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Aberrant TGFβ signaling is linked to metastasis and tumor immune escape of many cancers including metastatic triple negative breast cancer (mTNBC). Previously, we have found that oncolytic adenoviruses expressing a TGFβ signaling inhibitory protein (sTGFβRIIFc) induced immune activation in a mouse TNBC (4T1) immunocompetent subcutaneous model with intratumoral injection. Systemic administration of adenoviruses can be a superior route to treat mTNBC but faces the challenges of increased toxicity and viral clearance. Thus, we created a liver-de-targeted sTGFβRIIFc- and LyP-1 peptide-expressing adenovirus (mHAdLyp.sT) with enhanced breast cancer cell tropism. Its safety and immune response features were profiled in the 4T1 model. Our data showed that the systemic administration of mHAdLyp.sT resulted in reduced hepatic and systemic toxicity. mHAdLyp.sT was also effective in increasing Th1 cytokines and anti-tumor cell populations by cytokine analysis, spleen/tumor qRT-PCR, and flow cytometry. We further tested the therapeutic effects of mHAdLyp.sT alone and in combination with immune checkpoint inhibitors (ICIs). mHAdLyp.sT alone and with all ICI combinations elicited significant inhibition of lung metastasis by histological analysis. When mHAdLyp.sT was combined with both anti-PD-1 and anti-CTLA-4 antibodies, primary 4T1 tumor growth was also significantly inhibited. We are confident in advancing this new treatment option for mTNBC.

Abstract: Increased tumor infiltrating lymphocytes (TIL) are associated with improved patient responses to immunotherapy. As a result, there is interest in enhancing lymphocyte trafficking particularly to colon cancers since the majority are checkpoint blockade-resistant and microsatellite stable. Here, we demonstrate that activated T-cells (ATC) armed with anti-CD3 x anti-EGFR bispecific antibody increases TIL and mediate anti-tumor cytotoxicity while decreasing tumor cell viability. Furthermore, treatment induces endogenous anti-tumor immunity that resisted tumor rechallenge and increased memory T-cell subsets in the tumor. When combined with targeted tumor expression of the tumor necrosis factor superfamily member LIGHT, activated T-cell proliferation and infiltration were further enhanced, and human colorectal tumor regressions were observed. Our data indicate that tumor-targeted armed bispecific antibody increases TIL trafficking and is a potentially potent strategy that can be paired with combination immunotherapy to battle microsatellite stable colon cancer. Significance: Enhancing trafficking of tumor infiltrating lymphocytes (TILs) to solid tumors has been shown to improve outcomes. Unfortunately, few strategies have been successful in the clinical setting for solid tumors, particularly for “cold” microsatellite stable colon cancers. In order to address this gap in knowledge, this study combined TNFSF14/LIGHT immunomodulation with a bispecific antibody armed with activated T-cells targeted to the tumor. This unique T-cell trafficking strategy successfully generated anti-tumor immunity in a microsatellite stable colon cancer model, stimulated T-cell infiltration, and holds promise as a combination immunotherapy for treating advanced and metastatic colorectal cancer.

LIGHT, also known as tumor-necrosis factor (TNF) superfamily member 14 (TNFSF14), is predominantly expressed on activated immune cells and some tumor cells. LIGHT is a pivotal regulator both for recruiting and activating immune cells in the tumor lesions. In this study, we armed human telomerase reverse transcriptase (TERT) promoter controlled oncolytic adenovirus with LIGHT to generate rAd.Light. rAd.Light effectively transduced both human and mouse breast tumor cell lines in vitro, and expressed LIGHT protein on the surface of tumor cells. Both rAd.Null, and rAd.Light could replicate in human breast cancer cells, and produced cytotoxicity to human and mouse mammary tumor cells. rAd.Light induced apoptosis resulting in tumor cell death. Using a subcutaneous model of 4T1 cells in BALB/c mice, rAd.Light was delivered intratumorally to evaluate the anti-tumor responses. Both rAd.Light and rAd.Null significantly inhibited the tumor growth, but rAd.Light produced much stronger anti-tumor effects. Histopathological analysis showed the infiltration of T lymphocytes in the tumor tissues. rAd.Light also induced stronger cellular apoptosis than rAd.Null in the tumors. Interestingly, on day 15, compared to rAd.Null, there was a significant reduction of Tregs following rAd.Light treatment. rAd.Light significantly increased Th1 cytokine interleukin (IL)-2 expression, and reduced Th2 cytokines expression, such as transforming growth factor ß (TGF-ß) and IL-10 in the tumors. These results suggest rAd.Light induced activation of anti-tumor immune responses. In conclusion, rAd.Light produced anti-tumor effect in a subcutaneous model of breast cancer via inducing tumor apoptosis and evoking strong anti-tumor immune responses. Therefore, rAd.Light has great promise to be developed as an effective therapeutic approach for the treatment of breast cancer.

Out-of-Hospital Cardiac Arrest (OHCA) is a global public health problem. There is inadequate data on OHCA in India. The Warangal Area out-of-hospital Cardiac Arrest Registry (WACAR) was planned to understand OHCA in a regional setting in India.

Purpose: Anaplastic thyroid cancer (ATC) is rare but with poor prognosis. TRAIL can selectively induce apoptosis in cancer cells; however, resistance is quite common. Aim of our study was to evaluate TRAIL-induced apoptosis in ATC-derived cell lines, in vitro and in vivo, and the effect of combination with tyrosine kinase inhibitors (TKIs) selective for BRAF (vemurafenib) or Akt (MK-2206). Methods: Four ATC-derived cell lines were used: C643, CAL62, HTh7, with activating mutation of RAS and copy gain of PI3K (HTh7) and, 8505C with activating mutation of BRAF. Cells were treated with TRAIL alone or in combination with vemurafenib or MK-2206. The pro-apoptotic effect of TRAIL alone or combined with TKIs was, also, evaluated in two mouse xenograft models (HTh7 and 8505C). Results: C643, CAL62, and HTh7 cells were sensitive to TRAIL-induced apoptosis, whereas 8505C cells were resistant. Both in vitro and in vivo vemurafenib was able to increase the TRAIL-induced apoptosis in 8505C cells causing a slower tumor growth in 8505C xenograft compared to placebo, while MK-2206 did not have any additive effect on TRAIL treatment in HTh7 model. Conclusions: TRAIL is a promising therapeutic agent in ATC and in case of resistance vemurafenib may be a valid complementary therapy.

A system of care designed to measure and improve process measures such as symptom recognition, emergency response, and hospital care has the potential to reduce mortality and improve quality of life for patients with ST-elevation myocardial infarction (STEMI).

While expansion of natural Tregs requires OX40‐OX40L interaction, adaptive Treg induction requires TGF‐β and TCR signaling. In our earlier work, we had shown that GM‐CSF treatment of CBA/J mice can suppress ongoing thyroiditis by inducing tolerogenic CD8α−DCs, which helped expand and/or induce CD4+Foxp3+Tregs. To identify the primary cell type that was affected by the GM‐CSF treatment and understand the mechanism by which Tregs were induced, we compared the effect of GM‐CSF on matured spDCs and BMDC precursors in vitro. Matured spDCs exposed to GM‐CSF ex vivo induced only a modest increase in the percentage of Foxp3‐expressing T cells in cocultures. In contrast, BM cells, when cultured in the presence of GM‐CSF, gave rise to a population of CD11c+CD11bHiCD8α−DCs (BMDCs), which were able to expand Foxp3+Tregs upon coculture with CD4+T cells. This contact‐dependent expansion occurred in the absence of TCR stimulation and was abrogated by OX40L blockage. Additionally, the BMDCs secreted high levels of TGF‐β, which was required and sufficient for adaptive differentiation of T cells to Foxp3+Tregs, only upon TCR stimulation. These results strongly suggest that the BMDCs differentiated by GM‐CSF can expand nTregs and induce adaptive Tregs through different mechanisms.

Aim: The aim of the study was to investigate the expression and function of the IG20 gene in thyroid cancer cell survival, proliferation, and apoptosis. METHODS: We determined the expression levels of the major isoforms of IG20 by quantitative RT-PCR in normal and thyroid tumor tissues/cell lines. We evaluated the functional consequence of IG20 knockdown in WRO (follicular carcinoma) and FRO (anaplastic carcinoma) thyroid cancer cell lines by measuring spontaneous, TNF-related apoptosis-inducing ligand (TRAIL), and TNF-induced apoptosis. RESULTS: The IG20 gene expression levels were higher in benign and malignant thyroid tumors and in WRO and FRO cells relative to normal tissues. Predominantly, MADD and DENN-SV isoforms of IG20 gene were expressed. IG20 knockdown resulted in increased spontaneous, TRAIL-, and TNF-induced apoptosis in WRO, but not FRO, cells. FRO cell resistance to apoptosis is likely due to caspase-8 deficiency. CONCLUSION: IG20 knockdown renders WRO cells more susceptible to spontaneous, TRAIL-, and TNF-induced apoptosis and thus demonstrates the prosurvival function of the IG20 gene in thyroid cancer. These observations, combined with overexpression of IG20 noted in thyroid tumor tissues, may suggest a potential role in thyroid cancer survival and growth and indicate that IG20 may be targeted either alone or in conjunction with TRAIL or TNF treatment in certain thyroid cancers.

Dendritic cell (DC)-specific intercellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN) is expressed on the surface of DCs and specialized macrophages and can support T cell proliferation. Antibody-mediated co-ligation of CD3 and ICAM-3, the ligand for both DC-SIGN and leukocyte function-associated antigen-1, leads to T cell activation. Therefore, we tested to see whether DC-SIGN or a splice variant of dendritic cell-specific intercellular cell adhesion molecule-3-grabbing non-integrin (sDC-SIGN) can co-stimulate primary human T cells. The sDC-SIGN lacking the transmembrane domain encoded by exon 3 localizes to the cytoplasm of cells and is not secreted. Both B7 and DC-SIGN co-stimulated phorbol myristate acetate-stimulated CD4+ cells as compared with controls. However, unlike B7, both DC-SIGN and sDC-SIGN failed to co-stimulate CD4+ T cells treated with sub-optimal amounts of anti-CD3 (2 μg ml−1) as defined by a lack of CD69 and CD25 up-regulation, cell division and cytokine secretion. Instead, DC-SIGN, and not sDC-SIGN, induced a small but consistent down-regulation of IL-2 production by these CD4+ T cells. In contrast, DC-SIGN in the presence of 30 μg ml−1 of anti-CD3 modestly up-regulated cytokine production as compared with control. These results suggest that DC-SIGN can differentially modulate T cell stimulation.

The CTLA-4-mediated signal is a critical step in the down-modulation of immune responses. The therapeutic potential of this signal to induce tissue-specific tolerance was investigated by using an anti-CTLA-4 antibody that was coupled to an antibody specific for the thyrotropin receptor. After in vivo administration, this bispecific antibody (BiAb) accumulated in the thyroid and prevented development of experimental autoimmune thyroiditis (EAT) in mice immunized with mouse thyroglobulin (mTg). Lymphocytes from BiAb-treated mice showed a significant reduction in their ability to proliferate, and to produce IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha, in response to mTg re-stimulation compared to lymphocytes from untreated mice. Moreover, BiAb-treated mice showed suppressed anti-mTg antibody response, lymphocytic infiltration of the thyroid and follicular destruction. The BiAb targeted to the thyroid most likely facilitated engagement of CTLA-4, resulting in an increase in the number of CD4(+)CD25(+) T cells. These regulatory T cells suppressed in vitro mTg-specific T cell responses, which were associated with an enhanced transforming growth factor (TGF)-beta1 production. Neutralization of TGF-beta1 increased mTg-specific in vitro proliferation of, and IL-2 production by, T cells from BiAb-treated mice. Our data suggest that engagement of CTLA-4 expressed on activated autoreactive T cells in close proximity to the thyroid can increase the number of regulatory T cells and their ability to produce TGF-beta1, with a concomitant reduction in IFN-gamma and TNF-alpha, resulting in suppression of EAT.

CD80 and CD86 expressed on the surface of antigen-presenting cells interact with cytotoxic T lymphocyte antigen-4 [CTLA-4 (CD152)] expressed on activated T cells and mediate critical T cell inhibitory signals. CD80 and CD86 are type I glycoproteins, and are made up of two extracellular (EC) Ig-like domains-a transmembrane region and a cytoplasmic tail. The N-terminal (V domain) and membrane-proximal (C) domains share homology with the variable region (V) and the constant region (C) of Ig respectively. Co-crystallographic structures of both CD80 and CD86 bound to CTLA-4 indicate that there is no direct interaction of the C domain of either CD80 or CD86 with the CTLA-4. In contrast, previous mutagenesis studies have identified specific amino acids within the C domain of CD80 that are critical for CTLA-4 binding. To further understand the importance of C domains in the functioning of CD80 and CD86, we constructed chimeric human CD80 and CD86 molecules by swapping their respective C domains, and tested their ability to stimulate T cells. A Chinese hamster ovary (CHO) cell line expressing CD86 activated murine T cells. In contrast, CHO cells expressing either CD80 or a chimeric construct of the CD86 V domain and the CD80 C domain showed a significantly reduced activation. Our studies further demonstrated that the decreased activation by cells expressing the CD80 or a chimera containing CD80 C domain is most likely due to enhanced CTLA-4 binding. From these results we conclude that C domains play a critical, albeit indirect, role in determining CTLA-4 binding affinities and co-stimulatory properties.

We identified a novel cDNA (IG20) that is homologous to cDNAs encoding a proteindifferentially expressed in normal and neoplastic cells (DENN-SV) and human MADD (MAPK-activating deathdomain-containing protein). Furthermore, we show that the above variants most likely result from alternative splicing of a single gene. Functional analyses of these variants in permanently transfected HeLa cells revealed that IG20 and DENN-SV render them more susceptible or resistant to tumor necrosis factor α (TNF-α)-induced apoptosis, respectively. All variants tested could interact with TNF receptor 1 and activate ERK and nuclear factor κB. However, relative to control cells, only cells expressing IG20 showed enhanced TNF-α-induced activation of caspase-8 and -3, whereas cells expressing DENN-SV showed either reduced or no caspase activation. Transfection of these cells with a cDNA encoding CrmA maximally inhibited apoptosis in HeLa-IG20 cells. Our results show that IG20 can promote TNF-α-induced apoptosis and activation of caspase-8 and -3 and suggest that it may play a novel role in the regulation of the pleiotropic effects of TNF-α through alternative splicing.

CTLA-4 is a T cell surface molecule that binds to the costimulatory molecules CD80 and CD86 on antigen-presenting cells and downregulates T cell function. Therefore, we wanted to test whether antigen-specific activated T cells could be inhibited through directed CTLA-4 signaling using a bispecific antibody (BiAb) capable of simultaneously binding to CTLA-4 and a tissue-specific antigen. The BiAb was prepared by linking two separate monoclonal antibodies against CTLA-4 and the thyroid-stimulating hormone receptor (TSHR). The mouse B cell lymphoma line M12 (H2d) was used to induce alloreactive T cells in CBA/J mice (H2k); M12 cells stably transfected with the cDNA encoding murine TSHR (mM12) were used to restimulate the alloresponse in vitro.Results of assays for in vitroT cell proliferation, IL-2 production, and cytotoxicity in the presence of BiAb demonstrated that the BiAb could inhibit the T cell alloresponse when stimulated with mM12 cells but not with M12 cells. This effect was dependent on binding of TSHR-bound BiAb to CTLA-4, since the addition of soluble CTLA-4-Ig blocked the inhibitory effect. Injection of mM12 cells, along with the BiAb, not with antibodies against TSHR or CTLA-4 either separately or together, into CBA/J mice (H2k) downregulated alloreactive T cell responses. Our study demonstrated that the presence of CTLA-4 signaling molecules on the surface of target cells can protect those cells from immune attack by antigen-specific T cells and suggested that a similar approach could have potential therapeutic value in transplant rejection and tissue-specific autoimmune diseases.

The mouse and human thyrotropin receptors show greater than 87% homology in their amino acid sequences. However, glycosylated extracellular domains of mouse (mET-gp) and human (hET-gp) thyrotropin receptors showed differences in their ability to react with patient autoantibodies to thyrotropin receptor (TSHR). To test for potential differences in their immunogenicity, we immunized BALB/c mice with either gel pure non-glycosylated ectodomain of human TSHR (ETSHR II), or hET-gp (hET-gp III), or mET-gp (mET-gp III). Alternatively, mice were primed with gel pure hET-gp or mET-gp and subsequently immunized with insect cells expressing hET-gp (hET-gp II) or mET-gp (mET-gp II) respectively. All groups of mice immunized with TSHR developed high titers of antibodies against the respective immunogens. As shown earlier, sera obtained from mice immunized with ETSHR showed strong reactivity to peptide 1 (aa 22–41) and weak reactivity to peptides 23 (aa 352–371), 24 (aa 367–386), 25 (aa 382–401), and 26 (aa 397–415). Mice immunized with hET-gp or mET-gp showed comparable titers to peptides 1 and 23 and lower reactivity to other peptides. Mice immunized with hET-gp showed higher TBII reactivity (52.2%) compared to mice immunized with either ETSHR (20.9%) or mET-gp (34.5%). Peptides from the C-terminal region of ETSHR could neutralize the TBII activities of sera from mice immunized with ETSHR or hET-gp but not mET-gp. Compared to corresponding control mice, T4 levels in mET-gp II mice were only marginally higher. These data suggested that outcome of immunization with mouse ETSHR is comparable to that seen after immunization with human ETSHR.

Although cytokines and other soluble regulators of immunity are known to be involved in hematopoiesis, little is known about the signals that induce the synthesis of those mediators locally. Based on recent studies linking the neuroendocrine hormone thyrotropin [thyroid-stimulating hormone (TSH)] to immune cell function in other tissues, we investigated the capacity of TSH to activate cytokine responses from bone marrow cells. These studies reveal that stimulation of the TSH receptor on bone marrow cells—using highly purified or recombinant TSH or by direct stimulation with anti-TSH receptor antibodies—rapidly induces the synthesis of cytokines from bone marrow cells that are classically used in the regulation of inflammatory responses. Of 13 cytokines screened for activity by ELISA or by RNase protection assays for gene expression, IL-6, IFN-β, TNFα, TNFβ, TGFβ2, and lymphotoxin-β responses were reproducibly induced by TSH within 2–3 h of stimulation. Intracellularly, TSH stimulation of bone marrow cells caused rapid increases in cAMP levels and induced the phosphorylation of the Jak2 protein kinase, thereby defining a novel G-protein-coupled receptor/cytokine synthesis pathway. These findings demonstrate that TSH can serve as a primary inductive signal of cytokine production by bone marrow cells.

ABSTRACTA multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine. The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media. Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result. Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation. The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively. In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively. Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination. In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks. Shedding of type 3 virus ceased in ~70% of vaccinees within a week after immunization. In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Graves' disease (GD) is characterized by the presence of autoantibodies to the thyrotropin receptor (TSHr). These antibodies bind to the TSHr and stimulate thyroid cells, thus causing hyperthyroidism. To understand the regulation of TSHr-specific immune responses in Graves' disease, it is important to evaluate the T-cell response in patients with GD against TSHr. In this study we used 11 different peptides that were derived from two regions (i.e. amino acid, AA 12-46 and 316-397) unique to the TSHr when compared to other glycoprotein hormone receptors, and which also have the highest predicted immunogenicity. We evaluated both lymphocyte proliferation as a measure of T-cell response and antibody binding to each of these peptides in nine patients with GD and eight healthy subjects. Patients with GD showed considerable lymphocyte proliferative and antibody responses against several of these peptides. There was considerable heterogeneity in immune responses amongst the patients. Moreover, our data suggested that several peptides contained both T cell and antibody reactive epitopes and might represent some of the highly immunogenic regions of the TSHr.

Autoantibodies to the thyrotropin receptor (TSHR) have been shown to mediate the hyperthyroidism associated with Graves’ disease (GD). A number of hypotheses have been proposed which link an infectious agent to the mechanism(s) involved in the induction of GD. Several studies have suggested that the development of GD may be linked to infection with the enteric pathogenYersinia enterocolitica. We have recently identified two low molecular weight (5.5 and 8kDa) envelope proteins ofY. enterocoliticathat are cross-reactive with the extracellular domain of human TSHR (ETSHR). In this study, we have purified these ETSHR-crossreactiveYersiniaproteins (TSHR-CRP) and have further characterized their immunoreactivity. Both the 5.5 and 8kDa TSHR-CRPs were shown to be mitogenic for mouse spleen cells. This mitogenic activity was specific for B cells and was not due to lipopolysaccharide (LPS) contamination. TSHR-CRPs were mitogenic for LPS-non-responsive spleen cells obtained from C3H/Hej mice, and polymyxin B did not inhibit the mitogenic activity of the TSHR-CRPs. TSHR-CRPs also induced high levels of IL-6 production in B cells and induced production and secretion of significant levels of IgG and IgM. Finally, culture supernatants from TSHR-CRP-stimulated spleen cells were shown by Western blot analysis to contain antibodies that recognized the ETSHR. These results identify for the first time two envelope proteins ofYersiniathat have mitogenic activity and therefore could represent important proteins involved in the pathogenesis ofYersiniainfections. Because these mitogenic proteins also contain epitopes cross-reactive with the TSHR, they are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the TSHR.

Unstimulated whole saliva was collected from 21 HIV-positive men and women before and after dental treatment. The frequency of HIV detection did not increase after dental treatment. Infectious HIV was recovered from only one patient. Study findings raise the possibility that, in most cases, salivary inhibitors render the virus non-infectious.

Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbant assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1μg/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.

Hyperthyroidism in Graves' disease (GD) is mediated by antibodies to the thyrotropin receptor (TSHr). Patients that go into remission show a decline in antibody titer. However, upon cessation of treatment with anti-thyroid drugs a significant proportion of patients relapse and TSHr antibodies (TSHrAb) are present in their circulation. This suggests that B cells capable of producing TSHrAb persist despite treatment. To determine the frequency of these cells, B cells from six patients with GD and four healthy controls were infected with Epstein-Barr virus and cultured in 96-well plates at varying cell concentrations. A higher frequency of B cells capable of producing TSHrAbs was detected in patients with GD, relative to normal controls. For example, at 2 × 10⁵ cells per well, 100% of wells containing cells from either patients with GD or controls were positive for immunoglobulin (Ig) production. In contrast, 27% of the wells containing cells from Graves' patients, and only 3% from controls, were positive for TSHrAb. Higher titers of TSHrAbs were produced in cultures containing lymphocytes from patients with GD and were predominantly of IgG isotype. All patients with GD who had high thyrotropin binding inhibitory immunoglobulins also had higher frequencies of TSHr-specific B cells. These findings show that TSHrAb-producing B cells are present at a higher frequency in the peripheral circulation of patients with GD. Copyright 1994, 1999 Academic Press

Salivary analysis indicates rates of herpesviruses isolated from HIV-infected patients are no higher than in the general population.

Autoantibodies directed against a wide range of normal tissue antigens have been found in the sera of patients with autoimmune diseases1–8. It is generally thought that different and specific autoantibodies react with different tissues but the possibility exists that some autoantibodies may react with common antigens found in different tissues and organs. Recently, we showed that mice infected with reovirus developed a polyendocrine disease with autoantibodies to the pancreas, anterior pituitary, thymus and gastric mucosa9,10. Using hybridoma technology, we obtained a number of monoclonal autoantibodies11which reacted with antigens in single organs. We now report the production and pattern of reactivity of seven multiple organ-reactive monoclonal autoantibodies. By using antibody-affinity columns, autoantigens also have been isloated and their molecular weights determined. The results suggest that monoclonal multiple organ-reactive autoantibodies react either with the same molecule present in several organs or with common antigenic determinants on different molecules in multiple organs. In either case, the existence of multiple organ-reactive antibodies may be a partial explanation for multiple organ autoimmunity.

The expression and functional relevance of thyroid stimulating hormone (TSH) receptors on human immune cells were studied. Flow cytometric analysis was used to study the binding of biotinylated TSH to human peripheral blood mononuclear cells (PBMC) and various purified lymphoid populations. Our results indicate that the hormone binds well to monocytes and natural killer (NK) cells and marginally to purified tonsillar T and B lymphocytes. There was a significant increase in the binding of TSH to purified B cells that were activatedin vitro withStaphylococcus aureaus Cowan. In contrast, the binding of TSH to T cells was unaltered when they were stimulated with phytohemagglutinin (PHA). While TSH increases DNA synthesis and intracellular cAMP levels of FRTL-5 rat thyroid cells, it did not have such stimulatory effects on lymphocytes. However, there was a moderate increase in Ig production by activated B lymphocytes when they were cultured in the presence of the hormone. A possible function for TSH as a link between the immune system and the thyroid is discussed.

Autoantibodies to the thyrotropin receptor (TSHr) bind to the extracellular domain of the TSHr (ETSHr) and either stimulate of inhibit thyroid cell function and/or growth. In order to investigate the regulation and the specificity of the immune response to the TSHr, our laboratory recently produced recombinant human ETSHr protein by using the baculovirus expression system. In the present study, we used the recombinant ETSHr protein, a panel of overlapping synthetic peptides derived from the TSHr, and polyclonal rabbit antibodies produced against recombinant ETSHr and synthetic peptides to define a highly immunogenic region (aa 352-388) of the TSHr. Moreover, we used competitive inhibition studies to identify a dominant epitope (aa 367-372) within this region to which ETSHr antibodies react. This immunodominant epitope lies within a region unique to the TSHr when compared to the other glycoprotein hormone receptors. These data, together with the earlier observation that antibodies against aa region 357-372 can inhibit thyrotropin (TSH)-mediated activation of thyroid cells in culture, show that aa 367-372 represents, an immunodominant epitope within a functionally important region which is unique to the TSHr. Therefore, this region may play an important role in the induction or modulation of the specific immune response against the TSHr. Copyright 1994, 1999 Academic Press

Unstimulated whole saliva was collected from 21 HIV-positive men and women before and after dental treatment. The frequency of HIV detection did not increase after dental treatment. Infectious HIV was recovered from only one patient. Study findings raise the possibility that, in most cases, salivary inhibitors render the virus non-infectious.

Human peripheral B lymphocytes were transformed with Epstein-Barr virus and probed with an antiidiotypic antibody made against a human monoclonal autoantibody. Six cell lines were isolated that produced antibodies bearing a common idiotope. Despite the presence of this common idiotope, the antibodies showed antigen-binding specificities different from that of the parental antibody to which the antiidiotypic antibody was made. By probing Epstein-Barr virus-transformed cells with well-characterized antiidiotypic antibodies, it should now be possible to isolate and determine the frequency of B cells bearing specific idiotopes in the human repertoire and to study the antigen-binding properties of these antibodies.

The Coxsackie B virus group consists of six serotypes (B1–B6) that are antigenically distinct. In patients, the Coxsackie B4 serotype can produce a variety of clinical diseases (for example, pleurodynia, respiratory illness, meningitis, myocarditis, orchitis and diabetes)1,2. It is not known, however, whether the different clinical diseases are due to chance infection of particular organs by Coxsackie B4 or due to antigenic variants of Coxsackie B4 that are inherently different in their tissue tropism. The long-standing reference hyperimmune sera do not identify variants that may exist within each of the serotypes3. In an attempt to identify variants of the Coxsackie B4 serotype, we prepared a panel of 18 monoclonal antibodies that neutralized the prototype Coxsackie B4 strain. These monoclonal antibodies have been characterized as to their subclass, neutralization titres and reactivity patterns with recent clinical isolates. Here we have identified over 13 variants of CoxsacKie B4 virus and have shown that there are major antigenic differences among naturally occurring isolates. Using monoclonal antibodies as a selecting agent, we find that the frequency of antigenic mutation may be as high as 10−4.

Among inadequately immunized laboratory animals dying of rabies, a small but consistent proportion succumb after an incubation period shorter than that of any of the unvaccinated controls1–3,7and this phenomenon has been termed ‘early death’. It has also been shown that, after immunosuppression either with cyclophosphamide or by thymectomy and irradiation4, rabies-infected animals survive longer with decreased incidence of paralysis. It appears that ‘early death’ also occurs in humans who have been treated (unsuccessfully) with vaccine, with or without accompanying serum therapy5–7, after exposure to rabies. Collectively, these studies suggest that there is a immunopathological component in rabies virus infection. We have therefore investigated a model of lethal rabies infection in immunosuppressed mice and have concluded that B lymphocytes or antibody specific for rabies play a part in the causation of early death. Accordingly the immune response to rabies has a dual role8, sometimes favouring survival but sometimes enhancing the disease.

Peripheral blood lymphocytes from 29 patients with autoimmune diseases and eight normal controls were cultured in vitro. Several weeks later, lymphocyte proliferation was observed in many of these cultures. Cell lines were obtained from about half of the donors, at a frequency of ∼1 in 107 lymphocytes. Outgrowth occurred only in cultures from patients who had antibodies to the viral capsid antigen of Epstein-Barr virus (EBV). The proliferating cells also contained EBV nuclear antigen, an occurrence confirming that proliferation was a result of latent EBV infection of the donors. Supernatants from these cell lines were tested for autoantibodies by screening against normal tissues. About 30% of the cell lines made autoantibodies, many of which reacted with smooth muscle or epithelial cells. Because EBV is harbored in a latent form in the majority of the adult population, reactivation of latent EBV infection in vivo may explain the production of autoantibodies seen in a variety of immunoregulatory disorders.