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Biomedical subjects

R Pujol-Borrell

Publications and source records attributed to R Pujol-Borrell.

At least 55 records · Page 3Linked to original sources
PubMed1991

Cytotoxic effect of IFN-gamma plus TNF-alpha on human islet cells.

We have previously reported that the combination of IFN-gamma plus TNF-alpha is able to induce the de novo expression of HLA class II on human beta cells. In the present study, we have investigated the effect of these cytokines, alone or in combination, on the function and viability of human islet cells in vitro. Three hour insulin release was markedly reduced in human islet monolayer cultures after 4 days' exposure to 1000 U/ml of the combination TNF-alpha plus IFN-gamma (36.7 +/- 7.7, % of the control +/- SEM) or to TNF-alpha alone (49.5 +/- 7% of the control) while IFN-gamma had little effect. On direct inspection cell damage was clearly detected only in the cultures treated with TNF-alpha plus IFN-gamma in which staining by indirect immunofluorescence (IFL) for insulin revealed that the number of beta cells was also significantly reduced, thus suggesting a real cytotoxic effect of this cytokine combination. This effect was not beta cell specific since glucagon release and the number of alpha cells were also reduced in the cultures exposed to IFN-gamma plus TNF-alpha. 51Cr release experiments supported the cytoxicity of these cytokines to normal islet cells. There was a time course relationship between class II induction (2 days) and the cytotoxic effect of IFN-gamma plus TNF-alpha (4 days) on the same islet cells. In conclusion, these results indicate that the combination of IFN-gamma and TNF-alpha exerts a cytotoxic effect on human islet cells in vitro.

Cell Survival↗
PubMed1991

Transfection with SV40 gene of human pancreatic endocrine cells.

At present, only islet cell lines of animal origin have been successfully generated (e.g. RIN, HIT). A fully differentiated human beta cell line would be advantageous for diabetes research. We now report the generation of a human endocrine pancreatic cell line obtained by transfection using a plasmid containing the early region of SV40 viral DNA. Viral integration and transcription was assessed by Southern and Northern blotting. This cell line has been growing continuously for more than 2 years and maintains several of the characteristics of the parental cells from which they were generated. The presence of Neuron Specific Enolase, Protein Gene Product 9.5, cytokeratin, microvilli, cytoplasmic electrodense granules and the secretion of insulin, glucagon and somatostatin supports the neuroendocrine origin of this cell line. However, hormone production progressively decreased and finally stopped at passage 8. Flow cytometric analysis showed that HLA expression in this cell line is readily induced by IFN-gamma and modulated by TNF-alpha. The establishment of this human endocrine cell line indicates the feasibility of immortalizing human islets by transfection with viral oncogenes. To obtain a fully differentiated cell line it may be necessary to use other DNA constructs which immortalize the cells without fully transforming their phenotype.

Antigens, Viral, Tumor↗
PubMed1991

De novo HLA class II and enhanced HLA class I molecule expression in SV40 transfected human thyroid epithelial cells.

Human thyroid follicular cells normally synthesize and express HLA Class I molecules but in glands affected by autoimmune or neoplastic diseases they express Class II molecules. We have investigated the effect of SV40 virus transformation on the expression of MHC molecules in human thyrocytes. Primary cultures of human thyroid from two different glands were transfected with the plasmid pX-8, containing the 'early' region of SV40 virus, and two continuous lines of thyrocytes were obtained. The cell lines maintained features of parental thyroid epithelial cells showing the characteristic cytokeratin filament network, microvillar protrusion and tight junctions. In addition, the SV40-transfected cells responded to graded doses of TSH with increased c-AMP production. Thyrocytes from both cell lines hyperexpressed Class I molecules and a significant proportion of them also acquired constitutive Class II expression, as determined by indirect immunofluorescence (IFL), flow cytometry and Northern blotting hybridization using a DR beta probe. These cells were found to be normally up-regulated by interferon (IFN)-gamma. Indirect IFL and flow cytometry analysis were used to detect and quantify the expression of HLA-DR, DP and DQ subregions. A co-ordinated expression (DR greater than DP much greater than DQ), reminiscent of the inappropriate HLA expression found in thyroid autoimmune disease in vivo and of the in vitro regulation in normal thyrocytes, was observed. Clones derived from these cell lines differed in their level of constitutive Class II expression and in their sensitivity to Class II induction by IFN-gamma. In conclusion, these thyroid cell lines could provide a useful tool for further investigation of HLA gene regulation in thyroid cells and for elucidating on the mechanism involved in the inappropriate HLA expression described in autoimmune and neoplastic diseases of the thyroid.

Autoantigens↗
PubMed1991

Adhesion molecules in human islet beta-cells. De novo induction of ICAM-1 but not LFA-3.

Understanding how T lymphocytes recognize beta-cell autoantigens is essential for the elucidation of the pathogenesis of insulin-dependent diabetes mellitus. The increased and ectopic expression of HLA class I and II molecules detected in human beta-cells may facilitate this interaction. T-lymphocyte recognition of surface antigens also involves adhesion accessory molecules: intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 3 (LFA-3). These molecules not only allow cell contact but can also provide costimulatory signals for T-lymphocyte activation. Levels of ICAM-1 and LFA-3 expression in normal human islet cells and regulation of their expression by cytokines interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and IL-6 have been studied by two-color immunofluorescence staining of pancreatic cryostat sections and fluorescence-activated cell sorter analysis. Neither ICAM-1 nor LFA-3 could be demonstrated in sections or in fresh cell preparations, but after 18 h of culture, beta-, alpha-, and delta-cells expressed spontaneously moderate levels of ICAM-1 (but not LFA-3). IFN-gamma and TNF-alpha alone or in combination strongly enhanced this spontaneous expression of ICAM-1 in a time- and/or dose-dependent and additive manner but had no effect on LFA-3. An SV40-transformed islet cell line showed high basal levels of both ICAM-1 and LFA-3, but the response to cytokines followed the same pattern as primary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Surface↗
PubMed1990

HLA DR, DP, DQ induction in human islet beta cells by the cytokine combination IFN-gamma + TNF-alpha.

Human islet beta cells do not express HLA Class II normally, yet, in the diabetic pancreas, beta cells are selectively positive for Class II and this may facilitate their recognition by T cells. It has been demonstrated that human beta cells can be induced to express Class II when cultured with IFN-gamma + TNF-alpha or IFN-gamma + TNF-beta. To assess whether or not they can be induced to express the products of the Class II subregions, DR, DP and DQ, human islet cultures from 10 pancreas were supplemented with the combination of IFN-gamma + TNF-alpha using MoAbs specific for DR, DP and DQ products, and antibodies to insulin and glucagon. The combination IFN-gamma + TNF-alpha (100-1000 U/ml each) was able to induce the expression of the three subregions in both beta and alpha cells. The induction of subregion expression followed the hierarchy DR greater than DQ greater than or equal to DP. The capability of beta cells to express all three Class II subregions supports the possibility that these cells can present their self antigens to T cells.

Gene Expression Regulation↗
PubMed1989

Retrovirus-like sequences in Graves' disease: implications for human autoimmunity.

On Southern blotting of DNA extracted from thyroid glands of five patients with Graves' disease, two probes (720 bp and 942 bp) for gag human immunodeficiency virus type 1 (HIV-1) gave a positive hybridisation signal in all samples tested. DNAs from peripheral blood mononuclear cells hybridised with the 720 bp gag HIV-1 probe in three of the five patients, none of whom had antibodies to HIV-1. Negative results were obtained with DNA from normal thyroid glands, thyroid neoplasms, various unrelated normal tissues, and virus-infected human cell lines. The intensity of the signal and the pattern of bands observed with the DNA of Graves' patients were heterogeneous and, in general, were not the same in the thyroid glands and peripheral blood mononuclear cells of individual patients. Similarly, no correlation was found between the positive hybridisation signals and other genetic and immunological indices or the duration of anti-thyroid drug treatment at the time the patients were investigated. The findings suggest the presence of a novel retrovirus, and the retrovirus-like sequences seem to be closely associated with thyroid autoimmunity.

Adolescent↗
PubMed1989

Inappropriate expression of HLA class II molecules in endocrine epithelial cells: the phenomenon, the new experimental data and comparison with animal models.

Physiologically, HLA Class II molecules are primarily expressed on immunoregulatory cells. In recent years, it has been shown that tissues affected by autoimmunity, including beta cells of 'diabetic' pancreases, 'inappropriately' express these glycoproteins. Cytokines are potent Class II inducers on a variety of cells but they exert a heterogenous effect when incubated with different human endocrine cells, i.e. thyroid cells are readily inducible, beta cells are much more resistant. The fine modulation of Class II expression by cytokines has been extensively studied and recent information on their action on endocrine cells is reported here. The possibility that distinct environmental factors from those postulated until now may be responsible for triggering the inappropriate expression of MHC molecules in epithelial cells in vivo is also emphasized. Despite several similarities between human Type I diabetes and spontaneous animal models of the disease, major differences still exist. Transgenic models have now been produced. However, even if they offer interesting new insights, they have not so far provided the decisive answer to explain the pathogenesis of human autoimmune diseases.

Animals↗
PubMed1989

Pathogenesis of type I (insulin-dependent) diabetes: possible mechanisms of autoimmune damage.

By definition, autoimmunity is a circumvention of self-tolerance. Considering the complex network of control mechanisms which initiate and regulate immune responses to foreign agents and maintain the natural state of self-tolerance, autoimmunity may result from a weakening of this delicate equilibrium at various levels. Several hypotheses have therefore been put forward to explain the 'active' immunization process against self-constituents which are normally tolerated.

Autoantigens↗
PubMed1989

Influence of tumor necrosis factor-alpha on the modulation by interferon-gamma of HLA class II molecules in human thyroid cells and its effect on interferon-gamma binding.

Cytokines are important modulators of immunological reactions, but it has been postulated that they might act on other unrelated epithelial cells. We studied the effects of recombinant interferon-gamma (rIFN gamma) and recombinant tumor necrosis factor-alpha (rTNF alpha) on normal human thyroid cells. We found that the combination of these two cytokines enhanced HLA class II molecule expression on these cells compared with the effect of rIFN gamma alone. This was proven by both immunofluorescence as well as a more sensitive and quantitative RIA. rTNF alpha alone had no effect on HLA class II molecule induction on the same thyrocytes, suggesting a synergistic rather than an additive action in combination with rIFN gamma. The addition of 600 U/ml rTNF alpha to low dose rIFN gamma (10 U/mL) enhanced class II expression by 50%, as quantified by RIA. We also demonstrated that normal thyrocytes possess distinct receptors for the two cytokines and that rTNF alpha probably augments IFN gamma binding, since it increased when the cells were first incubated with rTNF alpha. This increased binding provides an explanation for the synergistic action of rTNF alpha in enhancing class II molecule expression by rIFN gamma. We conclude that the presence of receptors for these cytokines on human thyroid cells gives a direct demonstration of their potential biological action on cells normally not involved in the immunological circuit. The phenomenon might also explain their direct or indirect involvement in vivo, such as in influencing inappropriate HLA class II molecule expression in epithelial cells affected by autoimmunity.

Binding Sites↗
PubMed1989

Correlation between residual beta-cell function and islet cell antibodies in newly diagnosed type I diabetes. Follow-up study.

To establish whether there is a correlation between the autoimmune response to the islets and beta-cell function during the initial stages of type I (insulin-dependent) diabetes, and islet cell antibody (ICA) titer and C-peptide levels (fasting and glucagon stimulated) were determined in 39 newly diagnosed patients at onset of diabetes and every 3-6 mo for 2 yr. ICAs were detected in 74% of the patients, and beta-cell function was detected in 84% of the patients at onset. The ICA+ and ICA- groups had similar C-peptide values at diagnosis and at 3 mo, but from 6 mo on, the ICA+ group consistently showed a tendency to lose C-peptide secretory capacity more quickly when assessed by fasting and glucagon-stimulated C-peptide levels (ICA+ vs. ICA- fasting C-peptide levels at 18 and 24 mo, P = .013 and .017, respectively; ICA+ vs. ICA- glucagon-stimulated C-peptide levels at 6, 18, and 24 mo, P = .023, .007, and .028, respectively). The initial ICA titer had the highest predictive value on the outcome of beta-cell function (P = .04), and patients with complement-fixing ICAs did not behave differently from the general ICA+ group. This correlation between beta-cell function and ICA titer supports the role of autoimmunity in the pathogenesis of type I diabetes and has important implications for the design of immunotherapy trials.

Adolescent↗
PubMed1988

Differential expression and regulation of major histocompatibility complex (MHC) products in neural and glial cells of the human fetal brain.

The cells of the central nervous system (CNS) have the peculiarity of physiologically expressing very low levels of HLA molecules. In multiple sclerosis (MS), however, as in endocrine autoimmune diseases, there is a marked increase of HLA expression in the tissue (i.e. the plaques) and this is attributable not only to infiltrating cells but also to the astrocytes. To gain an insight into the regulation of HLA in the different cell types in the CNS and to compare it to that observed in the endocrine organs, we have studied the effect of the lympho/monokines interferon (IFN)-alpha and -gamma, tumour necrosis factor (TNF)-alpha, and interleukin (IL)-2 and other agents on this aspect of the biology of human fetal brain cells in culture. A two-colour immunofluorescence technique which combines antibodies to diverse CNS cell markers and monoclonal antibodies (MoAbs) to the non-polymorphic region of HLA molecules was used throughout this study. In control cultures, only astrocytes expressed MHC class I, but after incubation with either IFN-gamma or TNF-alpha oligodendrocytes acquired class I expression. Surprisingly, astrocytes became spontaneously class II positive in culture and this was greatly enhanced by IFN-gamma. Other agents such as IL-2, epidermal growth factor, phorbolmyristate acetate and lectins had no effect. The expression of HLA molecules in the cells of the CNS both in basal conditions and in response to lymphokines is therefore selective and highly heterogenous, thus reflecting their intrinsic biological diversity. These findings may help to explain the features of the immunopathology of MS and also of latent viral infections of neural cells.

Astrocytes↗
PubMed1988

Occurrence of thyrocyte HLA class II expression in a wide variety of thyroid diseases: relationship with lymphocytic infiltration and thyroid autoantibodies.

The proteins of the major histocompatibility system (HLA in humans) play an essential role in the regulation of immune responses due to their involvement in the presentation of antigen to T lymphocytes. Thyroid follicular cells (thyrocytes) from patients with Graves' disease and Hashimoto's thyroiditis demonstrate increased expression of HLA class I and aberrantly or inappropriately express class II antigens, a phenomenon that may play an important role in the pathogenesis of these autoimmune diseases. To establish if these changes in the expression of HLA molecules are characteristic of thyroid autoimmune disease, the immunopathological features (including class I and class II antigen expression) of 100 thyroidectomy specimens from patients with nonautoimmune thyroid disease were studied by indirect immunofluorescence, and the results compared with the findings in specimens from 14 patients with Graves' disease and 12 subjects undergoing laryngectomies for carcinoma. Increased class I product expression was found in 61% of all tissues studied, with maximal occurrence in papillary carcinomas (100%) and Graves' disease (86%), but it was also detected in 50% of the glands containing nodular lesions and in 16% of the control glands. Inappropriate class II molecule expression was found in Graves' disease (71%), hyperplastic nodules (53%), multinodular glands (44%), papillary carcinomas (38%), and 16% of the control glands. In summary, an increase in inappropriate HLA class I and class II expression was very common in nonautoimmune thyroid glands, but it generally occurred in the context of lymphocytic infiltration and thyroid autoantibodies (i.e. focal thyroiditis). Multiple correlation analyses of these 4 phenomena indicated heterogeneity in the mechanism leading to the inappropriate expression of thyrocyte class II antigens in the different conditions studied.

Adolescent↗
PubMed1988

Presence of insulin autoantibodies at clinical diagnosis of diabetes mellitus type I predicts loss of beta cell function.

Recently the spontaneous development of insulin autoantibodies (IAA) has been detected in patients at diagnosis of Type I diabetes mellitus before the beginning of insulin treatment. The present study was undertaken to investigate if the presence of IAA at clinical onset of IDDM may act as a new marker of the beta cell function. The results obtained showed that IAA were present in 44% of newly diagnosed diabetic patients before therapy. Patients without IAA displayed a higher C-peptide secretion than those with IAA, at six months (12.11 +/- 5.08 versus 5.88 +/- 3.25 ng/ml/10 min.)(X +/- SD) and at twelve months (10.45 +/- 3.05 versus 4.90 +/- 5.25 ng/ml/10 min)(X +/- SD) of the follow up period. HbA1 levels, and insulin requirements were similar in both groups (IAA+ and IAA-). We conclude that the presence of insulin autoantibodies at clinical diagnosis, before initiating insulin treatment, may well predict the loss of the beta cell function.

Adolescent↗
PubMed1987

New ideas in thyroid autoimmunity.

Endocrine epithelial cells do not normally express human leukocyte antigen (HLA) class II molecules, but do so in a variety of autoimmune diseases. This finding suggests the hypothesis that such inappropriate class II-positive expression may enable these cells to present autoantigens and thus contribute to autoimmune pathogenesis. Indeed, class II-positive thyrocytes can present both exogenous antigenic peptides and intrinsic autoantigens to the appropriate T cells. Class II expression by thyrocytes can be induced by interferon-gamma, and is positively and negatively regulated by thyroid-stimulating hormone and epidermal growth factor, respectively. Furthermore, heterogeneity of thyrocyte class II subregion expression appears to be related to the nature of the inducing stimulus. The complexity of regulatory signals is underlined by findings in type I diabetes: islet beta cells aberrantly express class II in this disease, but class II cannot be induced in normal beta cells by interferon-gamma.

Antigen-Presenting Cells↗
PubMed1987

Thyrocyte HLA class II expression and regulation in relation to thyroid autoimmunity.

The occurrence of HLA Class II expression by thyroid (and other endocrine) epithelia in autoimmune diseases suggests that these cells may facilitate their own destruction by immunogenically presenting autoantigens. This is supported by the findings that Class II+ thyrocytes can specifically stimulate virus-specific and autoreactive T cell clones, and that Class II expression by thyrocytes correlates with the occurrence of thyroid autoantibodies. A variety of factors may contribute to the regulation of Class II expression by thyrocytes: this is induced by interferon (IFN-gamma), and is enhanced by thyroid stimulating hormone (TSH) and by tumour necrosis factor (TNF). Conversely, epidermal growth factor (EGF) suppresses the induction of Class II in thyrocytes. This complex regulation is reflected in differences in HLA-D subregion expression between patients (DR greater than DP greater than DQ). The immune-based mechanisms of thyrocyte Class II regulation are clearly applicable to the on-going disease in an infiltrated thyroid, but the possibility of nonimmune Class II induction deserves attention, particularly in identifying factors which might contribute to the initial autoimmune attack. The possible involvement of such mechanisms in autoimmunity is supported by findings in Type I diabetes in which Class II+ islet beta cells can be found in the absence of infiltration. Further evidence is provided by the observation that a proportion of thyrocytes transformed with SV40 DNA constitutively express Class II molecules. Finally, the 'activated' state of capillary endothelial cells in organs subject to autoimmune attack suggests that they may play an important role in facilitating the autoreactive infiltration of the tissues.

Epithelium↗