Autoantibody profile in individuals with chronic hepatitis C

IntroductionAutoantibodies are often produced during infection with chronic hepatitis C virus (HCV), but it remains controversial whether they influence the biochemical profile and histological features of this disease. Therefore, this current study sought to describe these autoantibodies and evaluate their impact on the clinical and histological presentation of hepatitis C.MethodsThis cross-sectional analytical study assessed patients with HCV (RNA+) from October 2011 to July 2012.ResultsThis study included 66 patients, with a mean age of 53.2±10.5 years. Of these patients, 60.6% were male, and 54.3% presented with genotype 1. Non-organ-specific autoantibodies (NOSA) were detected in 24% of the patients; of these, 7.6% were anti-mitochondrial antibodies (AMA+), 26.7% were anti-smooth muscle antibodies (SMA+) and 6.8% were liver kidney microsomal type 1 antibodies (LKM1+). With respect to the thyroid autoantibodies, 7.4% were anti-peroxidase (ATPO+) antibodies, and none were anti-thyroglobulin (ATG+) antibodies. Regarding celiac disease autoantibodies, 5.8% were endomysial antibodies (EMA+), and no transglutaminase (TTG+) antibodies were detected. Cryoglobulins were found in 2.1% of patients. When NOSA+ individuals were compared to patients without the presence of NOSAs, they exhibited higher median alkaline phosphatase (0.7 vs. 0.6 xULN; p=0.041), lower median platelet counts (141,500.0 vs. 180,500.0/mm3; p=0.036), lower mean prothrombin activity (72.6±11.5% vs. 82.2±16.0%; p=0.012) and an increased prevalence of significant fibrosis (E≥2) (45.5% vs. 18.2%; p=0.012). There was also a tendency for a greater proportion of NOSA+ cases to have marked periportal activity (APP≥3) (44.5% vs. 15.6%; p=0.087).ConclusionsIn addition to the high prevalence of autoantibodies associated with HCV infection, it was observed that NOSA positivity was associated with a more severe histological and biochemical profile of hepatitis C infection.

Reduced development of CD4-8-B220+ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class I molecules.

The lpr gene has recently been shown to encode a functional mutation in the Fas receptor, a molecule involved in transducing apoptotic signals. Mice homozygous for the lpr gene develop an autoimmune syndrome accompanied by massive accumulation of double-negative (DN) CD4-8-B220+ T cell receptor-alpha/beta+ cells. In order to investigate the origin of these DN T cells, we derived lpr/lpr mice lacking major histocompatibility complex (MHC) class I molecules by intercrossing them with beta 2-microglobulin (beta 2m)-deficient mice. Interestingly, these lpr beta 2m-/- mice develop 13-fold fewer DNT cells in lymph nodes as compared to lpr/lpr wild-type (lprWT) mice. Analysis of anti-DNA antibodies and rheumatoid factor in serum demonstrates that lpr beta 2m-/- mice produce comparable levels of autoantibodies to lprWT mice. Collectively our data indicate that MHC class I molecules control the development of DN T cells but not autoantibody production in lpr/lpr mice and support the hypothesis that the majority of DN T cells may be derived from cells of the CD8 lineage.

Autoantibody production in chronic idiopathic urticaria is not associated with Helicobacter pylori infection

Chronic idiopathic urticaria (CIU) is a dermatological syndrome, characterized by raised erythematous skin lesions, that affects 20% of the general population and has been associated with autoimmunity. However, some reports have also suggested a close relationship between CIU and Helicobacter pylori infection, which is endemic in developing countries and associated with chronic gastritis, peptic ulcer disease, and gastric carcinoma. In the present study, we investigated the occurrence of autoantibodies in sera from 23 CIU subjects infected with H. pylori and from 23 CIU subjects without this infection. The presence of anti-thyroid antibodies was determined by indirect hemagglutination assay and the presence of autoantibodies to IgE and C1INH was determined by ELISA. Antibodies to thyroid antigens were detected at low titers from 100 to 400 in three of 23 (13%) CIU-infected subjects and in four of 23 (17%) CIU-noninfected subjects. The titers of anti-IgE autoantibodies were similar in these CIU groups, presenting absorbances of 1.16 ± 0.09 and 1.07 ± 0.16, respectively, while a titer of 1.14 ± 0.15 was detected in the healthy control group. The concentration of anti-C1INH autoantibodies was the same in the CIU-infected and -noninfected subjects (7.28 ± 1.31 and 7.91 ± 2.45 ng/ml, respectively), and was 7.20 ± 2.25 ng/ml in the healthy control group. However, the serum levels of complexed anti-C1INH antibodies were increased in CIU-infected subjects compared to CIU-noninfected subjects and healthy controls with an absorbance of 1.51 ± 0.21 vs 1.36 ± 0.16 and 1.26 ± 0.23, respectively (P < 0.05), indicating an impaired clearance of immune complexes in CIU-infected patients. In conclusion, no correlation was observed between H. pylori infection and autoantibody production in CIU patients consistent with reports of clinical studies.

Dermal dendritic cell number correlates with serum autoantibody titers in Brazilian pemphigus foliaceus patients

Pemphigus foliaceus (PF) is an autoimmune bullous disease endemic in Brazil. Since serum IL-12 is increased in patients with PF and Langerhans cells (LC) produce IL-12, we titrated serum autoantibodies by indirect immunofluorescence, and quantified epidermal dendritic cells, known as LC, and dermal dendritic cells (DC). Biopsies of blistering lesions were obtained from 22 patients, 13 of whom were submitted to biopsy of both injured and of apparently healthy skin. The control groups consisted of skin from 8 cadavers and from 12 women submitted to breast plastic surgery. LC and DC were identified with anti-CD1a antibody and quantified by morphometric analysis. LC number in the lesion and in apparently healthy skin from PF patients was similar to that of both control groups. DC number in the injured skin (median = 0.94 DC/mm basement membrane) was higher than that of the cadaver group (median = 0.13 DC/mm basement membrane). In the 13 patients with biopsies of both injured and apparently healthy skin, LC and DC were present in larger numbers in the lesion. There was a direct correlation between DC number in the lesion of the PF group and serum autoantibody titers. This correlation was not observed for LC number. The increased number of DC in the lesion, as well as its direct correlation with serum autoantibody titers suggest the participation of DC in the pathogenesis of PF. The relationship between increased DC number and IL-12 in PF needs to be clarified.

Autoantibody response to chromatographic fractions from oxidized LDL in unstable angina patients and healthy controls

Levels of autoantibodies to oxidized low-density lipoprotein (oxLDL) have been correlated to atherosclerosis; however, contradictory results have been shown. To better understand the role of autoantibodies to oxLDL in atherogenesis, and their potential to predict risk of developing coronary artery disease we investigated the antibody response of unstable angina (UA) patients and healthy controls against chromatographic separated fractions of oxLDL. Five major peaks were detected after chromatographic separation of oxLDL and 10 fractions were collected. Surprisingly, when the response to high molecular weight fractions was analysed, we observed a significant increase in the levels of autoantibodies in controls compared to UA. In contrast, when the autoantibody response to intermediate and low molecular weight fractions was analysed, we observed that the UA group showed consistently higher levels compared with controls. Our data demonstrates that within oxLDL there are major fractions that can be recognized by autoantibodies from either UA patients or healthy individuals, and that the use of total oxLDL as an antigen pool may mask the presence of some antigenic molecules and their corresponding antibodies. Further studies are needed, but the analysis of antibody profiles may indeed open up a novel approach for evaluation and prevention against atherosclerosis.

Yersinia enterocolitica O : 3 isolated from patients with or without reactive arthritis induces polyclonal activation of B cells and autoantibody production in vivo

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica . Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG 1 , IgG 2a , IgG 2b , IgG 3 , IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG 2a and IgG 3 isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.

IgG Autoantibody Response against Keratinocyte Cadherins in Endemic Pemphigus Foliaceus (Fogo Selvagem)

It is well established that autoantibodies against desmoglein 3 and desmoglein 1 (Dsg1) are relevant in the pathogenesis of pemphigus vulgaris and pemphigus foliaceus, including its endemic form fogo selvagem (FS). Isolated reports have shown that in certain patients with these diseases, autoantibodies against other desmosomal cadherins and E-cadherin may also be present. The goal of this investigation was to determine whether FS patients and normal individuals living in endemic areas possess autoantibodies against other desmosomal cadherins and E-cadherin. By testing a large number of FS and endemic control sera by ELISA, we found a consistent and specific autoantibody response against Dsg1 and other keratinocyte cadherins in these individuals, which is quite different from healthy individuals from the United States (US controls). Overall, the highest correlations among the autoantibody responses tested were in the endemic controls, followed by FS patients, and lowest in the US controls. These findings suggest that multiple, perhaps cross-reactive, keratinocyte cadherins are recognized by FS patients and endemic controls.

PReS-FINAL-2177: Safety and lack of autoantibody production following influenza H1N1 vaccination in patients with juvenile idiopathic arthritis (JIA)

Introduction Vaccination is an effective tool against several infectious agents including influenza. In 2010, the Advisory Committee on Immunization Practices (ACIP) recommended influenza A H1N1/2009 immunization for high risk groups, including juvenile idiopathic arthritis (JIA) patients and more recently the EULAR task force reinforced the importance of vaccination in immunosuppressed pediatric rheumatologic patients. We have recently shown that Influenza A H1N1/2009 vaccination generated protective antibody production with short-term safety profile among 93 JIA patients, but the possible impact of the vaccine in autoimmune response in JIA have not been studied. Therefore, we aimed to assess the production of some autoantibodies generated following influenza H1N1 vaccination in JIA patients. Objectives To assess the autoimmune response and H1N1 serology following influenza H1N1 vaccination in patients with JIA. Methods Cepa A/California/7/2009 (NYMC X-179A) anti-H1N1 was used to vaccinate JIA patients: 1 dose of immunization was given to all participants and those <9yrs of age received a second booster 3 weeks apart. Sera were analyzed before and 3 weeks following complete vaccination. Serology against H1N1 virus was performed by hemagglutination inhibition antibody assay, rheumatoid factor (RF) by latex fixation test, antinuclear antibodies (ANA) by IIF, IgM and IgG anticardiolipin (aCL) by ELISA.Results Among 98 JIA patients that were vaccinated, 58 sera were available for this study. Mean age of 58 JIA patients was 23.9 ± 9.5 yrs, 38 were females and 20 males with mean disease duration of 14.7 ± 10.1 yrs. JIA subtypes were: 33 (57%) poliarticular, 10 (17%) oligoarticular, 6 (10%) systemic and 9 (16%) other. Sixteen patients were off drugs while 42 (72%) were under different pharmacotherapy: 32 (55%) were on 1 DMARD/IS, 10 (17%) on 2 DMARDs/IS, 19 (33%) antimalarials, 29 (50%) MTX, 8(14%) sulfasalazine, 6 (10%) anti-TNFs, 4 (7%) abatacept; no patient was using prednisone >0.5 mg/kg/d. Seroprotection rates against H1N1 influenza increased from 23 to 83% and seroconversion rates were achieved in 78% JIA. Prior to vaccination, 31(53.4%) JIA patients were ANA+, 6(10.3%) RF+, and 4 (7%) IgM + IgG aCL+. After complete H1N1 vaccination, positivity for ANA remained the same whereas 1 patient became negative for IgG aCL, and another for RF, IgM and IgG aCL. One (1.7%) patient turned low titer IgG aCL+. Conclusion Vaccination of JIA patients against pandemic influenza A (H1N1) generated successful protective antibody production without the induction of autoantibody production, except for 1 patient that became positive for low titer IgG aCL, supporting its safety.

Pemphigus herpetiformis: analysis of the autoantibody profile during the disease course with changes in the clinical phenotype

Pemphigus herpetiformis (PH) is a rare dapsone-responsive variant of pemphigus, characterized by annular and vesiculopustular cutaneous lesions. Most PH serum samples contain autoantibodies against desmoglein (Dsg)1, but not Dsg3, and the presence of the latter is almost invariably associated with mucosal involvement, as predicted based on the 'Dsg compensation theory'. We describe a patient with features characteristic of PH with histologically eosinophilic spongiosis who repeatedly tested positive for anti-Dsg3 but not anti-Dsg1 autoantibodies by ELISA. To investigate whether the peculiar clinical phenotype was due to a distinct immunological profile, the patient's serum was tested by ELISA and immunoblotting using recombinant forms of Dsg3. Serum samples were found to have low and high reactivity against the EC1 and the EC4 domains of Dsg3, respectively, whereas the autoantibodies belonged predominantly to the IgG1 and IgG4 subclasses. The overall immunological profile was typical of pemphigus vulgaris. The patient finally developed isolated oral erosions 22 months after initial presentation, without significant changes in the autoantibody profile and of the targeted antigenic sites. Our patient presented features characteristic of PH. Although circulating anti-Dsg3 antibodies were present, the patient had only cutaneous involvement for a long period. Our findings indicate that the proposed Dsg compensation theory cannot always explain the clinical phenotype, changes in which may occur without apparent modification of the autoantibody profile and antibody specificity. Hence, additional factors, such as Fcgamma-dependent neutrophil activation, may critically affect the clinical presentation of pemphigus.

[Which clinical symptoms suggest distinct autoantibody measurements?]

The detection of autoantibodies is an important cornerstone in the diagnosis of systemic autoimmune disorders such as autoimmune connectivitides and small vessel vasculitides. Antinuclear antibodies (ANAs ) and antineutrophil cytoplasmic antibodies (c+p-ANCAs) are appropriate diagnostic screening tools for autoimmune connectivitides and small vessel vasculitides. For economic and intellectual/didactic reasons more extensive autoantibody diagnostics should be performed only in particular situations when patients history and clinical exam lead to strong diagnostic suspicion.

Toward an early diagnosis of lung cancer: an autoantibody signature for squamous cell lung carcinoma

Serum-based diagnosis offers the prospect of early lung carcinoma detection and of differentiation between benign and malignant nodules identified by CT. One major challenge toward a future blood-based diagnostic consists in showing that seroreactivity patterns allow for discriminating lung cancer patients not only from normal controls but also from patients with non-tumor lung pathologies. We addressed this question for squamous cell lung cancer, one of the most common lung tumor types. Using a panel of 82 phage-peptide clones, which express potential autoantigens, we performed serological spot assay. We screened 108 sera, including 39 sera from squamous cell lung cancer patients, 29 sera from patients with other non-tumor lung pathologies, and 40 sera from volunteers without known disease. To classify the serum groups, we employed the standard Naïve Bayesian method combined with a subset selection approach. We were able to separate squamous cell lung carcinoma and normal sera with an accuracy of 93%. Low-grade squamous cell lung carcinoma were separated from normal sera with an accuracy of 92.9%. We were able to distinguish squamous cell lung carcinoma from non-tumor lung pathologies with an accuracy of 83%. Three phage-peptide clones with sequence homology to ROCK1, PRKCB1 and KIAA0376 reacted with more than 15% of the cancer sera, but neither with normal nor with non-tumor lung pathology sera. Our study demonstrates that seroreactivity profiles combined with statistical classification methods have great potential for discriminating patients with squamous cell lung carcinoma not only from normal controls but also from patients with non-tumor lung pathologies.

Angiotensin receptor agonistic autoantibody is highly prevalent in preeclampsia: correlation with disease severity.

Preeclampsia (PE), a syndrome affecting 5% of pregnancies, characterized by hypertension and proteinuria, is a leading cause of maternal and fetal morbidity and mortality. The condition is often accompanied by the presence of a circulating maternal autoantibody, the angiotensin II type I receptor agonistic autoantibody (AT(1)-AA). However, the prevalence of AT(1)-AA in PE remains unknown, and the correlation of AT(1)-AA titers with the severity of the disease remains undetermined. We used a sensitive and high-throughput luciferase bioassay to detect AT(1)-AA levels in the serum of 30 normal, 37 preeclamptic (10 mild and 27 severe), and 23 gestational hypertensive individuals. Here we report that AT(1)-AA is highly prevalent in PE ( approximately 95%). Next, by comparing the levels of AT(1)-AA among women with mild and severe PE, we found that the titer of AT(1)-AA is proportional to the severity of the disease. Intriguingly, among severe preeclamptic patients, we discovered that the titer of AT(1)-AA is significantly correlated with the clinical features of PE: systolic blood pressure (r=0.56), proteinuria (r=0.70), and soluble fms-like tyrosine kinase-1 level (r=0.71), respectively. Notably, only AT(1)-AA, and not soluble fms-like tyrosine kinase-1, levels are elevated in gestational hypertensive patients. These data serve as compelling clinical evidence that AT(1)-AA is highly prevalent in PE, and its titer is strongly correlated to the severity of the disease.

Pneumocystis carinii pneumonia during immunosuppressive therapy for antineutrophil cytoplasmic autoantibody-positive vasculitis.

We describe two human immunodeficiency virus-negative patients who developed Pneumocystis carinii pneumonia (PCP) during immunosuppressive therapy for antineutrophil cytoplasmic autoantibody-positive vasculitis and review the literature regarding the pathogenesis and frequency of PCP. The recent application of DNA amplification techniques suggests that PCP developing in immunocompromised individuals does not necessarily result from reactivation of a dormant focus, but may arise as de novo infection after exposure to an exogenous source of P carinii. In addition, several reports about clusters of PCP cases raise concern about the risk of a nosocomial transmission of P carinii. Therefore, PCP should be added to the list of bronchopulmonary complications in patients with antineutrophil cytoplasmic autoantibody-positive vasculitis who are receiving long-term steroid therapy.

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.

Precursor B cells for autoantibody production in genomically Fas-intact autoimmune disease are not subject to Fas-mediated immune elimination

The Fas/Fas ligand (FasL) system participates in regulation of the immune system through the apoptotic process. However, the extent to which abnormalities in this system are involved in the loss of self-tolerance and development of autoimmune disease not associated with Fas/FasL mutations remains unknown. The present study addresses this issue in Fas/FasL-intact, systemic lupus erythematosus (SLE)-prone (NZB × NZW) (NZB/W) F1 mice. While splenic B cells from 2-month-old mice before overt SLE expressed Fas poorly, in vitro stimulation with an agonistic anti-CD40 mAb up-regulated their Fas expression, thus revealing the existence of two populations: one was Fashigh and highly susceptible to anti-Fas mAb-induced apoptosis, and the other was Faslow and apoptosis-resistant. The Faslow cells were included in the CD5+ B cell subpopulation and contained most of the cells that produced IgM anti-DNA antibodies. The isotype of anti-DNA antibodies switches from IgM to IgG in NZB/W F1 mice at ages beginning at about 6 months. These IgG anti-DNA antibodies were produced almost exclusively by a subpopulation of splenic B cells that spontaneously expressed low levels of Fas in vivo and were apoptosis-resistant. The findings indicate that precursor B cells for autoantibody production and presumably autoantibody-secreting cells in these mice are relatively resistant to Fas-mediated apoptosis, a finding supporting the concept that abnormalities of Fas-mediated apoptotic process are involved in the development of autoreactive B cells in Fas/FasL-intact autoimmune disease.