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This review of literature concerns the different autoantibodies directed against membrane receptors and ion channels. The target antigens, the associated pathologies, the pathogenesis and the methods of detection of these autoantibodies will be addressed. Some of these autoantibodies are thought to be closely related to the autoimmune disease whereas for some others their pathogenesis role is still unclear. Overall, the roles of antibodies are different between diseases, but the presence of such autoantibodies support the basis of intervening immunotherapy, antibody titers predicted the activity of the diseases and some of them are very specific and become the useful markers for the diagnosis. Some autoantibodies are detected routinely as the antiacetylcholine receptor, voltage-gated potassium and calcium channels autoantibodies whereas most of them are detected very rarely and only by specialized laboratories. This review will be divided in three parts with the following classification: the first group of autoantibodies directed against membrane receptors included receptors with an enzymatic activity (mostly tyrosine kinase) with one transmembrane domain, receptors associated to G protein with seven transmembrane domains, ion channels and receptors associated to the membrane by the glycosyl phosphatidyl inositol and the second group of intracellular receptor autoantibodies directed to the estrogens, androgens, lamin and kinesin receptors.

Background: Treatment of Crohn's disease (CD) by infliximab has been associated with the induction of antinuclear (ANA) and anti-double stranded DNA (dsDNA) autoantibodies. As yet, little is known about the effect of the humoral response induced by infliximab on the production of other organ-specific or nonorgan-specific autoantibodies., Methods: Thirty-five patients with CD treated with repeated infusions of infliximab were prospectively studied. Thirty-two patients with CD who had never received infliximab served as controls. A large panel of autoantibodies directed against phospholipid, beta2-glycoprotein I, mitochondria, smooth muscle, liver--kidney microsomes, filaggrin, thyroid, adrenals, and rheumatoid factor was tested along with ANA and anti-dsDNA autoantibodies during 1 year of intermittent treatment with infliximab. Autoantibodies were detected using the appropriate methods (i.e., indirect immunofluorescence, a radioimmunological technique, and ELISA assays)., Results: Induction of ANA and anti-dsDNA autoantibodies was observed in 53% and 35% of infliximab-treated patients with CD, respectively. Overall, no other organ-specific or nonorgan-specific autoantibodies were detected during follow-up. A single patient who developed ANA and anti-dsDNA autoantibodies showed clinical features consistent with drug-induced lupus, which quickly resolved after discontinuation of infliximab. The other patients with CD receiving infliximab did not develop any clinical symptoms of autoimmunity., Conclusions: The humoral response induced by infliximab was restricted to ANA and anti-dsDNA autoantibodies, which persist for up to 1 year of follow-up. We confirmed the significant prevalence of such autoantibodies induced by infliximab in CD, but they are not generally associated with clinical signs of autoimmunity.

An association between autoimmunity and hematological malignancies has been reported including the detection of antinuclear autoantibodies (ANAs) in patients suffering from non-Hodgkin's lymphoma (NHL), with a high prevalence of ANAs directed to components of the mitotic apparatus or the mitosis-associated proteins. Previous studies have demonstrated that one of the targets of such ANAs could be the CENP-F protein, especially in some carcinomas. The prevalence and specificity of anti-CENP-F autoantibodies (aAbs) thus were analyzed in 347 patients with different histological subgroups of NHL before any treatment of NHL, along with 150 controls. The detection of these aAbs was performed using two techniques: a radioimmunological assay (RIA) and an indirect immunofluorescence technique (IIF). Twenty-five (7.2%) NHL patients and 2 (1.3%) control patients displayed anti-CENP-F aAbs using RIA. This difference between the two groups was found to be significant (P < 0.01), with a higher prevalence of aAbs in the follicular (13%) and in the marginal zone B and MALT (10.2%) lymphoma subgroups. By IIF, 10 (2.9%) patients with NHL displayed aAbs with a CENP-F or CENP-F-like pattern, whereas none of the control group did. In conclusion, these data demonstrate that a significant incidence of anti-CENP-F aAbs is observed, before any treatment, in some histological subgroups of NHL patients. In addition to the usefulness of anti-CENP-F aAbs as a marker for some NHL subgroups, prospective studies may be important to evaluate the predictive value of anti-CENP-F aAbs for the development of carcinomas.

According to international criteria, autoimmune hepatitis (AIH) type 1 is characterized by the presence of antinuclear or anti-smooth muscle antibodies (SMA) with F-actin specificity. SMA have been found in 85% of AIH patients, but are not specific to this disease, and anti-F-actin specificity is not always verified when SMA are detected. The objective of this study was to determine the diagnostic value of anti-F-actin antibodies in a large population. A multicenter study involving 12 clinical centers was performed. Patients were selected on the basis of the presence of F-actin SMA detected by indirect immunofluorescence (IIF) on rat liver-kidney-stomach sections and was confirmed by IIF on Hep2 cells treated with colchicine, or F-actin dot-blot. The clinical status of patients was determined from their medical records. One hundred sixty-eight patients were included: 76% women, 24% men; mean age of 45 years (range, 2-88 years), with a bimodal age distribution. Sixty percent had AIH type 1, and 40% had another disease. In the group of women younger than 25 years, 90% had AIH type 1. Other pathologies associated with antiactin were other liver diseases (19%), including viral hepatitis C (7%), and non-liver diseases (21%), including connective tissue diseases (12%). Antibody titers were higher in AIH than in other diseases. Antiactin antibodies are of major diagnostic value in AIH, especially in young women; they may be found in other disease settings, but mostly at low levels.

Expression and transamidation activity of tissue transglutaminase (tTG) may be involved in the morphological modifications leading to the mucosal atrophy observed in coeliac disease (CD). We aimed to investigate the localization of tTG within the duodenal mucosa during the development of villous atrophy. The localization and level of expression of N epsilon-(gamma-glutamyl) lysine isopeptides which could reflect the transamidation activity of tTG were also analyzed. tTG and N epsilon-(gamma-glutamyl) lysine were localized using an immunohistochemical technique on duodenal biopsies obtained from 75 patients with CD and 51 subjects with normal mucosa (control group). The number of cases displaying tTG-expressing cells in the basement membrane and lamina propria was significantly higher in CD patients than in the control group. Moreover, the intensity of tTG staining in these areas was higher in CD. In contrast, the number of biopsies with tTG-expressing enterocytes was significantly lower in CD than in the control group. There was no difference in N epsilon-(gamma-glutamyl) lysine between the two populations. Tissue transglutaminase was differently expressed in the various areas of the mucosa according to the stage of atrophy, whereas the localization and the intensity of the labelling of N epsilon-(gamma-glutamyl) lysine isopeptides did not show any modification. The preferential localization in the basement membrane and lamina propria may reflect the involvement of tTG in the development of mucosal atrophy in CD.

Objective: The diagnosis of coeliac disease (CD) is often delayed because many children are free from the major symptoms characteristic of this enteropathy. The aim of the present study was to determine the efficacy of antibodies directed against tissue transglutaminase (tTG Abs) for early detection of CD in a population with few symptoms of the disease, as well as in children with an autoimmune disorder., Methods: This was a prospective study in a paediatric population including 638 patients with clinical symptoms frequently associated with CD, autoimmune diseases such as type 1 diabetes mellitus (DM1), autoimmune thyroiditis or hepatitis, and Turner's syndrome. Anti-endomysium, tTG Abs and antigliadin antibodies were analysed in these patients using an indirect immunofluorescence technique and enzyme-linked immunosorbent assay techniques. Intestinal biopsies were performed for some patients with positive or negative antibodies., Results: tTG Abs were detected in 2.6% of children with symptoms associated with CD, such as digestive signs and growth failure, and in 5.4% of children with DM1. No other autoimmune disease was positive for tTG Abs. Biopsies performed in the patients with positive tTG Abs showed mucosal atrophy confirming the diagnosis of CD in all cases., Conclusion: Children displaying minimal symptoms frequently associated with CD and children with DM1 should be systematically screened for tTG Abs.

We investigated the presence of autoantibodies (aAbs) directed against the parathyroid gland in 17 patients with spontaneous isolated acquired hypoparathyroidism. Fourteen patients with acquired hypoparathyroidism (AH) associated with type I or II autoimmune polyendocrinopathy syndrome were also tested in comparison with a control group of 68 subjects without AH, including patients with other autoimmune diseases and healthy blood donors. aAbs against parathyroid tissue were screened using an indirect immunofluorescence technique on primate parathyroid tissue and human parathyroid adenoma. aAbs against the calcium-sensing receptor (CaSR) were analyzed using an immunoblotting assay with the recombinant extracellular domain of the human CaSR as antigen. Seven of the 31 patients with AH were positive for CaSR aAbs. Five of the positive sera were obtained from the group with isolated AH. The two other positive sera were from patients with autoimmune polyendocrinopathy syndrome. The sensitivity of the immunoblotting technique was higher than that of both the radioimmunological test using the extracellular domain of the CaSR and the indirect immunofluorescence technique. There were no positive sera in the control group. In conclusion, using an immunoblotting assay, we demonstrate the presence of CaSR aAbs in about one third of the patients with isolated AH, pointing out the value of detecting such aAbs to assess the autoimmune origin of the disease.

Liver-Kidney Microsomes Type 3 (LKM3) autoantibodies (aAbs) have been described in chronic hepatitis D virus infection in 1983. The detection of such aAbs in autoimmune hepatitis (AIH) Type 2 was thereafter reported. The molecular targets of LKM3 aAbs have been identified as enzymes belonging to the UDP-glucuronosyltransferase family 1. Since 20-30% of suspected AIH are negative for the classical autoimmune serological markers, such as aAbs directed against antinuclear autoantibodies, smooth muscle autoantibodies and Liver-Kidney Microsomes Type 1 aAbs, LKM3 aAbs could be of great interest in the diagnosis of such negative AIH. In this review, we discuss the sensitivity and specificity of these aAbs in AIH in order to stress out their potential clinical use as a marker.

Treatment of rheumatoid arthritis (RA) with infliximab (Remicade) has been associated with the induction of antinuclear autoantibodies (ANA) and anti-double-stranded DNA (anti-dsDNA) autoantibodies. In the present study we investigated the humoral immune response induced by infliximab against organ-specific or non-organ-specific antigens not only in RA patients but also in patients with ankylosing spondylitis (AS) during a two-year followup. The association between the presence of autoantibodies and clinical manifestations was then examined. The occurrence of the various autoantibodies was analyzed in 24 RA and 15 AS patients all treated with infliximab and in 30 RA patients receiving methotrexate but not infliximab, using the appropriate methods of detection. Infliximab led to a significant induction of ANA and anti-dsDNA autoantibodies in 86.7% and 57% of RA patients and in 85% and 31% of AS patients, respectively. The incidence of antiphospholipid (aPL) autoantibodies was significantly higher in both RA patients (21%) and AS patients (27%) than in the control group. Most anti-dsDNA and aPL autoantibodies were of IgM isotype and were not associated with infusion side effects, lupus-like manifestations or infectious disease. No other autoantibodies were shown to be induced by the treatment. Our results confirmed the occurrence of ANA and anti-dsDNA autoantibodies and demonstrated that the induction of ANA, anti-dsDNA and aPL autoantibodies is related to infliximab treatment in both RA and AS, with no significant relationship to clinical manifestations.

The presence of antinuclear autoantibodies (ANA) was investigated in a large cohort of patients with non-Hodgkin's lymphoma (NHL) in order to assess their frequency, specificity and prognostic relevance. ANA were analysed in 347 patients with different histological subgroups of NHL and in 213 controls using an indirect immunofluorescence technique on HEp2 cells. As the appearance of autoantibodies may be found after treatment of NHL, samples were collected at the time of diagnosis of NHL before any therapy. Sixty-six (19%) NHL patients and 12 (5.6%) patients from the control group displayed ANA. The prevalence between the two groups was found to be significantly higher in NHL patients (P < 0.0001) with a marked increased prevalence in follicular and mantle cell lymphoma subgroups. Autoantibodies directed against mitotic proteins or mitotic-associated proteins were found in 6.9% of NHL patients versus 0.5% in the control group (P < 0.001), with a significantly increased incidence in follicular and mantle cell lymphoma subgroups (P < 0.0001). Some 28% of the patients with positive ANA displayed clinical symptoms that could correspond to classical autoimmune manifestations, this frequency appearing to be higher in the marginal zone/mucosa-associated lymphoid tissue lymphoma subgroup. These data demonstrate a significant incidence of ANA before any treatment in NHL occurrence, which seems to be higher in some histological subgroups with particular ANA, such as ANA directed against mitotic proteins or mitotic-associated proteins.

Systemic sclerosis is characterized by a fibrosis and a microvascular injury. These vascular lesions can affect internal organs causing severe visceral damages. The pathogenesis of this disease is complex but some immunological disorders with a production of antinuclear auto-antibodies can be useful to the clinicians, especially for an early diagnosis and to distinguish the scleroderma/myositis overlap syndromes. This review presents the target antigens and the methods of detection of the different antinuclear auto-antibodies. Sensitivity, specificity and clinical association between the antinuclear auto-antibodies and the different subclasses of scleroderma are also presented in detail.

Objective: To determine the clinical usefulness of measuring antistratum corneum (ASC) and antifilaggrin autoantibodies (AFA) to discriminate between rheumatoid arthritis (RA) and other rheumatic or autoimmune diseases, using an indirect immunofluorescence (IIF) assay, along with a complementary immunoblotting technique (IB) when IIF detection of ASC was negative., Methods: Sera from 346 patients were studied: 189 sera from patients with RA seen in the same clinic, 92 from patients with non-RA rheumatic diseases, 24 from nonrheumatic autoimmune diseases, and 41 from healthy blood donors. ASC and AFA were detected using IIF and IB, respectively., Results: ASC detection using IIF showed a specificity of 97.5% for RA with 44.4% sensitivity. When both IIF and IB techniques were used, sensitivity for RA increased significantly (up to 53.4%; p < 0.01) with no decrease in specificity (p < 0.01)., Conclusion: These data confirm the usefulness of 2 different techniques performed simultaneously for detecting ASC/AFA, and the usefulness of these biological markers for discriminating between RA and other rheumatic diseases in clinical practice.

USEFULLNESS OF DETECTION OF ANTIBODIES AGAINST THE ACETYLCHOLINE RECEPTOR IN THE DIAGNOSIS OF MYASTHENIA GRAVIS (MG): MG is an autoimmune disorder including a fatigability of skeletal muscles and the presence of antibodies against the acetylcholine receptor (AChRAbs). These Abs are pathogenic by blocking the acetylcholine receptor within the neuromuscular junction. A transient neonatal MG occurs in 12% of babies born to myasthenic mothers. AChRAbs have been found in 77 to 89% of systemic MG and in 47 to 60% in ocular form of MG. These Abs are generally directed against the "Main Immunogenic Region" (MIR) within the a subunit of the receptor; others are directed against beta, gamma and epsilon subunits. Eleven to 23% of the MG are negative for the Abs directed against the a subunit reinforcing the interest to detect the other AChRAbs, specially the anti-epsilon subunit Abs and the Abs directed against the muscle-specific receptor tyrosine kinase. MULTICENTER EVALUATION: In order to compare the results of AChRAbs for a same patient from various laboratories, a multicenter study was performed on 26 sera with different titers of AChRAbs using radioimmunoassays. Six french laboratories have participated to this study. The antigen was obtained from TE671 cells for 5 laboratories (3 used a commercial test) or from human muscle (1 laboratory). At the end of the study, the qualitative analysis of the results showed a concordance of 92.3%. The interpretation of AChRAbs assays can thus be done by clinicians whatever the test is used. A same quality control is nowadays used by the six laboratories in order to estimate quantitative results according to the assay performed. USEFULNESS OF THE OTHER ABS: The Abs directed against the striated muscle are good markers of thymoma in MG but are not specific for MG. The Abs directed against titin which is the major target of the anti-striated muscle Abs, are also good biological markers of thymoma specially in MG adults younger than 60 years. The detection of anti-myoid cells and thymic reticuloepithelial cells Abs is no more useful due to a lower sensitivity compared to the Abs directed against the striated muscle.

Concomitant acquired immunodeficiency syndrome (AIDS) and lupus nephritis is an exceptional feature in white patients. A white boy with maternofetal human immunodeficiency virus (HIV) infection had no medical follow-up until he presented at 12 years of age with a nephrotic syndrome, macrohematuria, renal failure, pancytopenia, and low CD4(+) cell count. A renal biopsy revealed severe lupus nephritis (World Health Organization class IV) with specific immune deposits in the absence of any clinical sign of systemic lupus erythematosus or specific autoantibodies at the time of diagnosis. The treatment consisted of methylprednisolone pulses followed by oral prednisone; antiretroviral triple therapy was started a few weeks later, which contributed to clinical and biologic improvement. To our knowledge, this is the first case report of lupus-like nephritis in a white child with AIDS, whose outcome might be improved significantly by a combination of steroids and antiretroviral therapy.

Background: Antibodies to glutamic acid decarboxylase (GAD-Ab) are described in patients with insulin-dependent (type 1) diabetes mellitus (IDDM), in stiff-man syndrome, and, recently, in a few patients with cerebellar ataxia., Objectives: To show a link between GAD-Ab and some patients with cerebellar ataxia and to clarify their clinical and immunologic profiles., Methods: Serum samples were selected from 9000 samples of 4 laboratories. The selection criterion was an immunohistochemical pattern compatible with GAD-Ab that was confirmed by radioimmunoassay. We identified 22 patients with stiff-man syndrome and 14 with cerebellar ataxia and GAD-Ab., Results: Thirteen of the 14 patients with cerebellar ataxia and GAD-Ab were women, and 11 had late-onset IDDM. Patients did not have clinical or radiologic evidence of brainstem involvement. Ten patients had oligoclonal IgG bands in the cerebrospinal fluid, and intrathecal GAD-Ab synthesis was observed in 5 of the 6 patients studied. The level of GAD-Ab of these patients was similar to those with stiff-man syndrome and significantly higher than those with IDDM or with polyendocrine autoimmunity (P<.001). However, the GAD-Ab levels of 6 of the 9 patients with polyendocrine autoimmunity overlapped with those of patients with cerebellar ataxia., Conclusions: These results suggest a link between high level of GAD-Ab and some cases of cerebellar ataxia, particularly women with IDDM. If high serum levels of GAD-Ab are detected, the cerebrospinal fluid should be evaluated for the presence of oligoclonal IgG bands and intrathecal synthesis of GAD-Ab to further prove an autoimmune origin of the syndrome.

The utilization of accurate and sensitive methods for the measurement of cytokines in body fluids is prerequisite for the proper use of these mediators in clinical practice. Many factors contribute to the complexity of cytokine quantitation. Bioassays historically preceded immunoassays, which are now very popular, but there is a need for standardization. Nevertheless, due to the local effects of cytokines, the study of their blood levels is of limited value for an understanding of the pathophysiology of these mediators. This explains the development of alternative approaches to assess the ability of cells to produce cytokines. These include the Enzyme-Linked Immuno Spot Assay (ELISPOT), the measurement of cell-associated cytokines by flow cytometry, and the study of cytokine secretion by isolated peripheral blood mononuclear cells or by whole blood test. All these techniques, associated with a local detection of cytokines by immunohistochemistry or in situ hybridization and reverse transcriptase polymerase chain reaction, appear to be complementary tools for a better understanding of the biology of cytokines. Selected examples of possible clinical applications related to infectious diseases, cancer, autoimmune diseases, allergy, transplantation and preclinical evaluation of drugs and biotechnology products are given.

Objective: To assess the implication of hepatitis C virus (HCV) infection in the development of autoimmune thyroid response, thyroid autoantibodies were studied in serum of HCV positive patients. Methods: Anti-microsomal, anti-thyroperoxidase (anti-TPO) and anti-thyroglobulin antibodies were evaluated in the sera of 100 patients with chronic hepatitis C (53 women and 47 men; mean age = 55&#x00B1;5 years). In parallel, thyroid autoantibodies were investigated in blood samples obtained from two separated control groups: age-matched HCV negative-HBV positive patients (25 women, 25 men; mean age= 47&#x00B1;6 years) and healthy blood donors (29 women and 21 men; mean age= 54&#x00B1;8 years). Results: Anti-thyroid antibodies were found more frequently in HCV positive women when compared to the men (8/53= 15.1&#x0025; vs 0/47= 0&#x0025;,.p<0.01).The prevalence of these autoantibodies was not statistically different between HCV positive and healthy female blood donors. However the investigation of thyroid autoantibody titers showed significantly higher levels of anti-TPO and anti-microsomal antibodies in HCV positive women in comparison with healthy women controls (respectively 1: 83200 vs 1: 1900 and 834 vs 23, p<0,01). Conclusions: This strong association between HCV infection and high levels of anti-thyroid autoantibodies in women outlines the interest of systematic detection of anti-microsomal and/or anti-TPO antibodies in this population.

The COBAS Core HEp2 ANA enzyme immune assay (EIA) was evaluated in a precision and a clinical sample study in comparison to indirect immunofluorescence assay (IFA) on HEp2-cells. In the precision study the COBAS Core EIA yielded intraassay coefficient variations (CVs) mostly below 9%, and interassay CVs between 4.7% and 10.4%. When comparing the COBAS Core EIA to IFA, the results corresponded well in healthy subjects, systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis. In the case of Sjögren's syndrome and scleroderma patients the COBAS Core EIA yielded a lower rate of positive results compared to IFA. This discrepancy may be explained by the lack of detection of autoantibodies to nuclear antigens that can be identified only by IFA due to their compartmentalization and higher localized antigen density in HEp2 cells. The discrepancies in the group of dermato/polymyositis patients are due to the fact that the EIA contains mainly nuclear antigens and was able to detect only antibodies against the cytoplasmic Jo1 antigen that was added to the HEp2 nuclear extract. Routine sera were also evaluated; good agreement was found in sera from patients attending tertiary reference centres for autoimmune diseases but a higher number of discrepancies was reported in sera from unselected populations., (Copyright 1999 Academic Press.)

The autoantibodies (aAbs) directed against the ribosomal P proteins (RPP aAbs) are known to react mainly against epitopes localized within the common C-terminal sequence of the three acidic ribosomal P proteins, P0, P1 and P2. In order to investigate the opportunity to select short recombinant peptides of this common C-terminal sequence to detect the RPP-aAbs, the location of the epitopes recognized by ribosomal proteins (RP) aAb(+)sera of systemic lupus erythematosus patients (SLE) was investigated. Immunoblotting and ELISA techniques using extracted or recombinant, entire or cleaved RPP showed that 55% of the RP aAbs were directed against the three ribosomal P0, P1, and P2 proteins. The epitopes recognized by the RPP aAbs are located not only within the C-terminal sequence common to the three proteins but also within the N-terminal sequence of the P2 or P1 protein. The other RP aAbs sera (45%) did not react with all three proteins but with some of them, and showed the following pattern: P0(+)P1(+); P1(+); P2(+); P0(+)and P1(+). They recognized epitopes located in the region of the C-terminal sequence of the protein but not common to the three proteins. In addition two out of the six monoclonal Abs produced by immunization of mice using the P1 protein did not react with the peptide N-65 or N-71 of the P2 protein or with the C-terminal sequence of the three proteins. In conclusion, this study showed that the RPP aAb in SLE patients are not only directed against epitopes within the C-terminal sequence shared by the three acidic ribosomal P proteins. In view of these data it seems necessary to be cautious in using only a C-terminal peptide of ribosomal P proteins in tests performed to detect RPP aAb in human sera., (Copyright 1999 Academic Press.)

Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inflammatory immune reactions against self- or non-self-antigens. Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease. We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals. We now report that feeding small amounts (2-20 microg) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice. The protective effect could be transferred by T cells from CTB-insulin-treated animals and was associated with reduced lesions of insulitis. Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration. These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.

To compare the protective effects of IGF-1 and insulin on the autoimmune process of beta-cell destruction, permissive NOD recipients were adoptively transferred with 7 x 10(6) autoreactive T cells from diabetic NOD mice and then administered either 10 micrograms of rhIGF-1 or 0.5 unit of regular insulin subcutaneously twice daily for three weeks. The final incidence of successful transfers of diabetes observed at day 22 was significantly reduced in 1/12 mice (8.3%) treated with IGF-1, while diabetes was observed in 4/10 (40%) receiving insulin and 7/11 (63.4%) controls. A marked reduction of insulitis during histological analysis of the pancreatic glands of IGF-1 or insulin-treated mice was also observed. Non-diabetic mice treated with rhIGF-1 had a higher mean +/- SD percentage of intact islets (68.9 +/- 36% vs 10.7 +/- 13%, p < 0.01) and a lower percentage of severely infiltrated islets (5.7 +/- 9.8% vs 30.3 +/- 21%) than non-diabetic control mice. Insulin reduced islet-cell infiltration, though to a lesser extent, with a high percentage of normal islets (55.2 +/- 31%, p < 0.02). Some mice developed diabetes and severe islet-cell infiltration despite rhIGF-1 or insulin, thus indicating that some committed T cells were still able to invade islets and cause beta-cell destruction. To evaluate the effects of rhIGF-1 and insulin on cell trafficking in recipient mice, T cells from diabetic NOD Thy-1, 2 mice injected into congenic NOD-N Thy-1, 1 mice were monitored three weeks after adoptive cell transfer. The percentage of Thyq-1.2+ T cells was significantly reduced in the spleen (10.8 +/- 1.3% vs 17.2 +/- 3.9%, p = 0.004) of rhIGF-1 treated mice as compared to the thymus (68.4 +/- 7.9% vs 72.87 +/0 6.2, p = 0.306). Similar experiments performed in mice treated with insulin revealed no significant differences in the percentages of Thy-1,2+ T cells compared to controls in the spleen (l4.3 +/- 1.4%), thymus (84 +/- 2.5%) or pancreatic lymph nodes (21.5 +/- 1.6% vs 23.4 +/- 1.5%) of treated animals. These results suggest that rhIGF-1, as compared to insulin, could influence T-cell trafficking to the lymphoid organs in addition to affecting beta cells. These findings may have important implications for new preventive strategies in human Type 1 diabetes mellitus.

This retrospective study was performed to evaluate the interest of cytology in the diagnosis of pulmonary carcinomas. Bronchial samples were collected from 330 patients known to display a macroscopic lesion which was detected by bronchoscopy. Cytological analysis of the bronchial brushings and washings, removed at the first fibroscopy, allowed the diagnosis of malignancy in 92% of the cases analyzed whereas the biopsy confirmed the malignancy in 77% of the cases. In conclusion cytological studies gave information on malignity and classification in 90% of the cases. However histological classification only could guarantee the choice of the best treatment regimen.

Objective: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias., Design: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations., Methods: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses., Results: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene., Conclusions: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours.

We have previously generated transgenic mice bearing a molecular construct obtained by fusing the rat thyroglobulin promoter with the human Kirsten ras oncogene (KRAS). These mice showed thyroid abnormalities, although at very low incidence and after a long latency period. A six-month thyrotropin (TSH) stimulation of thyroid glands, followed by a two-month suspension, induced a significant increase in the number of lesions in transgenic mice as compared with a nontransgenic control group. Our goal was to follow the progression and the reversion of the tumorigenesis process in relationship with the levels of expression of the KRAS in this experimental model. In situ hybridization was used to detect expression of KRAS mRNA in sections of thyroids of the various groups of mice. A positive hybridization was observed in follicular cells of TSH-stimulated transgenic mice, whereas no expression could be appreciated in control nontransgenic mice. A positive signal was also observed in thyroid glands excised from transgenic mice after the 2-month suspension of treatment; however, the number of expressing cells was decreased compared with transgenic mice killed immediately after 6 months of a goitrogen regimen. Finally, every cell in the single thyroid carcinoma observed after the two-month suspension was positive for the transgene mRNA. This study further strengthens the role of the expression of mutated KRAS in the early stages of thyroid follicular cell transformation and indicates that when the expression of the mutated KRAS becomes independent of exogenous TSH stimulation, this event coincides with a further progression towards tumorigenesis.

We investigated the in vivo effect of cyclosporin A (CsA) on mouse thymus and thymocytes. Administration of CsA (10 mg/kg of body weight) was found to induce a marked reduction in the size, weight and consistency of the thymus. These modifications were associated with thymic reticulo-epithelial cells (TREC) and thymocyte damage. Some of the damaged thymocytes displayed characteristic of cells undergoing apoptosis. Ultrastructural study of thymocytes and thymic tissue, as well as DNA electrophoresis of thymocytes, showed chromatin condensation, cellular shrinkage, and nuclear fragmentation in oligonucleosomal fragments. DNA labeling with propidium iodide (PI) of thymocytes from CsA treated mice cultured for 24 hrs showed an increased number of apoptotic nuclei. Furthermore, flow cytometric analysis using monoclonal antibodies (mAbs) specific for thymocyte subsets confirmed that CsA induces a large decrease in the relative number of mature single positive (SP) CD4+CD8- and CD8+CD4- thymocytes expressing high densities of CD3 and T cell receptor ab (TCR alpha beta) surface molecules, but also a decrease in the absolute number of the other thymocyte subsets. These results suggest that CsA causes macroscopic and ultrastructural modifications of the thymus, associated with an active process of cell death in mouse thymocytes in vivo. In line with these results we formulate a hypothesis concerning the stage of T-cell development at which CsA induces apoptosis.

The RET protooncogene encodes a transmembrane protein of the receptor-type tyrosine kinase family whose ligand has not yet been identified. Its activation in vivo is restricted to human carcinomas of the thyroid. In order to learn more about the possible role played by RET during normal development, we have examined its expression by performing in situ hybridization experiments on mouse embryos. Here, we show that the RET protooncogene is expressed during mouse embryogenesis in an unusual temporal and spatial manner. In fact, its expression was first detected around day 10 of gestation in the basal plate of the neural tube and in the developing encephalic ganglia, and later its pattern of expression was definitely established in neural structures, mostly in neural crest derivatives (spinal and encephalic ganglia). As far as the central nervous system is concerned, RET expression was confined to the ventral part of the midbrain from 12.5 days postcoitum (dpc) until birth. RET was also found to be expressed within structures of sensory organs such as the ganglial layer of the retina and the olfactory epithelium. A peculiar pattern of RET expression was clearly observed in the wall of the gut and in the nephrogenic zone of the developing kidney cortex, specifically in the metanephrogenic vesicles. Finally, RET was found to be expressed in the liver mostly between 12.5 dpc and 14.5 dpc. In conclusion, its expression in the early stages of embryogenesis suggests that RET may play a role in the differentiation of specific neural structures and the excretory system.

The in vivo effect of cyclosporin A (CsA) on the murine thymus was investigated by studying the ultrastructural cellular alterations, which are described for the first time, and the immunohistochemical modifications of thymocytes and thymic reticulo-epithelial cells (TREC). A marked reduction of the thymus size became apparent after 6 days of CsA treatment (10 mg/kg/day). Light microscopy studies using polyclonal antibodies (Ab) or monoclonal Ab specific to lymphoid sub-populations (anti-CD4, anti-CD5, anti-CD8), and specific to epithelial cells (anti-keratin: AKs, K8), to cortical TREC (TR4) and to subcapsular/medullary TREC (TR5, 3H9) showed that the number of CD4- 8+ or CD4+ 8- medullary thymocytes dramatically decreased and that the cortical TREC are affected, after 10 days of CsA treatment. These observations were confirmed by electron microscopy studies demonstrated that the medullary lymphoid population disappeared almost entirely. TREC, principally cortical type, presented signs of cellular lysis. A few cortical thymocytes showed some damage. At day 8 the medullary thymic tissue was disorganized, but no change was noted in the subcapsular area right up to the final day (day 10) of CsA treatment. These results suggest that CsA has a harmful effect on cortical TREC, which affects the development of immature thymocytes.

One of the difficulties in characterization of the oncogenes involved in thyroid carcinogenesis is the production of cell lines. Arising from a poorly differentiated thyroid papillary carcinoma we have established a cell line synthesizing the thyroglobulin and human chorionic gonadotropin (alpha and beta subunits) (HCG) hormones. These cells will allow research of the oncogenes involved or potentially involved in thyroid papillary carcinomas and evaluation of the role of the autocrine secretion of HCG.

Different hormones (thymulin, thymosin alpha 1, vasopressin), antigenic markers of cortical and subcapsular/medullary thymic areas and tumour associated antigens were studied on paraffin or frozen section and cultures of human epithelial thymic tumours ('thymomas'). Thymulin, thymosin alpha 1 and for the first time vasopressin are found in most tumours. The epithelial cells of five 'thymomas' had markers of both cortical (TE3) and subcapsular/medullary thymic regions (A2B5 and/or TE4 and/or anti-p19). Leu-7, a marker of subcapsular epithelial cells was positive only in two tumours. The histological classification into cortical and medullary tumours does not correspond to our immunofluorescence results. The presence of these markers does not support the theory of different embryologic origin of the cortical and subcapsular/medullary epithelial cells. Transferrin receptors were detected on only some epithelial cells of thymic 'carcinomas'. Adenocarcinoma related antigen and carcino embryonic antigen only stained a few epithelial cells of all the tumours. There is no expected correlation between the presence of epidermal growth factor receptors on cell membranes and the number of proliferative cells stained by the anti-Ki67 antibodies. Immunostainings were heterogeneous according to the epithelial thymic tumours, independent of histological classification and not yet useful for prognosis.

Hybridomas between spleen cells from BALB/c mice and Sp2/O-Ag14 mycloma cell line are performed by PEG fusion. An in vitro booster is realized with human thymic reticulo-epithelial cell cultures as antigen. A monoclonal antibody (3H9) directed against the thymic epithelium and stratified epithelia of mammals has been obtained and characterized.

Anti-thymosin alpha 1 monoclonal antibodies recognized, on immunoblots, 1 to 2 bands corresponding to molecules of 34 and 35 Kd when using aqueous extracts of thymus, spleen, kidney, liver, brain, pituitary and adrenal glands from rats or mice. Anti-bovine thymopoietin polyclonal antibodies, in the same conditions, labelled analogous 34, 35 and 35.5 Kd molecules in brain and thymus extracts but also a 40 Kd molecules in thymus and a 90 Kd in brain extracts. Anti-synthetic thymulin monoclonal antibodies recognized irregularly and poorly a 52 Kd molecule from thymus and brain extracts. These results suggest that thymopoietin, thymulin and specially Thymosin alpha 1 are first synthesized in large precursors. Finally, other organs seem capable of synthesizing thymosin alpha 1 and probably thymopoietin, but for thymulin, the results are too irregular to conclude.