: The subtractive proteomic selection technology called Phage Display (PD) has been extensively used by our group in the discovery of high affinity ligands to target tissues and molecules. We have selected specific ligands against IgG purified from BC tissues, which was successfully coupled to an electrochemical sensor to detect the tumor-specific immune response. Methods: After PD selections, all immunoreactive peptide ligands were further characterized by DNA sequencing, in vitro translated and submitted to bioinformatic analyses. Further validations were performed by ELISA. We then used one synthetic peptide (SF4) for the construction of an immunosensor, which was applied to patients and control samples for final validation. Electrochemical impedance spectroscopy (EIS) was performed. Results: We have selected the F4 peptide for final validation and sensor construction due to its capability of detecting IgG in the peripheral blood and the excellent ELISA ratio BC:BBD, discriminating more than 70% of BC patients. The synthetic peptide reached a good precision in BC diagnosis (68%), but surprisingly, the selected F4 clone presented the highest sensitivity and specificity, (77.8% and 85.7%, respectively), suggesting that it can be used as a diagnostic reagent for early BC screening prior to imaging and pathological analyses. The electrochemical sensor that was built with the epitope-based peptide discriminated all IgG from BC and healthy individuals. Results with the EIS sensor demonstrated that the presence of (SF4) peptide IgG generated higher resistivity (-Z ') compared to the system containing only the peptide or the control sera. This is justified by the fact that with the immobilized biological peptide layer was correctly conjugated with the IgG forming an antigen: antibody complex that led to an increased resistance to the charge transfer system, producing a decrease in electron transfer between the iron-coupled / ferricyanide redox and the electrode surface. Conclusions: An electrochemical sensor using an epitope-based biomarker was developed, which allows for the first time BC screening using a very simple platform that could be an important auxiliary tool to mammography imaging.

Anti-endothelial cell antibodies in connective tissue diseases associated with pulmonary arterial hypertension.

To investigate the prevalence of anti-endothelial cell antibodies (AECA) in connective tissue diseases (CTD) associated with pulmonary arterial hypertension (PAH) and to corroborate the pathologic function of AECA in PAH-associated CTDs. AECA were detected by cellular enzyme-linked immunosorbent assay (ELISA) in sera of 19 PAH-associated CTD patients, 22 CTD patients without PAH involvement, and 20 age- and sex-matched healthy individuals as controls. Using IgG purified from the sera of AECA-positive, AECA-negative, and healthy subjects, the effects of AECA on the expression of ICAM-1 and the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) in cultured endothelial cells were also evaluated. A total of 12 of the 19 (63.2%) CTD patients with PAH, 9 of the 22 (40.9%) CTD patients without PAH, and 1 of the 20 (5%) healthy controls were positive for AECA, which were calculated as ELISA ratio (ER) values. ER values in PAH-associated CTD patients were significantly higher than those with CTD without PAH (3.68±2.05 versus 1.67±1.07, P<0.001). IgG purified from AECA-positive sera induced a significantly increased level of ICAM-1 expression after 48 h incubation (795.2±32.5 pg/mL) compared with AECA-negative or healthy control IgG (231.5±27.1 and 192.8±33.4 pg/mL, respectively; P<0.001). In addition, RANTES production by cultured human pulmonary arterial endothelial cells (HPAECs) increased in both a time- and concentration-dependent manner in response to incubation with purified AECA-positive IgG. AECA could be involved in CTD and might participate in the pathogenesis of PAH-associated CTD.

Cross-Reactivity of Chicken Anti-Japanese Encephalitis Virus Serum and Anti-West Nile Virus Serum in Serological Diagnosis

The cross-reactivity of Japanese encephalitis virus (JEV)-immunized chicken sera and West Nile virus (WNV)-immunized chicken sera in serological tests, such as the IgG indirect ELISA (IgG-ELISA), hemagglutination inhibition test (HI) and plaque reduction neutralization test (PRNT), for JEV and WNV were examined. The mean JEV/WNV ELISA ratio in IgG-ELISA of JEV-immunized sera was significantly higher than that of WNV-immunized sera (P<0.01). JEV-immunized chicken sera did not cross-react in the WNV HI. However, all the WNV-immunized chicken sera cross-reacted in the JEV HI. JEV-immunized chicken sera did not show the WNV neutralization titer at 90% plaque reduction, and WNV-immunized chicken sera did not showed the JEV neutralization titer at 90% plaque reduction. Therefore, it is possible that chicken JEV serum can be distinguished from WNV serum by comparing the titers of IgG-ELISA, HI or PRNT respectively.

Analysis of Meat Juice ELISA Results and Questionnaire Data to Investigate Farm‐Level Risk Factors for Salmonella Infection in UK Pigs

The study set out to explore risk factors for Salmonella infection in pigs, based on seroprevalence amongst slaughtered pigs, using a large study population of holdings and a comprehensive list of farm characteristics. Farm data were collected from pig quality assurance schemes and supplemented by a postal questionnaire. These data were used with meat juice serology results from ongoing abattoir Salmonella surveillance, for a multivariable risk factor analysis, modelling the ELISA sample to positive ratio directly (ELISA ratio). The study population contained 566 farms, covering a geographically representative spread of farms within the United Kingdom, with a mean average of 224 sample results per holding over a 4-year period. The model highlighted that temporal factors (quarterly and yearly cycles) and monthly meteorological summaries for rainfall, sunshine and temperature were associated with Salmonella presence (P < 0.01). The ELISA ratio was found to be highest in autumn and lowest in spring and summer, whereas yearly averages showed a greater degree of variation than seasonal. Two feed variables (homemix and barley) were found to be protective factors, as was a conventional, rather than organic or freedom foods, farm enterprise type. The number of annual pig deliveries and dead stock collections, and the main cause of pig mortality on the farm were found to be associated with Salmonella infection. Scottish farms had a lower ELISA ratio than other regions, and an increased number of pig farms within a 10-km radius was associated with a higher ELISA ratio. The study demonstrated that the analysis of routinely collected data from surveillance and quality assurance schemes was cost-effective, with sufficient power to detect modest associations between Salmonella and exposure variables. The model results can be used to inform on-farm Salmonella control policies and could target-specific geographical regions and seasons to assist the efficiency of surveillance.

Anti-Endothelial Cell Antibodies in Patients With Sarcoidosis

Anti-endothelial cell antibodies (AECA) are circulating antibodies that bind to endothelial antigens and induce endothelial cell damage. These antibodies have been detected in patients with collagen vascular disease and systemic vasculitis. Sarcoidosis is a multiple granulomatous disorder of unknown etiology, and its clinical presentation, organ involvement, and prognosis are highly diverse. In this study, we aimed to investigate the expression of AECA in patients with sarcoidosis. We also examined whether these antibodies could serve as a marker for the activity, severity, and prognosis of sarcoidosis. Forty sarcoidosis patients, whose diagnosis was established by clinicoradiologic findings and histologic confirmation of noncaseating epithelioid cell granulomas, were studied. Serum and BAL samples were examined for AECA by cellular enzyme-linked immunosorbent assay (ELISA) using human umbilical vein endothelial cells. The findings were expressed in terms of an ELISA ratio (ER). Fifty-seven subjects without any clinical, radiologic, or serologic evidence of pulmonary disease or autoimmune disorders served as the reference population to judge the positivity of AECA. Patients with sarcoidosis had a significantly higher positivity rate for and levels of AECA in both serum and BAL fluid than the reference population. In addition, the ER of AECA was significantly elevated in patients with multiple lesions or who required corticosteroid therapy compared with that in patients without multiple lesions or who did not need corticosteroid therapy. Expression of AECA might be a useful marker to predict the course of sarcoidosis.

Biological responses to porcine respiratory and reproductive syndrome virus in pigs of two genetic populations1,2

One hundred pigs from the NE Index Line (NEI) and 100 Hampshire-Duroc cross pigs (HD) were inoculated intranasally with porcine respiratory and reproductive syndrome virus (PRRSV 97-7895 strain) at 26 d of age to determine whether genetic variation in response to PRRSV exists. An uninfected littermate to each infected pig served as a control. Pigs were from 163 dams and 83 sires. Body weight and rectal temperature were recorded, and blood samples were drawn from each pig on d 0 before inoculation and on d 4, 7, and 14 after inoculation. Pigs were sacrificed on d 14. Lung and bronchial lymph nodes were collected, placed in optimal cutting temperature compound, and frozen at -80 degrees C. The presence of PRRSV in serum and in lung tissue and bronchial lymph nodes was determined by isolation in cell culture. The presence of antibodies in serum collected on d 14 was determined by a commercial ELISA test. Lung tissue was examined microscopically and scored for incidence and severity of lesions (score of 1 to 3; 1 = no or few lesions, and 3 = severe interstitial pneumonia). Data were analyzed with a mixed model that included random sire and dam effects. The interaction of line x treatment was significant (P < 0.001) for weight change and rectal temperature. Un-infected HD pigs gained 0.67 kg more from d 0 to 14 and averaged 0.32 degrees C higher rectal temperature than uninfected NEI pigs (P < 0.001), whereas infected NEI pigs gained 0.34 kg more and had -0.54 degrees C lower temperature than infected HD pigs (P < 0.001). Viremic titer (cell culture infectious dose 50%/mL) was greater (P < 0.05) in HD than NEI at d 4 (10(4.52) vs. 10(4.22)), 7 (10(4.47) vs. 10(3.99)), and 14 (10(3.49) vs. 10(3.23)). Viral titer loads in lung (P = 0.11) and bronchial lymph nodes tended (P = 0.07) to be greater in HD than NEI pigs. Antibody signal-to-positive (S/P) ELISA ratios in infected pigs ranged from 0.18 to 3.38, and 88% had levels > or = 0.40, which is the positive threshold for this ELISA. The S/P range in uninfected pigs was 0 to 1.11, and 99% had levels < or = 0.40. Mean S/P ratio for infected pigs was 0.23 units higher in HD than in NEI (P < 0.001). The HD pigs had a greater incidence of interstitial pneumonia and 0.65 higher mean lesion scores than NEI pigs (P < 0.001). In summary, responses of pigs of the two lines to infection with PRRSV differed, indicating that underlying genetic variation existed.

Evaluation of a 40 day Assay for Testing Endocrine Disrupters: Effects of an Anti-Estrogen and an Aromatase Inhibitor on Sex Ratio and Vitellogenin Concentrations in Juvenile Zebrafish (Danio rerio)

In the present study, the effects of the anti-estrogen ZM 189,156 and the aromatase inhibitor fadrozole were evaluated in a 40-day juvenile assay developed for screening of endocrine disrupting chemicals. Juvenile zebrafish were exposed from 20 to 60 days post hatch (dph) to ZM 189, 154 (100 μg l−1 and 200 μg l−1) and fadrozole (10, 32, and 100 μg l−1). VTG concentrations were measured at 38 dph by the use of a direct non-competitive sandwich ELISA and sex ratios were determined at 60 dph by histological examination of the gonads. A small but significant increase in VTG concentrations was observed in fish exposed to ZM 189, 156 (100 and 200 μg l−1) and fadrozole (10 and 100 μg l−1) compared to control groups. In fish exposed to ZM 189, 156 and fadrozole, the percentage of females declined and the number of undifferentiated fish increased. These findings show that exposure of juvenile zebrafish to an aromatase inhibitor or an anti-estrogen during early development inhibits differentiation and development of female gonads. The data presented, furthermore, show that the 40-day juvenile assay may be suitable for screening endocrine disrupting chemicals acting as anti-estrogens and aromatase inhibitors.

Oral antigen delivery of varying doses of soluble or particulate antigens in the presence of adjuvant selectively induces Th1 or Th2 responses in systemic and mucosal compartments

Rationale Oral antigen delivery with a nonreplicating immunogen has been thought to primarily induce a Th2 response, although high doses of intranasal or systemic antigen delivery with adjuvant create a Th2 response and low doses create a Th1 response. We hypothesized that a similar effect applies to oral antigen delivery. Methods C3H or BALB/c mice were orally immunized weekly for 3 weeks with 2.5 (n=6) or 250μg/dose (n=6) HIV-1 reverse transcriptase (RT) and 1μg cholera toxin (CT) adjuvant or 2, 20, or 200μg recombinant Norwalk virus-like particles (rNV VLP) and 1μg labile toxin LT(R192G) adjuvant, respectively. Mice were sacrificed after two weeks, splenic and mesenteric lymph node mononuclear cells were isolated and serum collected. Results Serum ELISA ratios of IgG1:IgG2a (Th2:Th1) were 1:27 and 1:2 for the 2.5 and 250μg/dose RT groups, respectively (p<0.005), and for the VLP groups 0:48 and 1:1 for the 20 (p=0.02) and 200μg/doses (p<0.005), respectively. RT-stimulated culture supernatants from the 2.5μg/dose group showed IFN-γ, a Th1 cytokine, in splenic and mesenteric lymph node cells but not IL-4, a Th2 cytokine; both were present at 250μg/dose. RT-specific lymphoproliferation was observed in both spleen (p=0.007) and mesenteric lymph nodes (p=0.02) at 250μg/dose and the mesenteric lymph nodes at 2.5 μg/dose (p=0.015). VLP-specific lymphoproliferative responses were found in VLP-immunized mice spleens at 2, 20, and 200μg/dose (p<0.005, p=0.01, p<0.005, respectively). Conclusions Oral immunization with high or low antigen doses in the presence of adjuvant allows for the selective immunomodulation of Th1 or Th2 responses, respectively, in the systemic and mucosal compartments.

Effects of exposure to 17 α-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish ( Danio rerio)

To determine the critical stage of zebrafish development where exposure to xenoestrogens can affect sex ratio and vitellogenin induction, zebrafish ( Danio rerio) were exposed to 17 α-ethinylestradiol (actual concentration 15.4±1.4 ng EE2/l) during early development: from fertilisation to hatch; hatch to 10 days post hatch (dph); 10–20 dph; 20–30 dph; 20–40 dph; 20–60 dph; fertilisation to 25 dph; or hatch to 60 dph. Vitellogenin was measured in whole body homogenate 30 dph by ELISA and sex ratio was determined 60 dph by histological examination of the gonads. All exposure periods significantly induced vitellogenin synthesis and affected the sex differentiation leading to development of ovo-testis or complete feminisation of the exposed fish depending on exposure period. Complete sex reversal was obtained in groups exposed from 20 to 60 dph and hatch to 60 dph. The half-life for degradation of vitellogenin was calculated. Juvenile zebrafish were exposed to 15.4±1.4 ng EE2/l (actual concentration) from fertilisation to 25 dph and transferred to clean water, after which weekly measurements of vitellogenin concentration in whole body homogenate were performed until day 46 post hatch. The half-life of vitellogenin was 2.4 days.

Humoral immune response to mycobacterial heat shock protein (hsp)65 in the cerebrospinal fluid of neuro-Behçet patients.

Although systemic immune reactivity to 65-kD mycobacterial hsp65 (m-hsp65) has been shown previously in Behçet's disease (BD), local immune response was not investigated. We studied anti-m-hsp65 IgG, IgM and IgA antibodies in the serum and cerebrospinal fluid (CSF) of 25 BD patients with cerebral parenchymal involvement (p-NBD), seven BD patients with intracranial hypertension (ih-NBD), eight BD patients without central nervous system (CNS) involvement, 30 patients with multiple sclerosis (MS) and 24 patients with non-inflammatory CNS disorders (NIC). Significantly higher CSF IgG responses were detected in p-NBD patients (ELISA ratio 1.3 +/- 0.9) compared with NIC (0.7 +/- 0.4, P < 0.01). In p-NBD patients' IgG, IgM or IgA CSF anti-m-hsp65 positivity rate was 48% (12/25); this was significantly higher when compared with MS (3/30; P < 0.03) and NIC (3/24; P < 0.01). CSF anti-m-hsp65 IgG ratios correlated with the duration of BD (r = 0.4, P < 0.04) but not with the duration of neurological involvement. Serum IgM and IgA responses were elevated in ih-NBD, suggesting a different type of involvement than p-NBD. These results implicate an increased local humoral response to m-hsp65 in the CSF of p-NBD patients, which might be related to the pathogenesis of neurological involvement.

Factors associated with positive predictability of the anti-HCV ELISA method with confirmatory RT-PCR.

The positive predictability of anti-HCV ELISA is low, especially, in blood donors and in healthy populations. False positive anti-HCV results pose some difficulties in medical practice and in blood screening. The aim of this study was to identify the factors associated with true hepatitis C virus (HCV) infection among anti-HCV ELISA-positives. A case-control analysis was conducted using 354 subjects who were positive for anti-HCV ELISA. All subjects were tested for true HCV infection using the reverse transcriptase polymerase chain reaction (RT-PCR). Tests for serum alanine aminotransferase (ALT), fasting glucose, HBsAg, anti-HBc antibody, alpha-fetoprotein, platelet count and ultrasound of liver were also performed. Epidemiological data were obtained by self-administered questionnaires. Out of 354 subjects, 202 (57.1%) were positive for HCV by RT-PCR and 152 were negative and used as the control group. In multivariate analysis, blood transfusion (odds ratio, OR 2.3, 95% confidence interval, CI 1.3-4.0), elevated ALT (OR 2.2, 95% CI 1.2-4.3) and higher anti-HCV ELISA ratios (more than 3; OR 1.7, 95% CI 1.3-2.1) were associated with true HCV infection. Thrombocytopenia was also associated with the presence of HCV in univariate analysis. These results suggest that a history of blood transfusion, elevated ALT and a high score on anti-HCV ELISA ratios are associated with true HCV infection among anti-HCV ELISA-positives.