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We study two authentic cases of protein-losing enteropathy, the diagnosis of which was facilitated using Given M2A videocapsule endoscopy. The first case corresponded to a primary intestinal lymphangiectasia confirmed by jejunum biopsies and the second one to a protein-losing enteropathy with lymphatic abnormalities secondary to a chronic constrictive pericarditis. In the first case, the mucosa of jejunum presented with a diffuse oedematous aspect, whitish villi, white curved lines probably related to submucosal dilated lymphatics and lacteal juice. In the second case, capsule endoscopy showed oedematous aspect of jejunum mucosa associated with white curved lines similar to those observed in the first case. Videocapsule endoscopy is useful in cases of protein-losing enteropathy to identify presence of intestinal lymphangiectasia and to specify their localisation after ruling out other disorders liable to induce protein-losing gastrointestinal syndrome.

To investigate the role of retinoid X receptors (RXRs) in epidermal homeostasis, we generated RXRalphabeta(ep-/-) somatic mutants in which both RXRalpha and RXRbeta are selectively ablated in epidermal keratinocytes of adult mice. These mice develop a chronic dermatitis mimicking that observed in atopic dermatitis (AD) patients. In addition, they exhibit immunological abnormalities including elevated serum levels of IgE and IgG, associated with blood and tissue eosinophilia, indicating that keratinocyte-selective ablation of RXRs also generates a systemic syndrome similar to that found in AD patients. Furthermore, the profile of increased expression of cytokines and chemokines in skin of keratinocyte-selective RXRalphabeta-ablated mutants was typical of a T helper 2-type inflammation, known to be crucially involved in human AD pathogenesis. Finally, we demonstrate that thymic stromal lymphopoietin, whose expression is rapidly and strongly induced in RXRalphabeta-ablated keratinocytes, plays a key role in initiating the skin and systemic AD-like pathologies.

Autoantibody production during infections is considered to result from nonspecific activation of low-affinity autoreactive B cells. Whether this can lead to autoimmune disease remains uncertain. We show that chronic infection by Borrelia burgdorferi of Tg animals expressing human rheumatoid factor (RF) B cells (of low or intermediate affinities) in the absence or in the constitutive presence of the autoantigen (represented here by chimeric IgG with human constant region) breaks their state of immunological ignorance, leading to the production of RFs. Surprisingly, this production was more pronounced in intermediate-affinity RF Tg mice co-expressing the autoantigen. This overproduction was mediated by immune complexes and involved synergistic signaling between the B cell receptor and Toll-like receptors and T cell help. These findings indicate that chronic infection can activate autoreactive B cells with significant affinity and creates conditions that can drive them to differentiate into memory cells. Such cells may have some physiological yet undetermined role, but in autoimmune-prone individuals, this scenario may initiate autoimmunity.

X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder with impaired very-long-chain fatty acid (VLCFA) metabolism that produces a neurological disease with significant variability of clinical phenotypes even within kindred. The two most common forms are the cerebral form (CALD) with an important inflammatory reaction at the active edge of demyelinating lesions, resembling some aspects of multiple sclerosis pathology, and adrenomyeloneuropathy (AMN), which involves the spinal cord and in which the inflammatory reaction is mild or absent. One hypothesis is that the phenotypic variability is related to T cell-mediated immune mechanisms playing a primary role in the demyelinating pathogenic process of CALD. The present study aims to test the hypothesis that CSF of patients with the CALD form contains highly restricted T cell populations. The variable regions of the T cell receptor beta chains (TCR Vbeta) were studied in CSF from 29 ALD patients with different phenotypes. RNA was extracted and cDNA synthesized from CSF lymphocytes; TCR Vbeta gene segments were amplified from the cDNA by polymerase chain reaction (PCR) using 20 family-specific primers. PCR products were analyzed by Southern blot. Some amplified Vbeta products were sequenced. The majority of ALD patients (21/29), whatever their phenotype, exhibited oligoclonal T cell expansion. However the overexpression of some TCR Vbeta families was heterogeneous among the different patients without any preponderance of specific Vbeta families or any clustering according to clinical phenotype. In particular a dominant TCR Vbeta utilization was not found in patients with CALD., (Copyright 2005 John Wiley & Sons, Ltd.)

Antiphospholipid antibodies represent a heterogeneous group of autoantibodies directed against anionic phospholipids (PLs) usually linked to protein cofactors. Their presence during the antiphospholipid syndrome is associated with risks of thrombosis and fetal losses. Among 5 randomly selected monoclonal antiphospholipid antibodies, all originating from a single patient suffering from this autoimmune disease, only 1 induced fetal losses when passively injected into pregnant mice. Its antiphospholipid activity was dependent on annexin A5, and its variable regions contained mainly 3 replacement mutations. To clarify the role of these mutations in the pathogenicity of the antibody, they were in vitro reverted to the germ line configuration. The resulting "germ line" antibody reacted with multiple self-antigens and only partially lost its reactivity against PLs, but it was no more dependent on annexin A5 and, more importantly, was no more pathogenic. This study illustrates that the in vivo antigen-driven maturation process of natural autoreactive B cells can be responsible for pathogenicity.

Certain coagulation disorders can occur in patients with cancer and thromboembolic complications are frequent. We report the case of a 53-Year-old patient with metastatic adenocarcinoma of the lung treated with chemotherapy who presented several cerebral arterial thromboembolic events leading to death a few weeks after the initial diagnosis of cancer. This case illustrates the important role of certain satellite disorders related to coagulation activation: non-bacterial thrombotic endocarditis, disseminated intravascular coagulation, anti-phospholipid antibody syndrome. The role of anticancer chemotherapy as a favoring factor for thromboembolic events is also emphasized in patients with non-small-cell lung cancer.

Antiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL.

Objective: Arthritis in the K/BxN mouse model results from pathogenic immunoglobulins that recognize glucose-6-phosphate isomerase (GPI), a glycolytic enzyme residing in the cytoplasm of all cells. Antibodies directed against GPI can, alone, transfer arthritis to healthy recipients. Previous experiments have revealed significant titers of anti-GPI antibodies in the serum of many patients with rheumatoid arthritis (RA). We evaluated the generality of these observations in cohorts of patients with 12 different arthritic and chronic autoimmune diseases and in population-matched healthy control subjects., Methods: Anti-GPI antibodies were assayed in 811 individual serum samples by enzyme-linked immunosorbent assay with 2 forms of GPI, recombinant and native. Results were confirmed by immunoblotting., Results: Several patients had significantly elevated anti-GPI antibody titers, but without the prevalence or the specificity reported previously. Only 15% of RA patients had anti-GPI antibodies (range 12-29% in different cohorts), with a higher prevalence in patients with active disease. Psoriatic arthritis, undifferentiated arthritis, and spondylarthropathy patients also displayed anti-GPI antibodies at similar frequencies (12-25%). Similar titers were detected in a proportion (5-10%) of control subjects or patients with Crohn's disease or sarcoidosis. Very high titers were found in rare cases of RA and systemic lupus erythematosus., Conclusion: No disease-specific pattern of antibody positivity to GPI was apparent. While the antibody-mediated mechanism at play in the mouse model may exemplify a generic mechanism for some forms of arthritis in humans, GPI itself does not appear to be a target common to the majority of RA patients.

It is well established that autoreactive B cells undergo negative selection. This stands in paradox with the high frequency of so-called natural autoreactive B cells producing low affinity polyreactive autoantibodies with recurrent specificities, suggesting that these B cells are selected on the basis of their autoreactivity. We previously described two transgenic mouse lines (with and without IgD) producing a human natural autoantibody (nAAb) that binds ssDNA and human Fcgamma. In the absence of human IgG, nAAb-transgenic B cells develop normally. By crossing these mice with animals expressing knockin chimeric IgG with the human Fcgamma, we now show that the constitutive expression of chimeric IgG promotes the increase of nAAb-expressing B cells. This positive selection is critically dependent on the presence of IgD, occurs in the spleen, and concerns all mature B cell subsets, with a relative preferential enrichment of marginal zone B cells. These data support the view that soluble self-Ags can result in positive clonal selection.

Control of the immune reaction can become a major goal, particularly in patients with autoimmune diseases or who express alloreactivity after organ transplantation. The most important side effect of this control is an immunodeficiency, a consequence of the wide spectrum of activity of the treatment. Thus, in order to limit the infectious risks, it would appear reasonable to try to develop new more selective strategies. A better definition of the cellular and molecular mechanisms implicated in the initiation and effector phases of autoimmune diseases authorizes the development of new therapeutic approaches able to target precise points of the immune system. There are a large number of potential targets, mainly directed at orientating the cytokinic response toward an antiinflammatory profile, neutralizating proinflammatory cytokines or their receptors, inducing regulatory lymphocytes in order to normalize the state of T and B cell tolerance, and modulating cellular cooperation and lymphocytic homing by blocking adhesion molecules. Some of these new approaches have already been validated in autoimmune diseases, others will follow soon.

Under non-autoimmune conditions, rheumatoid factor (RF) B cells coexist peacefully with their antigen (IgG), or can be transiently activated during secondary immune responses because they can present xenoantigens to specific T cells captured in immune complex form. Such a situation should lead to affinity maturation of RF B cells and potentially dangerous production of high-affinity RF. We used two lines of transgenic mice expressing a somatically mutated pathological human RF in presence (IgM and IgD) or in absence (IgM only) of surface IgD, and confirm that RF B cell tolerance can result from an antigen-induced specific, but incomplete, deletion of naive RF B cells after antigen encounter. This deletion mainly concerns immature, transitional B cells. On the contrary, mature, IgM- and IgD-expressing RF B cells are resistant to such a deletion. These IgM and IgD RF B cells are functional and activable through both B cell receptor dependent (anti-IgM) and independent (LPS) pathways, but they are not fully responsive to human IgG either in vivo or in vitro. Taken together, these results suggest that another mechanism could be involved in the silencing of mature naive IgM and IgD RF B cells.

Introduction: Langerhans cell pulmonary histiocytosis is a rare disease, primarily enhanced by smoking, and of unclear mechanism., Observation: A 42 year-old man, smoking 25 packs-years, was infected by a type 1 human immunodeficiency virus (HIV-1). He successively developed pulmonary emphysema, Langerhans cell pulmonary histiocytosis and alveolar bronchial carcinoma of the lower right pulmonary lobe, which was fatal., Discussion: We discuss the concomitance of pulmonary histiocytosis and alveolar bronchial carcinoma, exceptional in the literature, and the eventual enhancing role of HIV-1 infection. The principal incriminating factor in pulmonary histiocytosis probably remains smoking, but the HIV-1 infection may have participated in the emergence of the neoplastic pathology.

The mechanisms which govern the production of autoantibodies and of tissue damage during systemic lupus (SLE) are still unclear. In the NZBxNZW F1 (BW) model of SLE glomerulonephritis, the activation and commitment of B cells are thought to play a major role in disease progression. Previous analysis has suggested that these mice have a substantial increase of marginal zone (MZ) B cells before the occurrence of the disease. Owing to the probable role of this B cell subset in autoantibody production, it was important to define the possible link between this abnormality and the occurrence of kidney damage. Using cytofluorometry analysis, we followed the splenic MZ B cell phenotype in different series of mice with shared autoimmune genetic background and histologically defined renal status. By comparing BW females and BW males, NZB and NZW mice, we confirm that BW mice have an increase in MZ B cells but this MZ B cells expansion is not directly linked to tissue lesions. Genetically modified BW female mice with a restricted repertoire of B and T cell antigen receptors, and which do not develop nephritis, exhibit the same increase of MZ B cells, suggesting that this increase does not depend on a specific set of antigens. Moreover, our analysis brings to light a pre-disease state in BW males, with autoantibody production and mesangial deposits.

The variable regions of the immunoglobulin (Ig) expressed on the surface of a malignant B cell can be considered tumor-specific antigens and, as such, could be targets for immunotherapeutic approaches. However, because until now the immunization procedures have been complex and have given only partial protection, it was necessary to find new methods of immunotherapy. Here, we present a successful method of vaccination against B-cell tumors in a murine model. We produced recombinant vaccinia viruses (rVV) expressing the heavy and the light chain of surface Ig of a patient's malignant B cells and we tested the ability of these rVV to protect immunized mice against tumor growth of transfectomas producing the same human Ig. The protection of the mice was complete and specific to the variable region of the immunizing heavy chain although specific lymphoproliferative and cytotoxic responses were not detectable in vitro. The protection was strictly dependent on the presence of CD4 T cells and asialo GM1+ cells. Furthermore, tumor protection clearly required gamma-interferon and was partially inhibited by blocking the Fas-Fas ligand interaction. We also show, in a murine syngeneic model, that rVV expressing a poorly mutated Ig protects against the growth of Ig-producing tumor.

The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis reveals the extreme heterogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune response and the mechanism of the prothrombotic state associated with these antibodies, but it could also shed some light on their possible origins. (Blood. 2001;97:3820-3828)

Naturally occurring autoreactive B cells are thought to be physically eliminated or rendered functionally silent through different mechanisms of tolerance. However, multireactive low affinity natural autoantibody-producing B cells seem to escape these mechanisms in normal adults and could constitute the B cell pool from which pathological autoantibodies can emerge. To analyze this apparent paradox to the clonal tolerance theory, we have made two transgenic mouse lines (mu(k), mudelta(k)) producing a natural low affinity multireactive human autoantibody. These models enable us to test both the central tolerance mechanisms (reactivity with single-stranded DNA) and the peripheral tolerance mechanisms after Ag administration. Not only are the multireactive B cells not deleted in the bone marrow, they circulate and remain in the periphery even after the prolonged administration of Ag, the presence of membrane IgD increasing the number of mature autoreactive B cells. Self-reactive B cells are shown to be autoantigen ignorant both in vivo and in vitro, but they are not anergic because they can be easily activated through both B cell receptor-dependent and -independent pathways. Thus, these mouse lines reproduce an apparent paradox to the clonal tolerance theory meriting further investigation of the biological significance of this phenomenon.

Objective: To assess the association between the occurrence first of preeclampsia and antiphospholipid antibodies., Methods: We conducted a prospective case-control study of 180 pregnant women with their first incidents of preeclampsia and no histories of thrombosis or systemic autoimmune diseases. Preeclampsia (n = 180) was defined as blood pressure (BP) at least 140/90 mmHg after 20 weeks' gestation and proteinuria at least 0.3 g per 24 hours. Two control subjects were matched to each case (n = 360). They were pregnant women without hypertension or proteinuria and without histories of thrombosis or systemic autoimmune disease. Lupus anticoagulant (activated partial thromboplastin time, diluted thromboplastin time, platelet neutralization procedure) and anticardiolipin antibodies (immunoenzymatic assays) were assessed in both groups, and the coagulation state (levels of thrombin-antithrombin III complexes, fragments 1 + 2 of prothrombin) was also evaluated. The analysis design was a sequential plan with 5% type I error and 95% power., Results: There was no association between antiphospholipid antibodies and preeclampsia. The odds ratio for the association was 0.95 (95% confidence interval 0.45, 2.61). Antiphospholipid antibodies were detected in eight of 180 preeclamptic women and in 19 of 360 controls. In contrast, there was a clear, confirmed activation of coagulation during preeclampsia., Conclusion: Despite evidence of a prothrombotic state during preeclampsia, it is unlikely that antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies) represent risk factors for preeclampsia among women with no previous preeclampsia and no histories of thrombosis or systemic autoimmune disease.

Purpose: Considering the importance to public health and the frequency with which drug addicts are imprisoned, we studied the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV), as well as drug addiction of patients admitted to the Elsau prison in Strasbourg (France)., Methods: The prospective study included all entering inmates from 1 September to 31 October 1997 (270 persons) to whom HIV, HBV and HCV blood tests were offered as well as a questionnaire on their drug addiction., Results: Thirty-six percent of the entering inmates were drug addicts, of whom 1% were HIV positive, 11.2% HBV positive and 30% HCV positive, compared to, respectively, 0.6, 9.9 and 6.4% for non-drug addicts. Ninety-five of the 98 patients used several drugs, including buprenorphine for 53 patients. At the beginning of this study, buprenorphine had been available in France for 9 months., Conclusion: The results are to be taken seriously regarding the misuse of this product in this selected population (intravenous use, multiple drug use, dealing).

Objective: Patients with Sjögren's syndrome (SS) have an increased risk of developing monoclonal B cell non-Hodgkin's lymphomas (MNHL), which frequently occur in the salivary glands (SG). The transition from the benign lymphocyte infiltrate of the gland that characterizes SS to MNHL is not well understood. Previous sequence analyses of the expressed variable (V) region genes have supported the theory that the surface Ig (sIg) plays an important role in the initial expansion of nonmalignant B cell clones and in lymphomagenesis. However, the antigenic specificities of these B cells were unknown. We describe the specificities of the Ig expressed by 2 cases of MNHL that developed in the SG of 2 patients with SS., Methods: The expressed V genes were amplified by polymerase chain reaction from biopsy specimens, sequenced, and subcloned into eukaryotic expression vectors. The constructs were transfected into P3X63-Ag8.653 cells to obtain 2 monoclonal cell lines, each secreting 1 of the sIg expressed by the MNHL. These IgM were tested by enzyme-linked immunosorbent assay and immunofluorescence against a panel of antigens potentially implicated in SS., Results: Our main finding was that the Ig products of the neoplastic B cells were rheumatoid factors (RF). Contrary to expectations, they did not react with nuclear or cytoplasmic antigens, double-stranded DNA, self antigens commonly bound by natural autoantibodies, or SG tissue., Conclusion: Previous analyses of V gene use have provided indirect evidence that SG MNHL may frequently express RF. We demonstrate that this hypothesis is true in the 2 patients we studied. Large-scale studies will be needed to establish the exact frequency of RF specificity among SS-associated MNHL.

Rheumatoid arthritis is a common and debilitating autoimmune disease whose cause and mechanism remain a mystery. We recently described a T cell receptor transgenic mouse model that spontaneously develops a disease with most of the clinical, histological, and immunological features of rheumatoid arthritis in humans. Disease development in K/BxN mice is initiated by systemic T cell self-reactivity; it requires T cells, as expected, but B cells are also needed, more surprisingly. Here, we have identified the role of B cells as the secretion of arthritogenic immunoglobulins. We suggest that a similar scenario may unfold in some other arthritis models and in human patients, beginning with pervasive T cell autoreactivity and ending in immunoglobulin-provoked joint destruction.

AIDS-associated primary central nervous system lymphomas are late events that have an extremely poor prognosis. Despite different hypotheses, the brain localization of these B cell lymphomas remains an enigma. To better define the cell origin of the lymphomas and the possible role of the B cell receptor (BCR) in the brain localization and/or in the oncogenic transformation, we analyzed the V region genes of the Ig heavy chain expressed by lymphoma cells in five randomly selected patients. After amplifying the rearranged VHDJH DNA by PCR, cloning, and sequencing of the amplified products, we observed that: 1) of the five lymphomas analyzed, four were clearly monoclonal; 2) there was no preferential use of one peculiar VH family or one peculiar segment of gene; 3) the mutation analysis showed that an Ag-driven process occurred in at least two cases, probably before the oncogenic event; and 4) there was no intraclonal variability, suggesting that the hypermutation mechanism is no longer efficient in these lymphoma B cells. Taken together, our results suggest that distinct Ags could be recognized by the BCR of the lymphoma cells in different patients and that, if the Ags are responsible for the brain localization of these B cells bearing mutated BCR, other factors must be involved in B cell transformations in primary central nervous system lymphoma.

Terminal deoxynucleotidyl transferase (TdT) enzyme activity in lymphocytes generates diversity in the Ag receptor repertoires by adding template-independent N nucleotides and disrupting homology-directed rearrangements. The importance of this diversity in vivo and the significance of the suppression of TdT during fetal life remain uncertain. Previous studies have shown that in TdT knockout mice (TdT(0)) 1) the T cell repertoire is less peptide oriented; and 2) natural autoantibody, particularly anti-DNA autoantibodies, are less polyreactive, and their mean affinities are reduced. Consequently, the suppression of TdT during early T/B cell ontogeny may participate in controlling autoimmunity. To study the impact of TdT suppression in autoimmune-prone mice, we introduced the TdT null mutation into the (NZB x NZW)F1 (B/W) mouse strain. We show that TdT deficiency significantly reduces the incidence of autoimmune nephritis and prolongs survival compared with those in control mice. Surprisingly, the long-term survivor TdT(0) mice produced amounts of anti-ADN and anti-histone autoantibodies similar to those of their TdT+ littermates. However, these TdT(0) mice showed no evidence of renal inflammation, and the immune deposits were restricted to the mesangium, whereas basal membrane deposits were clearly correlated with overt renal disease. The present study supports the idea that the absence of TdT enzyme activity in lymphocytes protects mice against autoimmunity and could offer a therapeutic approach to autoimmune diseases. Moreover, our results may help to unravel the mechanisms of lupus nephritis.

Objective: To study the variations of type II soluble receptors for tumor necrosis factor (sR-TNF) in patients with systemic lupus erythematosus and investigate their use in the clinical setting., Patients and Methods: Twenty-six patients with systemic lupus were followed for a mean 3 years. sR-TNF and other immunological parameters (C reactive protein, anti-DNA antibodies, C3 and C4 complement fractions, soluble receptors for interleukin 2) were measured in sera at different points of the disease course. The systemic lupus activity measure (SLAM) was determined at each point, and confronted with the biology results. The study was cross sectional for the group and longitudinal for the patients., Results: sR-TNF was the immunological parameter which correlated best with SLAM. It also correlated with sedimentation rate, C reactive protein, thrombopenia, anemia, creatinine level, anti-DNA antibodies and sR-IL2. The longitudinal study pointed out however that this finding is not consistent for each patient., Conclusion: A rise in sR-TNF related to systemic lupus activity but is of limited practical interest for individual patient follow-up.

The generation of terminal deoxynucleotidyl transferase knockout mice (TdT0) has demonstrated that TdT is the only major activity involved in N region addition. This enzyme generates diversity by adding random nucleotides at the V-D-J junctions and by disrupting the formation of repetitive "homology-directed" junctions. Several studies have demonstrated that the Ig heavy chain third complementarity-determining region (H-CDR3) and the N region play a critical role: 1) in distinguishing between polyreactive and monospecific combining sites in natural and Ag-induced Abs; and 2) in the specificity and polyreactivity of natural autoantibodies (autoAbs) and in particular of anti-DNA Abs. To examine the impact of the lack of TdT on the natural autoAb repertoire in adult mice, we have stimulated TdT0 and TdT+ littermates with LPS. Serum studies demonstrate that TdT is not critical for the generation of B cells expressing autoAbs including anti-DNA Abs and rheumatoid factors. However, the generation of a large collection of hybridomas indicates that the frequencies of these cells are reduced in TdT0 mice mainly due to a lower incidence of polyreactivity; also, the lack of N region diversity seems to negatively affect the affinity of anti-DNA Abs. The physiologic relevance of these data is discussed.

The plasma membrane remodeling, including the early transverse redistribution of phosphatidylserine, is a general feature occurring in cells in which a death program has been induced. In most cases, studies of this kind have focused mainly on cells. In this study, we report a clear correlation between the degree of apoptosis induced by a variety of agents in several types of cultured cells and the amount of shed membrane microparticles captured in the corresponding supernatants by insolubilized annexin V, a protein showing a strong affinity for phosphatidylserine. Such particles carry membrane antigens specific of the cells they stem from, and through which capture is also feasible. Homologous circulating microparticles were captured in peripheral blood from individuals with HIV-1 infection. A substantial proportion bore CD4 antigen. In some cases, CD4+ particles could be detected even in the absence of circulating CD4+ T cells, testifying to the presence of such resident cells in lymphoid tissues. These results suggest that shed membrane particles are one of the hallmarks of programmed cell death, of particular interest when the corresponding cells are hardly accessible.

Steroid-induced lipomatosis usually presents as a localized hypertrophy of the adipose tissue and seems more common than previously thought. Most patients develop this phenomenon after prolonged administration of moderate to high doses of oral corticosteroids. The localizations are numerous and determine the clinical presentation. Often asymptomatic, they can also be revealed by worrying symptoms usually due to a compressive syndrome. The most frequently reported localizations (spinal epidural, retro-orbital, mediastinal) are also the most clinically apparent. The cessation or reduction of steroid therapy, when medically possible, inconsistently results in the decrease or disappearance of the lipomatosis deposits. Computerized tomography or magnetic resonance imaging are the most helpful diagnostic means. Interestingly, these lipomatoses have rarely been reported in patients with Cushing disease. Their pathophysiology remains poorly elucidated and may imply an inhibition of the brown adipose tissue lipolysis.

Aim: To compare the peroperative blood loss and the postoperative systemic inflammatory reaction in patients receiving either a Vasculour II Albumin pre-impregnated prosthesis (VA group, n = 32) or a preclotted Vasculour II prosthesis (V group, n = 33) for elective surgery of the abdominal aorta., Setting: University Hospital., Design: Prospective, randomised study., Methods: Peroperative blood loss was measured over two different periods: Phase I from the beginning of the operation to the completion of the proximal anastomosis, when blood loss cannot be related to the model of prosthesis implanted and phase II after the completion of the proximal anastomosis to the end of the operation. Postoperative blood loss was evaluated by the determination of the retroperitoneal drainage volume over a period of 2 days immediately following the operation. The presence of periprosthetic fluid was measured with echography at days 4, 9, 30 and 60. The postoperative systemic inflammatory reaction was evaluated by measuring the sedimentation rate and the C reactive protein levels daily from day 1 to day 9, and at days 14, 21, 28, 45, and 60, and by measuring the body temperature daily from day 1 to day 9., Results: No significant differences of peroperative blood loss were observed. The same proportion of patients (35%) in both groups received homologous transfusion. The mean number of units of homologous blood transfused per patient was respectively 0.77 and 0.91 for the VA and the V group. The retroperitoneal drainage volume and the percentage of patients with periprosthetic fluid did not differ significantly. No significant differences in systemic postoperative inflammatory reaction were observed., Conclusion: There were no benefits in using albumin-impregnated prosthesis as opposed to preclotted prosthesis in terms of peroperative and postoperative blood loss, or by looking at the incidence of homologous blood transfusion. However, the glutaraldehyde cross-linked albumin did not induce any systemic inflammatory reaction.

Antiphospholipid antibodies (APL) are usually detected using solid-phase immunoassays, where cardiolipin is the most common capture antigen. Phosholipids are believed to adopt a monolayer organization when coated onto polystyrene after evaporation of the solvent. However, bilayer phospholipids are probably those evidenced as microparticles or cell fragments circulating in vivo under various pathological circumstances. The surface density of monolayer phospholipids on polystyrene is six times lower than that of bilayer phospholipids. In order to assess the influence of phospholipid organization on the detection of APL, we prepared glass microspheres coated with bilayer phospholipids (cardiolipin, phosphatidylcholine, cholesterol). Such lipospheres enabled us to study the binding of antibodies in 1:100 diluted plasma samples from patients with anticardiolipin antibodies of IgG isotype previously diagnosed by ELISA. Among the 39 plasma samples analysed by flow cytometry, 17 showed positive IgG binding to lipospheres. Only four additional samples became positive when adding 20 micrograms/ml apolipoprotein H. The specificity of the binding was demonstrated by complete reversibility with 1.4 microM annexin V and with a large excess of liposomes of the same composition. The absence of correlation between liposphere and ELISA results suggests that different subgroups of antibodies are detected depending on the method. The detection of APL using bilayer phospholipids is an original assay and may represent a more physiopathological approach to the specificity of APL.

Monoreactive high affinity pathologic autoantibodies were supposed previously to derive through somatic mutation from polyreactive low affinity autoantibodies that are encoded by a small set of unmutated V region genes in fetal and neonatal B cells. However, recent data exploring the physiologically expressed Ab repertoire and the importance of the stochastically generated heavy chain CDR3 (H-CDR3) in autoreactivity suggest that this scheme is incomplete. Here we analyzed via gene-swapping experiments and site-directed mutagenesis the relative contributions of the mutations in the light chain variable region (VL) and the heavy chain variable region (VH) domains and of the H-CDR3 in the autoreactivity of two IgM rheumatoid factors (RF), one a polyreactive low affinity Ab, the other a monoreactive high affinity Ab. These two RFs derived from the same V kappa III (humkv325) and VH1 (51p1) genes, but differed from each other by a few mutations and by the structure of the H-CDR3. The analysis of the reactivity patterns of different combinations of wild-type and in vitro engineered hybrid gene products clearly demonstrates the main influence of the H-CDR3 in the autoAb activity profiles. The results directly demonstrate the previously proposed hypothesis, namely, that the H-CDR3 plays a critical role in distinguishing poly- from monospecific RF. However, the data also indicate that self polyreactivity is a very fragile property and is dependent upon the primary structure of the VH segment.

Background: Endogenous posterior uveitis (PU) can be associated with systemic diseases, and certain forms have strong association with HLA antigens. Much less is known regarding intermediate uveitis (IU). The purpose of this study was to determine whether IU is associated with the HLA system and whether it can be associated with systemic symptoms., Methods: In 179 consecutive patients consulting for uveitis, a detailed history was obtained and a physical examination performed. HLA typing for 71 HLA-A, B, DR and DQ antigens, laboratory tests, and radiography of the chest, sinuses, and sacroiliac joints were systematically performed., Results: Thirty-two patients (18%) had IU; 51 (28.5%) had PU and constituted our internal control group. Nine of the patients with IU (28%) had the HLA-A28 antigen, compared with 8.1% of a healthy control population and 8.6% of the patients with PU (P < 0.001). An associated disease was found in four patients with IU (12.5%) (none was HLA-A28) and in 45% of the patients with PU (P < 0.01). Some 67% of HLA-A28 patients with IU had arthralgias affecting the knee(s), compared with 17% of non-HLA-A28 patients and 18% of patients with PU (P < 0.05 and P < 0.01 respectively); 55% had gonalgias and hypocomplementemia compared with 9% and 2% respectively (P < 0.01 and P < 0.001)., Conclusions: IU is significantly associated with HLA-A28; patients having this antigen may represent a subset of the disease characterized by an increased prevalence of arthralgias and hypocomplementemia.

The treatment of severe lupus nephritis remains problematic. We have analysed retrospectively 17 patients with corticoresistant lupus nephritis treated with pulse cyclophosphamide. Single monthly doses (500 mg/m2) were given by intravenous infusion with a mean of 10.4 infusions per patient (3 to 18). A comparison of parameters at entry and at the end of the treatment revealed an improvement in proteinuria (4.8 vs 1.9 g/24 h; p < 0.013) whereas mean serum creatinine level and SLAM (Systemic Lupus Activity Measure) remained stable. The results were identical at follow up (mean: 14.5 months). Most of the therapeutic effect was achieved as soon as the 6th pulse. Further treatment was beneficial for four patients only. None of the studied parameters (serum creatinine level, renal biopsy, SLAM) was predictive of a response to an extended course of pulse cyclophosphamide. The infusions were definitively stopped in one patient and delayed in two others because of serious adverse effects. The data indicate that, in mean term, monthly intravenous cyclophosphamide was associated with a substantial amelioration of 24 hours urinary protein level. An amelioration of the renal function was however, uncommon.

Mixed cryoglobulinemia is usually considered to be a nonmalignant human B cell proliferation that produces a monoclonal IgM rheumatoid factor (RF). Important immunologic similarities and differences were described between the monoclonal B cells during mixed cryoglobulinemia and during malignant chronic lymphocytic leukemia (CLL):high frequency of the same VH and V kappa gene usage by both types of monoclonal B cells producing IgM with RF activity, apparent intraclonal homogeneity, but different expression of the pan T cell CD5 Ag. The description of an unusual CD5-negative B cell CLL case secreting a mutated IgM RF led the authors to suggest that the usage of non-mutated germline Ig genes is a property of cells derived from the CD5 lineage or stage of differentiation, rather than an intrinsic property of CLL or of IgM RF-producing cells in general. Because mixed cryoglobulinemia cells are usually CD5-negative, it was of interest to test for the existence of mutations in the VH and V kappa regions, as well as for the intraclonal homogeneity of the expressed Ig genes. In this study, we used the PCR technique to analyze the monoclonal rheumatoid factor (mRF) V genes from a patient with mixed cryoglobulinemia. We show that the CD5-negative monoclonal B cells express a slightly mutated V kappa 3 gene, but a more mutated VH1 gene whose genomic counterpart was shown to be the 51p1 germline gene. The sequence analysis of several independent clones shows some degree of intraclonal diversity, suggesting the existence of a clonal filiation. These results are discussed in terms of the origin of the monoclonal B cell during mixed cryoglobulinemia and CLL.

SMI is a previously characterized IgM kappa polyreactive (natural) autoantibody. The variable regions of the heavy and light chains of SMI are respectively encoded by a nonmutated VH1 gene, designated 51p1, and a conserved nonmutated V kappa gene, designated Humkv325. These V genes seem to be over-represented in the autoimmune and fetal B cell repertoires, and to be frequently expressed in malignant B cells during certain lymphoid proliferations such as chronic lymphocytic leukemia. Polyreactive natural autoantibodies are thought to rely mainly on the use of such V genes in germ-line configuration. However, this model underestimates the contribution of the somatically generated heavy chain third complementarity-determining region (HCDR3) to autoantibody specificity. We used oligonucleotide site-directed mutagenesis to permute the sequence of the SMI-HCDR3 to generate a family of mutant proteins, each of which differed from the original SMI-IgM kappa by one amino acid residue. This allowed us to examine the relative contribution of selected amino acid residues in this region to the binding affinity of SMI against a panel of self-Ags. We found that a single amino acid substitution within the HCDR3 could dramatically alter the specificity of this autoantibody. Some substitutions abrogated the reactivity with all the tested Ags, whereas others changed the affinity or spectrum of reactivity for certain self-Ags. These results demonstrate that the autoantibody-binding activity of these conserved autoantibody-associated germ-line V genes is dependent upon heavy chain junctional sequences that are generated somatically during Ig gene rearrangement.

Several genetic mechanisms have been shown to diversify the expressed antibody repertoire of committed B lymphocytes. These include V gene replacement, ongoing gene rearrangement and somatic hypermutation. These mechanisms may be operational at discrete points in the B cell differentiation pathway and generate idiotype diversity in various malignant B cell tumors. In particular, V region mutations have been established as a major mechanism of tumor escape from anti-idiotype immunotherapy in some lymphoma. On the other hand, previous studies on a few selected cases have shown that this mutation process does not affect the B cell clone during chronic lymphocytic leukemia. However, to what extent this intraclonal stability is a general phenomenon during B cell CLL is not clear. Therefore, we randomly selected 6 patients suffering from classical B cell CLL (sIgM (+), CD5 (+), CD19 (+)) at different stages of the disease and analysed the intraclonal variability of the expressed variable region of the heavy chain (VH). After PCR amplification of the cDNA corresponding to the rearranged VDJ regions, the products were cloned and sequenced. In five cases, multiple clone analysis did not show any intraclonal variability whatever the stage of the disease. Furthermore, in a single case, this intraclonal stability was confirmed during a three year period of time when the disease progressed. The sixth case behaved differently since we found multiple nucleotide substitutions, apparently accumulating as the malignant clone expanded. Besides the theoretical difficulties that these changes can induce during immunotherapy, two findings merit further discussion: 1) the distribution of the ongoing mutations affecting the VH region was not suggestive of an antigen driven selection, 2) this intraclonal variability was specific for the VH region, since the VL region showed no intraclonal variation.

There is evidence that in certain situations the expressed antibody repertoire is dominated by small subsets of V gene segments. They include fetal, CD5+, and autoantibody-forming B cells as well as low grade B cell malignancies. For instance, inside the V kappa III family of approximately 10 members, only 3 (humkv325, 328, and Vg) are used recurrently for autoantibody production. However, the significance of this recurrence is difficult to interpret without a clear vision of the actual repertoire in normal subjects. To address this, we have sequenced and compared two sets of rearranged V kappa III genes generated by cDNA PCR amplification from a normal newborn, a normal adult, and from CD5+ B cells of the same adult donor. The results show that: (a) only four V kappa III gene segments are used by neonatal and total adult B cells (humkv325, humkv328, Vg, and kv305), humkv325 being overexpressed in both repertoires; (b) there is no significant difference in terms of V kappa III gene usage between the adult and newborn repertoires; (c) regarding the junction regions, there is a favored use of the most 5' JK gene segments (Jk1-Jk2); approximately 20% of the newborn and adult junction sequences was characterized by one or two additional codons, most probably resulting from a nontemplate addition of nucleotides; (d) adult clones, in contrast to most newborn clones, show sequence divergences from prototype sequences with patterns which suggest antigen-driven diversity; (e) regarding the adult CD5+ B cell library, it is most probable that the 78 clones analyzed derived from no more than nine different VK-JK rearrangements. Humkv325 is used by at least six of them, and most of the expressed V genes were in exact or very near germline configuration. Collectively these results suggest that the expressed antibody V kappa III repertoire in the adult represents only a fraction of the potential genetic information and that it resembles the preimmune repertoire of the neonate. The data, which also suggest that the adult peripheral blood CD5+ B cell population may be dominated by a small number of B cell clones, are discussed with regards to the V kappa III usage in pathological situations.

The authors report a case of granulomatous mastitis associated with erythema nodosum and oligoarthritis. The skin and joint symptoms improved with potassium iodide. The breast lesion clinically simulated a tumor of high malignant grade. Granulomatous mastitis is a benign and rare disease. Its interest lies in the possible association with systemic manifestations and in its steroid responsiveness. This condition has been recently described as a distinct entity. When associated with systemic manifestations, sarcoidosis should be considered.

Organ-specific autoimmune diseases are usually considered to be mediated by autoreactive T cells which infiltrate the target tissue. Conceivably, these T cells could represent pathogenic autoreactive cells which recognize their specific antigen (peptide or superantigen) within the pathological tissue. Extensive studies dealing with the clonality of the infiltrating autoreactive cells gave conflicting results both in humans and animals. One possibility for explaining these contradictory data could rely on the stage of the disease when the T cell population is studied. Here, we report on this parameter by analyzing T cell receptor beta-chain variable regions of infiltrating T cells involved during one of the most frequent human organ-specific autoimmune disease, the primary Sjögren's syndrome. Six patients were selected on the basis of the duration of the disease before the biopsy procedure (two early and four late stages) to analyze initial and late T cell waves within the abnormal tissue. Using short-term interleukin-2-stimulated T cells, polymerase chain reactions, Southern and sequence analysis, we conclude that: (a) there is a clear restriction in the V beta usage by the infiltrating T cells only during the early stage of the disease, (b) this V beta restriction is related to a monoclonal T cell expansion, (c) the expanded V beta families are different from one patient to the other, and (d) there is no clear homology in length or amino acid composition in the CDR3 of the analyzed V beta regions. These results could provide an explanation to conflicting results on the V beta restriction usage during autoimmune diseases and could indicate time limitations in anti-V beta treatment. Furthermore, the monoclonal expansion of particular V beta-bearing T cells argues against a role for a superantigen during this disease.