Total Immunoglobulin Production Research Articles

Abstract 226: Recellularization of Xenograft Heart Valves Reduces the Xenoreactive Immune Response in an In Vivo Rat Model

Background: Current xenograft valve constructs provoke an intense immune response that may lead to valve dysfunction. Our aim was to address the role of autologous mesenchymal stem cell (MSC) recellularization of xenogenic valves on the activation of the xenoreactive immune response in an in-vivo rat model. Methods: Explanted aortic valve constructs from female Hartley guinea pigs were procured and decellularized, followed by recellularization with syngeneic Sprague-Dawley rat MSCs. The recellularized aortic valve xenografts were then implanted into the infrarenal aorta of recipient female Sprague-Dawley rats. Grafts were implanted as either syngeneic grafts, non-decellularized (fresh), decellularized and recellularized xenografts. Rats were euthanized after 7-days, exsanguinated and the grafts explanted. Total serum immunoglobulin was quantified and histological analysis perfomed to analyze the immune response. Results: Overall survival to endpoint was significantly lower in the decellularized xenograft group (67%; 4/6), compared to fresh (100%; 6/6) and recellularized grafts (100%; 6/6). Similarly grafts in the decellularized group were more likely to have completely thrombosed (50%; 2/4), compared to fresh (33%; 2/6) and recellularized grafts (0%; 0/6). Decellularized guinea pig xenografts, when implanted into rats in-vivo , result in significantly reduced total serum immunoglobulin production and significantly reduced graft cellular infiltrate when compared to fresh xenografts. Moreover, when decellularized guinea pig xenografts were recellularized with syngeneic rat MSCs there was an additional decrease in total serum immunoglobulin production and graft cellular infiltrate when compared to both fresh and decellularized xenografts. Importantly, recellularized guinea pig xenografts had an equivalent total immunoglobulin production and graft cellular infiltrate when compared to syngeneic rat aortic valve controls. Conclusions: Autologous MSC recellularization of xenogenic valves reduces the xenoreactive immune response in an in-vivo rat model and may be an effective approach to decrease the progression of xenograft valve dysfunction.

Chronic exercise modulates the cellular immunity and its cannabinoid receptors expression.

The impact of performing exercise on the immune system presents contrasting effects on health when performed at different intensities. In addition, the consequences of performing chronic exercise have not been sufficiently studied in contrast to the effects of acute bouts of exercise. The porpoise of this work was to determine the effect that a popular exercise regimen (chronic/moderate/aerobic exercise) has on the proportion of different immune cell subsets, their function and if it affects the cannabinoid system with potentially functional implications on the immune system. A marked increase in several immune cell subsets and their expression of cannabinoid receptors was expected, as well as an enhanced proliferative and cytotoxic activity by total splenocytes in exercised animals. For this study male Wistar rats performed treadmill running 5 times a week for a period of 10 weeks, at moderate intensity. Our results showed a significant decrease in lymphocyte subpopulations (CD4+, Tγδ, and CD45 RA+ cells) and an increase in the cannabinoid receptors expression in those same cell. Although functional assays did not reveal any variation in total immunoglobulin production or NK cells cytotoxic activity, proliferative capability of total splenocytes increased in trained rats. Our results further support the notion that exercise affects the immunological system and extends the description of underlying mechanisms mediating such effects. Altogether, our results contribute to the understanding of the benefits of exercise on the practitioner´s general health.

Physalis peruviana L. inhibits ovalbumin‑induced airway inflammation by attenuating the activation of NF‑κB and inflammatory molecules

Physalis peruviana L. (PP) is well known for its various properties, including its antioxidant property. In our previous study, the protective effects of PP against cigarette smoke-induced airway inflammation were confirmed. The purpose of the present study was to evaluate the anti-inflammatory effect of PP against ovalbumin (OVA)-induced airway inflammation. Treatment with PP inhibited the numbers of eosinophils and the levels of inflammatory cytokines, including interleukin (IL)-4, IL-5 and IL-13, in the bronchoalveolar lavage fluid (BALF) of animal models with OVA-induced allergic asthma. PP also significantly decreased the production of total immunoglobulin E in the serum. Lung sections stained with hematoxylin and eosin revealed that the influx of inflammatory cells was decreased in the lungs of mice treated with PP compared with cells in the OVA group. The increased expression levels of monocyte chemoattractant protein-1 (MCP-1) and T cell marker KEN-5 were also reduced following PP treatment in the lung tissues compared with those in the OVA group. The PAS staining results showed that PP attenuated the overproduction of mucus in the lung. Additionally, western blot analysis revealed that PP significantly downregulated the activation of nuclear factor-κB/p38 mitogen-activated protein kinase/c-Jun N-terminal kinase, and upregulated the expression of heme oxgenase-1 in the lungs. In an in vitro experiment, PP effectively reduced the levels of LPS-stimulated MCP-1 in a concentration-dependent manner. Taken together, these results indicate that PP has considerable potential in the treatment of allergic asthma.

Changes in production of total immunoglobulin E but not food allergen-specific IgE or IgG4 in at-risk children after exposure to aluminum adjuvant.

Changes in production of total immunoglobulin E but not food allergen-specific IgE or IgG4 in at-risk children after exposure to aluminum adjuvant.

P079 Association of gut microbiota with mucosal inflammation in ulcerative colitis

Background: Disturbance in gut microbiota (dysbiosis) is a characteristic feature of ulcerative colitis (UC). However it remains unclear whether this dysbiosis contributes to disease pathogenesis by driving immune dysregulation or is merely secondary to mucosal inflammation and/or a result of host immune response. We aimed to determine whether microbiota dysbiosis varied between areas of inflamed and non-inflamed colon in patients with UC and whether this was associated with a humoral immune response. Methods: We collected colonic biopsies from histologically confirmed areas of inflamed and non-inflamed colon from 15 patients with active left sided ulcerative colitis or proctosigmoiditis. DNA was extracted using the FASTSpin Kit and gut microbiota was characterized using 16s rRNA based analysis of the V3–V4 region (Illumina MiSeq). Quality control and operational taxonomic unit classification of sequences was executed using QIIME. As a marker of mucosal humoral immune responses, inflamed and non-inflamed colonic biopsy samples were cultured in media (RPMI + 10% FCS) for 1 to 3 days prior to measurement of immunoglobulin production (IgA, IgG and IgM) by ELISA. Results: Consistent with previous observations patients with UC demonstrated reduced bacterial diversity with an increase in Proteobacteria, Bacteroides and Clostridiales species along with a decrease in Firmicutes to Bacteroides ratio. No differences were found in the microbial diversity nor phylae and genera in mucosally adherent gut microbiota between inflamed and non-inflamed colonic segments in patients with active UC. This observation was also seen when patients were subdivided based on disease activity as defined by Mayo scoring. Total immunoglobulin production did not differ between inflamed (n=6, mean 4966 ± sd 3670 ng/ml) and non-inflamed tissue (n=11; mean 5756 ± sd 8989 ng/ml; p>0.05) suggesting that equal numbers of antibody-producing B cells are present regardless of inflammation. Conclusions: We have demonstrated that the dysbiosis observed in patients with UC is consistent and is not influenced by mucosal inflammation or disease activity. Mucosal immunoglobulin production was not upregulated at sites of inflammation possibly suggestive of a uniform humoral response across the colon; although future work may uncover differences in antibody specificity to UC-associated dysbiosis. The mechanism for the complex interplay between the host immune system and gut microbiota in contributing to mucosal inflammation remains to be understood.

Biological Studies of high caloric foods supplemented with some probiotic bacteria

Biological Studies of high caloric foods supplemented with some probiotic bacteria Effect of different percent of high caloric food components (Lupine,chickpea and soyprotien) with probiotics bacteria on some biological and histological parameters of albino rats were studied. Thirty adult male rats were distributed into six groups, the first one was kept as control group, while the other five groups were fed on high caloric diet and supplemented with some probiotic bacteria for 6 weeks. Results showed that body weight gain markdely was higher especially in the mixture ofgroup(6) 1:1:1:1%) compared to control group and group which fed without probiotics. Also, this mixture with high probiotics lowered concentrations of total cholesterol, triglycerides, LDL-c, VLDL-c and decreased activity of AST and ALT enzymes to normal range. Furthermore, all mixture caused significant increase in HDL-c and total immunoglobulin production (IgG, IgM, IgE, IgA) were higher than the mixture (1%lupine, chickpea, soyprotien) without probiotics . Histopathological examination showed amelioration of histopathological lesions seen in liver of rats received the mixture of seeds at different levels with probiotics. So, it could be recommended that intake of the mixture of lupine , chickpea , soyprotien and prpbiotics especially at 1:1:1:1 respectively may be useful for high caloric foods. Key words: Malerats, lupine, chickpea, soyprotien, probiotics ,histopathological structure. الدراسات البيولوجية للأغذية العالية الطاقة المدعمة ببعض انواع من بکتريا البروبيوتک د/شريف صبري د/ نهاد رشاد الطحان, دعاء السيد عبدالرحمن المقدم قسم التغذية وعلوم الأطعمة، کلية الاقتصاد المنزلي، جامعة المنوفية، مصر تم فى هذا البحث دراسة الاغذية عالية الطاقة (الترمس والحمص وبروتين الصويا)المدعمة ببعض انواع من البربيوتک على بعض العوامل البيولوجية والنسيجية في ذکور الفئران الطبيعية الالبينو. وتم استخدام ثلاثين من الفئران البالغة تم تقسيمها إلى ستة مجموعات، المجموعة الاولى مجموعة ضابطة في حين أعطيت 5 مجموعات الاخرى يوميا خليط من (الترمس و الحمص وبروتين الصويا بنسبة 1:1:1) ونسبة من البروبيوتک بنسبة 0.5 , 1% لمدة 6أسابيع، وأظهرت النتائج أن الزيادة في وزن الجسم کان اکثر بشکل ملحوظ خاصة في خليط من( الترمس والحمص وبروتين الصويا و البروبيوتک المضافة) بنسبة 1:1:1:1مقارنة مع المجموعة الضابطه السالبة و أيضا حدث خفض ترکيزات الکوليسترول الکلي والدهون الثلاثية، LDL، VLDL وانخفض نشاط الانزيماتAST و ALT. وعلاوة على ذلک، حدثت زيادة کبيرة في ترکيز HDL- و کذلک زيادة واضحة فى دلالات المناعة خاصة مع المجموعات التى تغذت على البروبيوتک. وأظهر فحص الأنسجة تحسن الخلايا النسجية في کبد الفئران التى تغذت على خليط من الحبوب والبروبيوتک على مختلف المستويات. وتوصي هذه الدراسة أن تناول خليط الترمس والحمص وبروتين الصويا والبروبيوتک وخصوصا بنسبة 1:1:1:1 قد يکون من مفيدا هفى تحسين الهضم وزيادة الوزن بصورة صحية. الکلمات الرئيسية: الفئران الذکور من نوع الالبينو- الترمس- الحمص - بروتين الصويا -البروبيوتک(لاکتوباسليس والبيفيدوبکتريا) والترکيب التشريحي.

The immunopotentiator effects of nefopam

The effects of opiate analgesics and non-steroidal anti-inflammatory drugs on the immune functions have been reported. The effect of the non-opiate analgesic nefopam on the immune functions has not yet been investigated. Male Swiss albino mice were treated with either heat killed E. coli or saline. They were classified into 12 groups. The effects of subacute (15 mg/kg/12 h S.C. daily for one week) and chronic (10 mg/kg/12 h S.C. daily for one month) treatment with nefopam on the levels of interferon-gamma (IFN-γ) and total immunoglobulins were examined in both normal and immunized mice. Also, the effect of the chronic administration of nefopam on the phagocytic activity of peritoneal macrophage was evaluated in both normal and immunized mice. Subacute and chronic administration of nefopam induced no significant raise in the level of interferon-gamma (IFN-γ) or in the level of total immunoglobulins in non-immunized animals, while subacute and chronic treatment with nefopam augmented markedly the immunization induced increase of level of interferon-gamma (IFN-γ). Furthermore, chronic treatment with nefopam potentiated significantly the production of total immunoglobulin induced by heat killed E. coli. Chronic treatment with nefopam also was associated with significant enhancement of innate immune response reflected in the pronounced increase in the phagocytic activity of macrophages in non-immunized and immunized animals. The enhancement of phagocytic activity of macrophages by nefopam in immunized animals was significantly higher than that of non-immunized animals. These findings revealed that nefopam has the ability to trigger the immune response for bacterial antigen. The mechanism behind the immunostimulatory effect of nefopam requires further investigation, but it may be due, at least in part, to the inhibitory effect of nefopam on the serotonin and norepinephrine reuptake at nerve endings. In conclusion, our findings postulated that nefopam stimulated the immune functions and improved the defence mechanism. This information may be of future therapeutic value in diseases that need immunologic enhancement.

Generation and functional analysis of an octameric C3d construct to act as a molecular adjuvant in vaccine design

This thesis investigated two separate theoretical advancements for vaccine design: (1) present antigen in a self-forming natural C4b binding protein-based octameric array (to engage multiple adjacent B-cell receptors or BCR) and (2) combined with fusion to a molecular adjuvant C3d or C3dg, to cross-link C3d-receptor (complement receptor 2 (CR2) CD21). Initially, yellow fluorescent protein test antigen was evaluated, but only to monitor intracellular and extracellular protein levels for a range of C3 forms: C3dg (smallest covalently bound CR1/factor I processed form of activated C3) or C3d (non-complement enzyme processed C3), as well as consequence of mutating the internal thioester bond (C988S, 'C3dsg7 C3ds) that may confound recombinant production of C3dg/C3d fragments. The test antigen was then switched to hen egg lysozyme (HEL), with the hope that spider-like HEL octamer arrays would be expressed with the C3dg/C3d fused proteins at the outermost reach. Mutating the thioester bond increased expression for both C3 forms, and C3dsg-fusion octamers expressed at the highest levels, although C3ds had the highest binding for human CR2. Receptor binding was considered more important for vaccine function, leading to the prototype vaccine, hC3ds-HEL-Oct. Further dissection of the CR2-C3d interaction utilised cell-based assays and surface plasmon resonance (SPR) analysis, using dimeric immunoglobulin Fc-fusion recombinant forms of human C3d, C3dg and murine C3d. All C3dg/C3d Fc proteins bound strongly, although cross-species murine C3d Fc binding to human CR2 was 2-3 times higher than human counterparts. In vivo production of total immunoglobulin G (IgG) and IgM levels was measured, following injection of hC3ds-HEL-Oct, either as DNA or purified protein, and showed successful initiation of a HEL-specific immune response. Data suggested that CpG sequences may have a detrimental impact on vaccine function. These results highlight the power and flexibility of the octamer approach as well as C3d and their applications in vivo and in vitro.

Immunologic rebound.

Evidence for accelerated specific antibody (Ab) rebound or overshoot after single or multiple therapeutic plasmapheresis (TP) is fragmentary but suggested by both clinical and experimental evidence. In vitro studies showing increased peripheral blood lymphocyte turnover and total immunoglobulin production after a series of TP without immunosuppression may signify a generalized immunostimulation through removal of regulatory molecules by TP. It is known that IgG class Ab can down regulate B cells by cross linkage of their Ag and Fc receptors. Cyclophosphamide and other cytotoxic immunosuppressive agents effectively delete proliferating lymphocytes. TP could particularly foster deletion of lymphocytes actively mediating autoimmunity, since they would be more readily stimulated to proliferation by removal of Ab or other inhibitory factors than the generally resting normal immune system. This is supported by a relatively greater reduction of autoantibody levels than total immunoglobulin after treatment with TP and cytotoxic immunosuppressives.

Studies of immunomodulating actions of carotenoids. II. Astaxanthin enhances in vitro antibody production to T-dependent antigens without facilitating polyclonal B-cell activation.

Previously we have shown that astaxanthin, a carotenoid without provitamin A activity, enhances in vitro antibody (Ab) production to sheep red blood cells in normal B6 mice. In this study, we further attempted to examine the mechanisms of this enhancing action of carotenoids on specific Ab production in vitro in relation to different antigen (Ag) stimuli, cytokine production, and T- and B-cell interactions in both normal and autoimmune strains of mice. When the actions of carotenoids were tested in normal strains of mice, we found that astaxanthin enhanced in vitro Ab production to T cell-dependent Ag, but not to T-independent Ag, and did not augment total immunoglobulin production. Astaxanthin exerted maximum enhancing actions when it was present at the initial period of Ag priming. This action of astaxanthin was abolished when T cells were depleted from spleen cell suspensions and appeared to require direct interactions between T and B cells. The results also indicated that carotenoids may modulate the production of interferon-tau in this assay system. When the actions of carotenoids were tested in autoimmune-prone MRL and NZB mice, the enhancing action of astaxanthin on in vitro Ab production was less significant. Furthermore, carotenoids did not potentiate or augment spontaneous Ab and immunoglobulin production by spleen cells in these strains. Taken together, carotenoids without provitamin A activity may be able to augment in vitro specific Ab production to T cell-dependent Ag partly through affecting the initial stage of Ag presentation without facilitating polyclonal B-cell activation or autoantibody production.

Specific suppression of anti-DNA productionin vitro

To investigate the regulation of anti-DNA antibody production, we generated anti-DNA-specific suppressor cells by exposing normal human T cells and a small percentage of adherent cells to high concentrations of DNA. These cells suppressed the production of anti-DNA by both autologous peripheral blood mononuclear cells (PBMC) and allogeneic PBMC derived from systemic lupus erythematosus (SLE) patients. Anti-DNA production was suppressed significantly more than anti-RNA, antitetanus, or total immunoglobulin production. Specific suppression was enhanced by increasing the numbers of DNA-primed CD8+ cells and was obliterated by irradiation of the DNA-primed cells. In contrast to T cells from normal individuals, T cells obtained from two intensively studied SLE patients were unable to generate specific suppressor cells for anti-DNA production in both autologous and allogeneic test systems. Despite this defect, these patients were still capable of generating specific suppressor cells for antibody production directed against an exogenous antigen, tetanus toxoid.

The rate of Wallerian degeneration in the absence of immunoglobulins. A study in chick and mouse peripheral nerve

Sciatic nerves undergoing Wallerian degeneration are subject to massive invasion by monocytes bearing Fc receptors. The present experiments were done to explore the potential role of the Fc receptors. The effect of suppressed immunoglobulin levels on the rate of Wallerian degeneration was studied in the n. medioulnaris of the chick and in murine sciatic nerve. In the avian model, total immunoglobulin production was eliminated by bursectomy. The mice were injected after birth with antibodies against IgM, causing a selective reduction of immunoglobulin M levels. Myelin degeneration was measured in both groups by computer-assisted line sampling and corrected for infrafascicular edema. In both models, there were only minor differences with the controls. Immunoglobulins, accordingly, do not seem to play a significant role in the efficiency of myelin phagocytosis by monocytes during Wallerian degeneration. The study also includes data on the degree and the distribution of edema in nerves undergoing Wallerian degeneration.

Polyclonal activation of salmonid B lymphocytes

The process of in vitro polyclonal activation of coho salmon ( Oncorhynchus kisutch ) lymphocytes was examined with respect to the induction of mitogenesis, total immunoglobulin production, and the production of specific antibodies or plaque forming cells. These studies demonstrate that antigen specific stimulation of antibody production is not linked to mitogenic activity, or total immunoglobulin production, while the polyclonal activation of specific antibody production is closely linked to these functions. Stimulation of immunoglobulin production by phytohemagglutinin suggests that this mitogen may not be limited to T cell activation in salmonids or, alternatively, it may induce the production of lymphokines capable of polyclonally activating B cells. Further, fetal calf serum was found to cause production of large amounts of immunoglobulin in without antigenic stimulation.

Intrahepatic, nucleocapsid antigen-specific T cells in chronic active hepatitis B.

Hepatitis B core antigen (HBcAg)-specific T cell lines were established from hepatic lymphomononuclear cells derived from five patients with chronic active hepatitis B. No hepatitis B virus envelope antigen-specific cell lines were established. Proliferation in response to recombinant and native HBcAg, but not to native hepatitis B surface antigen containing the pre-S(2) region, confirmed the specificity of the five T cell lines. All cell lines represented mixed populations of CD4+ and CD8+ T cells. The CD4+ subset provided antigen-specific help to autologous B cells with respect to anti-HBc production and to CD8+ cells with regard to HBcAg-induced proliferation and suppressor activity. The CD8+ subset contained suppressor cells that selectively inhibited the proliferative response of autologous HBcAg-specific CD4+ cells without inhibiting CD4+ cells of unrelated specificity (tetanus toxoid). Moreover, the CD8+ cells were also capable of suppressing HBcAg-stimulated antibody to HBcAg production without showing inhibition of total immunoglobulin production stimulated by pokeweed mitogen. The cytotoxic potential of the T cell lines was established in a lectin-dependent cytotoxicity system; natural killer cytotoxicity was completely absent. Our data suggest that the lesional T cells present at the site of hepatocellular injury in chronic active hepatitis B are primarily HBcAg-specific lymphocytes of the helper and suppressor/cytotoxic phenotypes and that both are functionally competent.

Inhibition of in vitro anti-DNA B-cell responses by cyclosporine

The action of the immunosuppressive agent cyclosporine (CsA) on anti-DNA B-cell responses was investigated in an in vitro system. Spleen cells from autoimmune MRL- lpr lpr or control BALB/c mice, when cultured at high cell density, spontaneously produced significant amounts of IgM and IgG, including anti-DNA. IgG levels, both total and anti-DNA, were much higher for MRL- lpr lpr cells compared to BALB/c cells, suggesting similarity of this model system to the in vivo response. For cells of both strains, the production of IgM and IgG anti-DNA was 10- to 100-fold more sensitive to the inhibitory activity of CsA than total immunoglobulin production. The effect was not manifest, however, in cultures stimulated with the B-cell mitogen, lipopolysaccharide. These observations suggest that CsA in certain dose ranges preferentially inhibits anti-DNA production, with efficacy determined by the mechanisms promoting B-cell hyperactivity.

Spontaneous and egg antigen-induced syntheses of immunoglobulin and antibody by spleen cells and hepatic granulomas of mice infected with Schistosoma japonicum.

In the absence of egg antigen (SEA), spleen cells from C57BL/6 mice infected for 22 weeks with Schistosoma japonicum were spontaneously induced in vitro to synthesize total immunoglobulin and antibody to SEA. Hepatic granulomas from mice infected for 12 weeks, but not for 22 weeks, also showed spontaneous syntheses of total immunoglobulin and antibody to SEA, but these syntheses were not enhanced upon the addition of SEA. Total immunoglobulin production was enhanced when SEA was added to spleen cells from mice infected for 4 to 7 weeks but not at any other time point.

In Vitro Humoral Immune Response of Human Peripheral Blood Lymphocytes to Tetanus Toxoid Sepharose 4B

The in vitro immune response of unfractionated human peripheral blood lymphocytes (PBL) from immune donors who had not been re-immunized with tetanus toxoid (TT) prior to donation was investigated. In this study we were able to stimulate PBL with tetanus toxoid coupled to Sepharose 4B (STT) for production of anti-tetanus toxoid antibody (Ab). Soluble tetanus toxoid or STT alone did not stimulate production of specific Ab. Pokeweed mitogen (PWM) and STT were required for optimal production of IgG and IgM antibodies specific to tetanus toxoid. Specific Ab responses were reduced in low and high concentrations of STT. Depletion of monocytes had no effect on either total IgG or specific IgG synthesis, but decreased the synthesis of both total and specific IgM. Depletion of E-rosette-forming cells decreased the production of specific Ab, suggesting T-dependency of the immune response to STT. Simultaneous production of total immunoglobulin and specific Ab by Sepharose 4B was negligible in the absence of PWM. In the presence of PWM, total immunoglobulin production was optimal, and specific anti-TT Ab production was undetectable. The specificity of the antiTT Ab was studied by absorption of the culture supernates with an STT column which removed all measurable specific Ab.

Use of plaque assays to study thyroglobulin autoantibody synthesis by human peripheral blood lymphocytes.

Plaque assays have been used to study thyroglobulin autoantibody synthesis and total immunoglobulin production by cultures of peripheral blood lymphocytes from patients with Hashimoto's thyroiditis. Freshly isolated Hashimoto or normal lymphocytes contained small numbers (7-1300) of total IgG plaque-forming cells (PFC) and total IgM PFC, but specific thyroglobulin antibody PFC was undetectable. After 5-8 days' culture with pokeweek mitogen (PWM), total IgG and IgM PFC were markedly increased to a geometric mean of 10,140 (8414-12,220) IgG PFC and 3450 (163-7534) IgM PFC per 10(6) cultured lymphocytes (95% confidence limits in parentheses). Furthermore, a mean of 63 (22-287) specific IgG thyroglobulin antibody PFC per 10(6) lymphocytes were detectable in cultures of Hashimoto lymphocytes. The IgG thyroglobulin antibody PFC were stimulated by Epstein-Barr virus (EBV) infection, suggesting that EBV infection may be useful in obtaining monoclonal thyroid autoantibodies.