The severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) omicron sub-variants in Bangladesh cause mild COVID-19 and associate with similar antibody responses irrespective of natural infection or vaccination history.

Objective: Genomic surveillance and seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) in Bangladesh is paramount for COVID-19 pandemic preparedness yet lagging the high-income countries due to limited resources. Methods: SARS-CoV-2 variants, COVID-19 symptoms, and serology were prospectively evaluated in a cross-sectional study of Bangladeshi adults testing RT-PCR positive in 2021 and 2022. Results: SARS CoV-2 Omicron variants of asymptomatic or mild COVID-19 in 2022 replaced Delta variant infections requiring hospitalization and oxygen support. The omicron XBB became predominant in July 2022 and associated with cough, headache or body ache and loss of smell; 47 of 68 (69 %), 30 of 68 (44 %) and 27 of 68 (40 %) respectively at higher frequency than BA.1/BA.2; 16 of 88 (18 %), 13 of 88 (15 %) and 0 of 88 (0 %) p < 0.01, p < 0.01 and p < 0.0001. Linear regression analysis reveals no associations between the number of previous infections and the number of symptoms, r = -0.084, p = 0.68. The anti-nucleoprotein (N)-protein IgG post COVID-19 and anti-Spike (S) protein IgG post-COVID-19 vaccination were similar between BA.2, BA.4/BA.5 and XBB and significantly lower than the levels in delta variant infections (p < 0.001). Conclusions: Omicron XBB subvariants emerged in Bangladesh two months prior to previous reports and include unique patterns of S-protein mutations not assigned in PANGO lineage. The SARS CoV-2 omicron break-through infections persist in the presence of sustained antibody responses and vaccinations, underscoring the importance of molecular surveillance in low-income countries.

Clinical Presentation of COVID-19 and Antibody Responses in Bangladeshi Patients Infected with the Delta or Omicron Variants of SARS-CoV-2.

The clinical presentation of COVID-19 and the specific antibody responses associated with SARS-CoV-2 variants have not been investigated during the emergence of Omicron variants in Bangladesh. The Delta and Omicron variants were identified by post-PCR melting curve analysis of the spike (S) protein receptor binding domain amplicons. Anti-S-protein immunoglobulin-G anti-nucleocapsid (N)-protein immunoglobulin-G and immunoglobulin-A levels were measured by ELISA. The Delta variant was found in 40 out of 40 (100%) SARS-CoV-2 RT-PCR positive COVID-19 patients between 13 September and 23 October 2021 and Omicron variants in 90 out of 90 (100%) RT-PCR positive COVID-19 patients between 9 January and 10 February 2022. The Delta variant associated with hospitalization (74%, 80%, and 40%) and oxygen support (60%, 57%, and 40%) in the no vaccine, dose-1, and dose-2 vaccinated cases, respectively, whereas the Omicron COVID-19 required neither hospitalization nor oxygen support (0%, p < 0.0001). Fever, cough, and breathlessness were found at a significantly higher frequency among the Delta than Omicron variants (p < 0.001). The viral RNA levels of the Delta variant were higher than that of the Omicron variants (Ct median 19.9 versus 23.85; p < 0.02). Anti-spike protein immunoglobulin-G and anti-N-protein immunoglobulin-G within 1 week post onset of Delta variant COVID-19 symptoms indicate prior SARS-CoV-2 infection. The Delta variant and Omicron BA.1 and BA.2 breakthrough infections in the Dhaka region, at 240 days post onset of COVID-19 symptoms, negatively correlated with the time interval between the second vaccine dose and serum sampling. The findings of lower anti-spike protein immunoglobulin-G reactivity after booster vaccination than after the second vaccine dose suggest that the booster vaccine is not necessarily beneficial in young Bangladeshi adults having a history of repeated SARS-CoV-2 infections.

Molecular and Serological Characterization of the SARS-CoV-2 Delta Variant in Bangladesh in 2021.

Novel SARS-CoV-2 variants are emerging at an alarming rate. The delta variant and other variants of concern (VoC) carry spike (S)-protein mutations, which have the potential to evade protective immunity, to trigger break-through infections after COVID-19 vaccination, and to propagate future waves of COVID-19 pandemic. To identify SARS CoV-2 variants in Bangladesh, patients who are RT-PCR-positive for COVID-19 infections in Dhaka were screened by a RT-PCR melting curve analysis for spike protein mutations. To assess the anti-SARS CoV-2 antibody responses, the levels of the anti-S -proteins IgA and IgG and the anti-N-protein IgG were measured by ELISA. Of a total of 36 RT-PCR positive samples (75%), 27 were identified as delta variants, with one carrying an additional Q677H mutation and two with single nucleotide substitutions at position 23029 (compared to Wuhan-Hu-1 reference NC 045512) in the genome sequence. Three (8.3%) were identified as beta variants, two (5.5%) were identified as alpha variants, three (8.3%) were identified as having a B.1.1.318 lineage, and one sample was identified as an eta variant (B.1.525) carrying an additional V687L mutation. The trend of higher viral load (lower Cp values) among delta variants than in the alpha and beta variants was of borderline statistical significance (p = 0.045). Prospective studies with larger Bangladeshi cohorts are warranted to confirm the emergence of S-protein mutations and their association with antibody response in natural infection and potential breakthrough in vaccinated subjects.

Predicting the Course of Graves' Orbitopathy Using Serially Measured TSH-Receptor Autoantibodies by Automated Binding Immunoassays and the Functional Bioassay.

The aim of the study was to investigate the use of serial measurements of TSH-receptor autoantibodies (TRAb) with the newest available assay technology to predict the course of Graves' Orbitopathy (GO) during the first 24 months from disease onset. Serial serum samples from patients with GO (103 mild/135 severe) were collected between 2007 and 2017 and retrospectively analyzed. The course of GO were classified into mild/severe 12 months after manifestation (severe: NOSPECS≥5; mild<5). TRAb were measured with automated binding immunoassays (IU/l): TRAb Elecsys (Cobas, Roche), TRAb bridge assay (IMMULITE, Siemens), and a cell-based bioassay (percent of specimen to reference ratio - SRR%) (Thyretain, Quidel). Variable cut off levels of measured TRAb were calculated at specificity of 90% from receiver operator curve (ROC) analysis for several timepoints during the course of GO. To select one: 5-8 months after first GO symptoms, which is the timepoint for usual referals for treatment mild course could be predicted at cut offs of 1.5 IU/l (Elecsys), 0.8 IU/l (Immulite) and 402% SRR (Thyretain) and the risc of severe course has to be anticipated if TRAb are above 11.6 IU/l (Elecsys), 6.5 IU/l (Thyretain), and 714% SRR (Thyretain). The Thyretain bioassay showed the highest diagnostic sensitivity (using the commercial cut off's) over the entire follow up period. TRAb measurements during the 24-month follow up of GO provide added value to the GO clinical activity and severity scores and should be used especially in the event of an unclear decision-taking situation with regard to therapy.

Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection.

BACKGROUND: The assessment of antibody responses to severe acute respiratory syndrome coronavirus-2 is potentially confounded by exposures to flaviviruses. The aims of the present research were to determine whether anti-dengue antibodies affect the viral load and the detection of anti-coronavirus nucleocapsid (N)-protein antibodies in coronavirus infectious disease 2019 (COVID-19) in Bangladesh. METHODS: Viral RNA was evaluated in swab specimens from 115 COVID-19 patients by real-time reverse transcription polymerase chain reaction (rT-PCR). The anti-N-protein antibodies, anti-dengue virus E-protein antibodies and the dengue non-structural protein-1 were determined in serum from 115 COVID-19 patients, 30 acute dengue fever pre-COVID-19 pandemic and nine normal controls by ELISA. RESULTS: The concentrations of viral RNA in the nasopharyngeal; Ct median (95% CI); 22 (21.9-23.3) was significantly higher than viral RNA concentrations in oropharyngeal swabs; and 29 (27-30.5) p < 0.0001. Viral RNA concentrations were not correlated with-dengue IgG levels. The anti-nucleocapsid antibodies were IgA 27% positive and IgG 35% positive at days 1 to 8 post-onset of COVID-19 symptoms versus IgA 0% and IgG 0% in dengue patients, p < 0.0001. The levels of anti- nucleocapsid IgA or IgG versus the levels of anti-dengue IgM or IgG revealed no significant correlations. CONCLUSIONS: Viral RNA and anti-nucleocapsid antibodies were detected in COVID-19 patients from dengue-endemic regions of Bangladesh, independently of the dengue IgG levels.

Predicting the Relapse of Hyperthyroidism in Treated Graves' Disease with Orbitopathy by Serial Measurements of TSH-Receptor Autoantibodies.

The aim of this study was to investigate the potential of the new TSH-receptor antibody (TRAb) assays to predict remission or relapse of hyperthyroidism in patients with Graves' disease (GD) and Graves' orbitopathy (GO). TRAbs were measured retrospectively in sera from a cohort of GD patients with GO (n=117; remission n=38 and relapse n=79-Essen GO biobank) with automated binding immunoassays: TRAb Elecsys (Cobas Roche) and TRAb bridge assay (IMMULITE, Siemens), and the TSAb (thyroid stimulating Ab) cell-based bioassay (Thyretain, Quidel Corp.). To identify relapse risk/remission of hyperthyroidism patients were followed up at least 10 months after the end of antithyroid drug therapy (ATD) therapy. ROC plot analysis was performed to calculate cut-off levels of TRAb and TSAb for prediction of relapse and remission of hyperthyroidism. Cut-off serum levels are provided for timepoints around 3, 6, 10, and 15 months after the beginning of ATD. Repeated measurements of TRAb increase the rate of relapses predictions to 60% (Elecsys), 70% (IMMULITE), and 55% (Thyretain). Patients with remission have consistently TRAb levels below the cut off for relapse in repeated measurements. The cell-based bioassay was the most sensitive - and continued to be positive during follow up [at 15 months: 90% vs. 70% (IMMULITE) and 65% (Elecsys)]. Identification of relapsing hyperthyroidism is possible with automated immunoassays and cell-based bioassay especially with serial TRAb measurements during the course of ATD therapy. Patient who need eye surgery may profit from an early decision towards definitive treatment.

Hepatitis E Virus Capsid Antigen (HEV-Ag) - A practical diagnostic biomarker in the HEV outbreak scenario.

BACKGROUND: The increased global incidence of hepatitis E virus (HEV) infections, warrants accurate and affordable diagnostics across different geographical regions. The soluble and highly conserved HEV open reading frame 2 (ORF2) capsid antigen (HEV-Ag) is detectable in self-limited acute enteric hepatitis by HEV-Ag ELISA which is a promising serological assay in settings where HEV-RNA testing is not feasible. Our aim was to assess the HEV-Ag biomarker in an HEV outbreak in a low income country. METHODS: A prospective single center longitudinal study during HEV outbreaks in the Chittagong, Bangladesh region between October 2018 and October 2019 was conducted based on recruitment of acute jaundice cases with clinical signs and symptoms of suspect HEV infections. Acute HEV infection was defined as a positive test result for anti-HEV IgM antibodies. RESULTS: Forty four of the 51 enrolled enteric hepatitis cases (86 %) were confirmed HEV by anti-HEV IgM ELISA at day 0 hospital entry. The anti-HEV-IgM and IgG were positive in all patients and did not reveal significant differences; neither between the time points day 0 and follow-up hospitalization on day 2-6 or day 7-10 nor between RNA-positive (n = 36) versus RNAnegative (n = 8) HEV groups. The HEV-Ag positivity was higher in viral RNA-positive (29/36, 81 %) than the viral RNA-negative (1/8, 12 %) group, p < 0.001 and the HEV-Ag levels positively correlated with viremia, r = 0.77, p < 0.0001. All non-HEV cases; n = 7 tested negative anti-HEV IgM and HEV-Ag and 5 of 7 (71 %) tested anti-HAV IgM positive. CONCLUSIONS: The HEV-Ag ELISA is a reliable and practical diagnostic tool in this acute HEV outbreak.

Predominant secondary dengue infection among Vietnamese adults mostly without warning signs and severe disease.

BACKGROUND: The morbidity in dengue fever is dependent on the dengue virus (DENV) serotypes, the patient age, predisposing immunogenic markers and the frequency of primary and secondary infections. This study aims to distinguish acute primary from secondary dengue infections of Vietnamese adults and to assess the association of viremia and anti-dengue immunoglobulin levels with clinical outcomes. STUDY DESIGN: Viral RNA, dengue serotypes and levels of anti-dengue IgM and IgG of hospitalized adult cases were determined in EDTA-plasma samples prospectively collected during three consecutive years of dengue infection in Hanoi. Patients admitted to hospital within 7 days of their 1st reported fever were included. Primary infections were anti-dengue IgG enzyme-linked immunosorbent assay (ELISA) negative on both day of hospital entry (day 0) and day two or three of hospitalization (day 2 or 3) with a positive anti-dengue IgM on either day 0 or day 2 or 3 hospitalization. The secondary infections were anti-dengue IgG ELISA positive on both day 0 and day 2 or 3 with positive anti-dengue IgM ELISA on either day 0 or day 2 or 3. RESULTS: The hospitalized dengue fever cases between October 2016 and March 2019 were predominantly secondary infections (74%, 68% and 77%, respectively) with DENV-1 (60% and 65%) and DENV-2 (22% and 26%) serotypes determined in the latter two years. The viremia in primary infection was significantly higher than that in secondary infection (P < 0.01) and positively correlated with the days of hospital stay. In secondary infections, platelet counts were lower than in primary infections (P = 0.04) and IgG levels in secondary infection negatively correlated with platelet counts (Spearman's r = -0.22, P < 0.01). CONCLUSIONS: Our results indicate high rates of secondary infection with DENV1 and DENV2 serotypes. Anti-dengue immunoglobulins negatively correlate with hospital stay and platelet counts with few warning signs or severe disease. Further investigations of specific antibodies in adults which predict auto-inflammatory activity after the recovery from dengue infection are warranted.

Neopterin levels and Kyn/Trp ratios were significantly increased in dengue virus patients and subsequently decreased after recovery.

OBJECTIVES: During dengue fever, a pronounced gamma-interferon immune response produces neopterin and promotes tryptophan degradation by the enzyme indoleamine-2,3-dioxygenase 1 (IDO-1). Activated IDO-1 is indicated by an increased kynurenine to tryptophan ratio (Kyn/Trp) in patients. METHODS: Plasma levels of neopterin, kynurenine, and tryptophan were measured in 72 hospitalized dengue virus (DENV) patients and 100 healthy individuals. Plasma levels of neopterin, kynurenine, and tryptophan were also measured prospectively in a second cohort of 13 DENV patients; on the day of hospitalization, on day 2-3 at discharge, and 7-10 days after discharge. DENV RNA positivity was determined by qualitative and quantitative methodologies. RESULTS: DENV RNA-positive patients presented significantly higher levels of neopterin (mean 36.5nmol/l) and Kyn/Trp ratios (mean 102µmol/mmol) compared to DENV RNA-negative individuals. A significant correlation between neopterin levels and Kyn/Trp ratios was observed in both DENV RNA-positive (Spearman's rho=0.37, p< 0.01) and DENV RNA-negative (Spearman's rho=0.89, p<0.001) patients. Kyn/Trp ratios were negatively correlated with platelet counts (Spearman's rho=-0.43, p<0.01) and positively correlated with liver enzymes: AST (Spearman's rho=0.68, p<0.01) and ALT (Spearman's rho=0.51, p<0.05). In addition, the follow-up data presented a significant decrease in neopterin levels and Kyn/Trp ratios within 10 days after hospital entry. CONCLUSIONS: Neopterin levels and Kyn/Trp ratios were significantly increased in DENV patients and subsequently decreased after recovery.

Tryptophan-kynurenine profile in pediatric autoimmune hepatitis.

The impairment of regulatory T cells (Tregs) is a characteristic feature of autoimmune hepatitis (AIH), and the degradation of tryptophan (Trp) to kynurenine (Kyn), by gamma interferon-induced indoleamine-2,3-dioxygenase-1 (IDO-1), is a central metabolomics check point in the differentiation of Tregs. For this reason, we investigate whether or not Kyn and IDO activity is potentially useful biomarkers in pediatric AIH.Between January 2016 and January 2017, children of AIH type-1 (AIH-1, n = 37), AIH type-2 with liver kidney microsome-1 autoantibodies (AIH-2-LKM-1, n = 8), and autoantibody-negative Wilsons Disease (WD, n = 8) and alpha-1 anti-trypsin deficiency (AATD, n = 10), were enrolled in a cross-sectional survey of Kyn and Trp levels and Kyn/Trp ratios (IDO activity) by HPLC, and neopterin levels by ELISA.The mean Kyn and mean Kyn/Trp ratios of AIH-1 with smooth muscle antigen (SMA) 1.85 µM and 27 µmole/mmole, and AIH-2-LKM-1; 1.7 µM and 28.6 µmole/mmole were lower than that of the WD; 2.2 µM p = 0.03 and 33 µmole/mmole p = 0.02 and of AATD; 2.3 µM, p = 0.02 and 55 µM, p = 0.001. Kyn/Trp ratios of AIH relapse; 23.6 µmole/mmole were lower than Kyn/Trp ratios of AIH remission; 27.6 µmole/mmole (p < 0.05). The stage of liver disease and grade of liver biopsies in AIH-1 patients negatively correlated with the Kyn/Trp ratios.The serum Kyn levels and Kyn/Trp ratio of AIH patients, within or below the normal range, indicate a trend of IDO activity lower than non-autoimmune WD or AATD. Prospective monitoring of serum tryptophan metabolomics in larger cohorts of pediatric AIH patients is required to confirm the apparent paradigm of weak IDO activity contributing to the Treg deficit and pathogenesis of pediatric AIH.

Autoantibody Profile of Adult Patients With Childhood Onset Type 2 Autoimmune Hepatitis.

BACKGROUND: Anti-liver kidney microsome (anti-LKM) autoantibodies are a distinguishing feature of type II autoimmune hepatitis (AIH-2). However, the levels of anti-LKM-1 in adult AIH-2 patients and their role in liver immunopathology remain equivocal. The aim of the study was to survey the autoantibody profile and the activity of liver disease in adult patients diagnosed with AIH-2 at childhood. METHODS: The autoantibody profile of adults was compared with the autoantibodies of the pediatric period. Liver function test, Immunoglobulin G (IgG), and gamma globulins were evaluated at the AIH presentation, at the age of 18 years, and at the current adult visit. RESULTS: All ten patients tested positive for LKM-1 at least once during the pediatric period. At the adult visit, four patients lost autoantibody positivity. LKM-1 was positive in four, liver cytosol antigen 1 (LC-1) in two, soluble liver antigen in one, and antinuclear antigen in one patient. Additionally three patients with LKM-1 and one patient without LKM-1 were positive for AMA-M2 (where AMA is antimitochondrial antibodies) Immunoglobulin M (IgM). Liver function markedly improved at 18 years and adult visit compared with initial diagnosis of AIH with only a mild decrease of IgG. The six adult patients positive for at least one autoantibody had statistically lower aspartate aminotransferase (AST) and gamma-glutamyltranspeptidase (GGTP) than the four patients autoantibody negative (AST: 52 vs. 88 IU/l, P < 0.05; GGTP 19 vs. 163 IU/l, P < 0.05). CONCLUSION: LKM-1 positivity is not a stable condition in all patients with AIH-2. Patients who remained autoantibody positive had better liver function tests than those who lost their positivity. The presence of AMA-M2 autoantibodies suggest that development of AIH/Primary Biliary Cirrhosis (PBC) overlap syndrome should be considered.

Thyroid-stimulating immunoglobulins as measured in a reporter bioassay are not detected in patients with Hashimoto's thyroiditis and ophthalmopathy or isolated upper eyelid retraction.

Although ophthalmopathy is mainly associated with Graves' hyperthyroidism, milder eye changes are also found in about 25% of patients with Hashimoto's thyroiditis (HT). The recent finding of negative thyrotropin receptor (TSHR) antibodies, as measured in the Thyretain™ thyroid-stimulating immunoglobulin (TSI) reporter bioassay, in patients with euthyroid Graves' disease raises the possibility that TSHR antibodies are not the cause of ophthalmopathy in all situations. Here, we have tested serum from patients with HT with and without ophthalmopathy or isolated upper eyelid retraction (UER) for TSHR antibodies, using the TSI reporter bioassay and collagen XIII as a marker of autoimmunity against the orbital fibroblast. Study groups were 23 patients with HT with ophthalmopathy, isolated UER, or both eye features and 17 patients without eye signs. Thyretain™ TSI results were expressed as a percentage of the sample-to-reference ratio, with a positive test being taken as a sample-to-reference ratio of more than 140%. Serum collagen XIII antibodies were measured in standard enzyme-linked immunosorbent assay. TSI tests were positive in 22% of patients with HT with no eye signs but in no patient with eye signs. In contrast, TSI tests were positive in 94% of patients with Graves' ophthalmopathy. Tests were negative in all normal subjects tested. Collagen XIII antibodies were detected in 83% of patients with ophthalmopathy, UER, or both eye features, but in only 30% of patients with no eye signs. Our findings suggest that TSHR antibodies do not play a major role in the pathogenesis of ophthalmopathy or isolated UER in patients with HT. Moreover, the role of TSHR antibodies in the development of ophthalmopathy in patients with Graves' disease remains to be proven. In contrast, collagen XIII antibodies appear to be a good marker of eye disease in patients with HT.

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen.

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.

Neo-epitope tissue transglutaminase autoantibodies as a biomarker of the gluten sensitive skin disease--dermatitis herpetiformis.

BACKGROUND: The deamidated gliadin peptides (DGP) cross linked to human tissue transglutaminase (tTg) comprises a novel neo-epitope structure (Neo-tTg) for serological screening of celiac disease (CD). Our aim is to verify anti-Neo-tTg IgA and IgG in adults with dermatitis herpetiformis (DH). METHODS: Multi-centric retrospective evaluation of the IgA/G autoantibodies in sera of DH patients on a regular diet (n=40) and a gluten restricted diet (GRD, n=53) and control adults with autoimmune skin diseases (n=107) by ELISA. RESULTS: The sensitivities of Celicheck Neo IgA/G (76%, 95% CI 67-84%) and the Neo tTg-A (85%, 95% CI 70-97%) ELISA were significantly greater than that of tTg-A (56%, 95% CI 46-67%), eTg-A (62%, 95% CI 52-72%), DGP-A (55%, 95% CI 55-65%), DGP-G (61%, 95% CI 51-71%), Glia-A (55%, 95% CI 45-65%) and Glia-G (56%, 95% CI 46-66%) ELISA. The specificities of all 8 ELISA were in the range of 90-100%. The area under the curve (AUC) of receiver operator characteristic curve (ROC) for the two Neo-tTg ELISA (0.863 and 0.949) were higher than the AUCs for ROCs of tTg, DGP and eTG ELISA (range between 0.657 and 0.783). The autoantibody levels of DH patients on a normal diet were significantly higher than those on GRD in the Celicheck Neo IgA/IgG, NeotTg-A; tTg-A and the eTg-A; ELISA (p<0.01) and of no significance in the DGP and Gliadin ELISA. CONCLUSION: Neo-epitope IgA autoantibodies represent a new and sensitive serological marker of DH.

Anti-T lymphocyte globulin (ATG) induces generation of regulatory T cells, at least part of them express activated CD44.

We show here that the anti-T lymphocyte immunoglobulin (ATG) can induce Treg cells following 24-h incubation in human peripheral blood mononuclear cells (PBMCs). The ATG-induced Treg cells express known cell surface markers (e.g., CD25, FoxP3) and suppress the proliferation of autologous responder PBMCs, stimulated with allogeneic PBMCs, when added into the mixed lymphocyte culture (MLC) at zero time point or 48 h later. We expanded the characteristics of the ATG-induced human Treg cells by showing that they express a novel biomarker designated "activated CD44". ATG-induced Treg cells retain their suppressor function after freezing and thawing or irradiation. Suppression of MLC by ATG-induced Treg cells is consistently seen when the Treg cells and the responder cells were derived from the same donor, but not when they derived from different donors. Finally, patients undergoing stem cell transplantation and conditioned with ATG generate in vivo Treg cells that suppress MLC.

Regulatory T-cells in Graves' orbitopathy: baseline findings and immunomodulation by anti-T lymphocyte globulin.

BACKGROUND AND AIM: Graves' orbitopathy (GO) is characterized by orbital T cell infiltration and local release of proinflammatory cytokines. We aimed to evaluate the involvement of baseline regulatory T (Treg) cells and rabbit anti-T lymphocyte globulin (rATG)-induced Treg cells in GO. DESIGN: Peripheral blood mononuclear cells (PBMCs) from seven patients with Graves' disease (GD) without eye manifestations, 29 patients with GO, and 15 healthy controls were incubated with rATG, washed, and analyzed for expression of Treg cell markers and for ability to suppress mixed lymphocyte reaction. RESULTS: Elevation of CD4 to CD8 ratio and enhanced secretion of IL-6, IL-10, and TNFα were detected in PBMCs of GO patients compared with controls (both P < 0.01). Despite this abnormality, the frequencies of CD4(+)CD25(+)FoxP3(+) of GO and control PBMCs were similar and remained unchanged after 24 h incubation with control rabbit IgG (rIgG). Incubation with polyclonal rATG increased the frequency of PBMCs of GO patients, expressing Treg cell markers (CD25, FoxP3, and the IL-7 receptor CD127(low/-)) by 2.5-8 fold over corresponding rIgG-incubated cells (P < 0.05). FoxP3/CD4 rATG-induced Treg cell marker expressed more intensively on GO peripheral blood leukocytes (PBLs) than on GD (P < 0.01) or normal (P < 0.05) PBLs, yet its expression on normal PBLs was stronger than on GD PBLs (P < 0.05). GO rATG-incubated PBMCs, but not rIgG-incubated PBMCs, suppressed (P < 0.05) proliferation of autologous responder cells stimulated with allogeneic irradiated cells in mixed lymphocyte reaction. Such rATG-induced suppressive activity was not detected in GD. CONCLUSION: This study is the first to show that PBMCs of patients with GO substantially increase Treg cells in both frequency and potency after in vitro incubation with rATG.

Bioassays for TSH-receptor autoantibodies: an update.

Immunoglobulins in patients with Graves' disease (GD) that modulate the thyroid stimulating hormone receptor (TSH-R) do so via stimulating cAMP dependent signals (TSI), blocking TSH or inhibition of TSH-receptor activation (TBI) or inducing apoptotic signals. These functional immunoglobulins represent powerful biomarkers of anti-self reactivity in the thyroid and systemic tissues that harbor TSH-R expressing target cells. TSI on thyrocytes induce hyperthyroidism, and TSI on TSH-R fibroblasts of orbital muscles, skin and heart provoke the release of cytokines and antigen-specific T-cell responses leading to systemic inflammation. Bioassays of anti-TSH-R autoantibodies provide decisive information on GD activity. This review examines the past and present bioassays in GD. The critical goal of cell-based anti-TSH-R autoantibody bioassays, to identify the pathogenic immunoglobulins in GD under robust and standardized conditions suitable for routine clinical laboratory practice, is discussed.

Treatment of autoimmune disease with rabbit anti-T lymphocyte globulin: clinical efficacy and potential mechanisms of action.

The rabbit anti-T lymphocyte globulins (rATGs) are immune-suppressive anti-T cell agents with beneficial effects in solid organ and hematological transplantation. The present review evaluates the potential mechanisms of rATGs and their impact on pilot and exploratory studies of diffuse cutaneous systemic sclerosis (scleroderma-SSc), inclusion body myositis (IBM), vasculitis, and type 1 diabetes mellitus (T1DM). The rATGs are associated with improvements in well-defined parameters of clinical autoimmunity: insulin usage, tissue inflammation, and systemic organ functions. Meta-analysis of a retrospective database of SSc, N = 196 and two prospective randomized pilot studies; IBM, N = 11 and T1DM, N = 17 shows a two- to ninefold increase in the relative response to treatments with intravenous infusions of rATG. The rATGs deplete T cells and are associated with increases in the percentage of CD25+ T cell subsets. This may underlie the apparent long-lasting immunomodulation associated with these agents. The future optimization of rATG adjunct therapy requires statistically powered-controlled prospective trials of rATG dose-finding and timing of administration. The potential mechanisms of rATGs:depletion of autoreactive T cells, generation CDCD25+Foxp3+ regulatory T cells (Tregs), and the acquisition of regulatory immune cell functions, need to be examined in patients prior to rATG infusion and at time intervals following rATG treatment to identify those mechanisms relevant to the improvement of their clinical outcome.

Production of ammonium by Helicobacter pylori mediates occludin processing and disruption of tight junctions in Caco-2 cells.

Tight junctions, paracellular permeability barriers that define epithelial cell polarity, play an essential role in transepithelial transport, cell-cell adhesion and lymphocyte transmigration. They are also important for the maintenance of innate immune defence and intestinal antigen uptake. Ammonium (NH4+) is elevated in the gastric aspirates of Helicobacter pylori-infected patients and has been implicated in the disruption of tight-junction functional integrity and the induction of gastric mucosal damage during H. pylori infection. The precise mechanism of the effect of ammonium and the molecular targets of ammonium in host tissue are not yet identified. To study the effects of ammonium on epithelial tight junctions, the human colon carcinoma cell line Caco-2 was cultured on permeable supports and the transepithelial resistance (TER) was measured at different time intervals following exposure to ammonium salts or H. pylori-derived ammonium. A biphasic response to treatment with ammonium was found. Acute exposure to ammonium salts or NH3/NH4+ derived from urea metabolism by wild-type H. pylori resulted in a 20-30 % decrease in TER. After 24 h, the NH4Cl-treated cells showed a partial recovery of TER. In contrast, the control culture, or cultures that were exposed to supernatants derived from urease-deficient H. pylori, showed no significant decrease in TER. Occludin-specific immunoblots revealed the expression of a low-molecular-weight form of occludin of 42 kDa upon NH3/NH4+ exposure. The results indicate that modulation of tight-junction function by H. pylori is ammonium-dependent and linked to the accumulation of a low-molecular-weight and detergent-soluble form of occludin.

Antigen/MHC class II/Ig dimers for study of uveitogenic T cells: IRBP p161-180 presented by both IA and IE molecules.

PURPOSE: Detection and modulation of effector T cells specific to immunodominant epitopes is a central issue in autoimmune diseases. Experimental autoimmune uveitis is a model for human autoimmune uveitis, induced in B10.RIII mice with interphotoreceptor retinoid binding protein or with its immunodominant epitope encoded by residues 161-180. METHODS: The authors generated a dimer composed of p161-180 fused in frame to IA(r) and mouse IgG1, and studied its effects on a CD4(+) uveitogenic T-cell line specific to p161-180 and on a T-cell clone derived from that line. RESULTS: Immunofluorescent staining of the T-cell line with the peptide/IA(r)/Ig dimer revealed that about 90% of the cells bound the reagent, and 10% did not. The T-cell clone failed to bind the reagent. Consistent with this, the line proliferated when stimulated with the reagent plus anti-CD28, and the clone did not. Conversely, after being incubated with the reagent without CD28 cross-linking, the line showed decreased proliferation on subsequent stimulatory exposure to p161-180, whereas the clone was unaffected. Antigen-specific proliferation of splenocytes from B10.RIII mice primed with p161-180 was inhibited by anti-IA as well as anti-IE antibodies; proliferation of the T-cell line was inhibited strongly by anti-IA and poorly by anti-IE, and the clone showed the opposite pattern. Finally, the line, but not the clone, proliferated to p161-180 presented on a B-cell lymphoma expressing IA(r) as its only restriction element. CONCLUSIONS: Uveitogenic T cells can be detected as well as functionally modulated with their cognate peptide-class II reagent, suggesting the potential of such reagents for diagnostic and therapeutic use in uveitic disease; p161-180 can be presented by IA(r) as well as IE(r) major histocompatibility complex (MHC) class II molecules. The possibility that the same immunodominant fragment might be presented by more than one class II molecule should be taken into account when diagnostic or clinical use of peptide-MHC reagents is considered.