Impairment of quality of life in patients with antiphospholipid syndrome.

OBJECTIVES: Health-related quality of life (HRQoL) has not been fully explored in antiphospholipid syndrome (APS); therefore, we compared HRQoL between APS patients and the general population and assessed the impact of thromboembolic history. METHODS: HRQoL was measured in a multicentre cohort study by the Medical Outcomes Study Short-Form 36 (MOS-SF-36) questionnaire. HRQoL scores were compared to the French general population norms. Factors significantly associated with an impaired HRQoL were identified. RESULTS: A total of 115 patients with aPL and/or systemic lupus erythematosus (SLE) were included (mean age 42.7 ± 14.1 years old, 86 women). In 53 patients APS was diagnosed. Compared to general population norms, patients with APS had an impaired HRQoL. SLE-associated APS patients had the worst HRQoL scores (physical component summary (PCS)=40.8 ± 10.6; mental component summary (MCS)=40.6 ± 16.5) in comparison with SLE or aPL patients without thromboembolic history. In APS patients, history of arterial thrombosis significantly impaired HRQoL (PCS score: 42.2 ± 9.4 vs 49.2 ± 8.5; MCS score: 33.9 ± 13.7 vs 44.6 ± 10.3). CONCLUSION: Compared to the general population, APS patients experienced a lower HRQoL. In these patients, a history of arterial thrombosis significantly impaired HRQoL. Therefore, measurements of HRQoL should be included in APS patient management to assess the burden of the disease from a patient's perspective and to provide patients with the support they need.

Value of (18)F-FDG-PET/CT in patients with fever of unknown origin and unexplained prolonged inflammatory syndrome: a single centre analysis experience.

OBJECTIVE: The aim of our study was to evaluate the diagnostic contribution of (18)F-fluoro-deoxyglucose ((18)F-FDG)-positron emission tomography (PET)/computed tomography (CT) in patients with fever of unknown origin (FUO) or unexplained prolonged inflammatory syndrome (UPIS) in real life. PATIENTS AND METHODS: We performed a retrospective study including 14 patients with FUO or UPIS hospitalised in our institution (Strasbourg University Hospital, France) between January 2005 and July 2006. (18)F-FDG-PET/CT was considered helpful when abnormal results allowed an accurate diagnosis. RESULTS: (18)F-FDG-PET/CT was helpful in half the patients (7/14) for final diagnosis. A diagnosis was reached in 87.5% of the patients (7/8) with an abnormal (18)F-FDG-PET/CT but only in 50% of the patients (3/6) with a normal (18)F-FDG-PET/CT. Conventional chest and abdominal CT was performed in 13 patients before ordering (18)F-FDG-PET/CT. We considered that (18)F-FDG-PET/CT was essential to establish the final diagnosis in only 23% of the patients (3/13) since neither chest nor abdominal CT identified abnormalities consistent with the final diagnosis. However, among the three patients, two were diagnosed with large vessel vasculitis and one patient with local prosthetic infection. CONCLUSIONS: Our study supports the potential interest of (18)F-FDG-PET/CT in the diagnostic workup of FUO and UPIS as it helped establish a fine diagnosis in half of the cases. However, (18)F-FDG-PET/CT appeared to be essential to the final diagnosis in only 23% of the cases. In our opinion, this protocol should be performed as a second level test, especially when conventional CT is normal or is unable to discriminate between active and silent lesions.

Evidence that the V kappa III gene usage is nonstochastic in both adult and newborn peripheral B cells and that peripheral CD5+ adult B cells are oligoclonal.

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.

Inheritance of immunoglobulin M rheumatoid-factor idiotypes.

The idiotypic determinants on IgM rheumatoid factor (RF) from a single family have been analyzed. Rabbit Fab'2 antiidiotypic antibody was prepared against purified IgM-RF from a patient with rheumatoid arthritis. As measured by radioimmunoassay, the antiidiotype reacted with at least 90% of the patient's RF, but not with non-RF immunoglobulins from the same serum, nor with 10 of 11 polyclonal and monoclonal RF from unrelated individuals. Cross-reacting idiotypes were detected on RF in four of the patients' first degree relatives, spanning three generations, without apparent relation of HLA type or clinical rheumatoid arthritis. These results suggest that IgM-RF associated idiotypes were inherited in this family.

The significance of shed membrane particles during programmed cell death in vitro, and in vivo, in HIV-1 infection.

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.

Videocapsule endoscopy is useful for the diagnosis of intestinal lymphangiectasia.

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.

Recombinant vaccinia viruses expressing immunoglobulin variable regions efficiently and selectively protect mice against tumoral B-cell growth.

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.

Fractionation of human lymphocyte subpopulations on immunoglobulin coated Petri dishes.

This report describes a simple solid-phase technique for the positive selection of lymphocytes labeled with fluoresceinated antibodies. B lymphocytes were labeled with fluoresceinated anti-human Ig or monoclonal anti-human Ia (L243), and then were bound to plastic culture dishes coated with affinity purified goat anti-fluorescein specific antibody. Bound cells were eluted at 37 degrees C with 1 mM fluorescein-L-lysine phosphate-buffered saline. Functionally the eluted Ig positive cells responded to pokeweed mitogen (PWM) by in vitro secretion of IgM, as measured by radioimmunoassay of culture supernatants. The secretion of IgM was dependent on the addition of T lymphocytes. Moreover, the isolated B cells were functionally receptive to 'help' and 'suppression' by T cells with and without Fc receptors for IgG respectively. T cell subsets were fractionated on plastic culture dishes coated with heat aggregated rabbit or human IgG. the non-bound cells (enriched T(gamma-)) provided collaborative 'help' in the PWM induced IgM secretion response by human B lymphocytes. The bound cells (enriched T(gamma+)) eluted with 0.01 M EDTA in phosphate-buffered saline, suppressed IgM secretion. This method can be adapted to fractionate subsets of lymphocytes for which a fluoresceinated antibody is available. For routine functional studies, the isolation of cell types with conventional or monoclonal antibodies does not require the use of a fluorescence activated cell sorter, but only an antifluorescein labeled Petri dish. In conclusion, a rapid solid-phase technique enables us to prepare enriched populations of functionally active lymphocytes.

Immunogenicity and epitope mapping of a recombinant soluble gp160 of the human immunodeficiency virus type 1 envelope glycoprotein.

The human immunodeficiency virus 1 envelope glycoprotein is synthesized as a precursor, gp160, which is subsequently cleaved to generate the external gp120 and the transmembrane gp41. Both of these cleavage products are known to mediate critical functions of the virus. In order to define the best strategy for the development of a vaccine against human immunodeficiency virus 1, it could be important to map the crucial epitopes on gp160. This entire gp160 is uneasy to purify because it is readily subjected to proteolytic cleavage. Furthermore, it is anchored on the cell membrane and needs detergent treatment for purification. We thus used a recombinant gp160 which was engineered to remove the cleavage sites between gp120 and gp41 and the hydrophobic transmembrane in order to investigate the murine immune response. We selected a panel of 8 monoclonal antibodies which recognize different epitopes on the immunizing recombinant soluble gp160. The reactivity of the monoclonal antibodies was checked on virus-derived gp160, gp120, and gp41. Three antibodies reacted only with gp120 but the others were shown to react with gp41 epitopes or with discontinuous epitopes bridging gp120 and gp41. One subregion of these epitopes was located using a synthetic peptide corresponding to the sequence of gp41. This epitope is apparently part of an immunodominant site since it is recognized by three different monoclonal antibodies. We used competitive inhibition experiments to map the epitopes on recombinant gp160; therefore, the results are probably indicative of the folding of the recombinant soluble gp160 used for immunization.

Permissivity of primary cultures of human Kupffer cells for HIV-1.

Kupffer cells (liver macrophages) represent the largest reservoir of fixed macrophages in the body. Accordingly, we have undertaken a study to evaluate their susceptibility to human immunodeficiency virus type 1 (HIV-1). Five-day-old primary cultures of Kupffer cells (KC) were infected with HIV-1, and as the infection progressed, syncytia appeared. Within the cells, viral proteins were detected by immunofluorescence using monoclonal antibodies directed against gp120 and p24. Electron microscopic examinations revealed the presence of typical Lentivirinae particles. The particles released from KC in the extracellular medium showed reverse transcriptase activity and p24 antigen; they could infect lymphocytic cells and were neutralized by a HIV+ patient's serum or an anti-gp120 monoclonal antibody. Our results thus demonstrate that the interaction of HIV-1 with KC in vitro leads to a productive infection. They suggest that the KC may be involved in the pathogenesis of HIV-1 infection and may (i) participate in the transmission of the infection to the peripheral blood cells, (ii) play a role in the depletion of uninfected CD4+ cells.

Analysis of the V kappa III variable regions of polyclonal rheumatoid factors arising during Epstein Barr virus induced infectious mononucleosis.

The mechanisms that govern autoantibody production are still under debate. In particular, auto-antibodies can appear as a consequence of a polyclonal activation of B cells or as a consequence of an antigen driven B cell expansion. The molecular analysis of the variable regions of auto-antibodies arising during different clinical situations can help to understand the origin of auto-antibodies. We recently described the main light chain variable regions of polyclonal rheumatoid factors occurring during rheumatoid arthritis and suggested that the mutation pattern of these regions could reflect an antigen driven process. Using the same approach, we now report the molecular analysis of the same light chain variable region containing a VKIII segment of rheumatoid factors originating from a polyclonal activation of B cells during an in vivo Epstein-Barr virus infection, infectious mononucleosis. The cDNA derived from rheumatoid factor synthetizing cells were amplified by two sets of polymerase chain reaction. The amplified products were cloned in M13mp19 phages and sequenced. The nucleotide analysis of the VKIII containing VK regions shows that: 1) the rheumatoid factor activity is associated with the 3 VKIII genes (Kv 325, Kv 328 and Vg) already known to encode for monoclonal and polyclonal rheumatoid factors, 2) there is a preferential use of Kv 328 and Vg, each one of these genes being poorly mutated, 3) the CDR mutation rates of these genes is no higher than the framework mutation rates, 4) there is a restriction of the JK usage; Kv 328 derived gene segments rearrange exclusively with JK1, Vg preferentially rearranges with JK1 and JK4. These results mainly suggest that naturally occurring polyclonal activation of autoreactive B cells produces poorly mutated autoantibodies.

A minor group of rheumatoid factors isolated from a patient with rheumatoid arthritis is derived from somatically mutated Vk1 genes further evidence that rheumatoid factors during autoimmune diseases undergo an antigen driven maturation.

To better understand the structural basis for rheumatoid factor [RF] activity and the origin of autoantibodies in human autoimmune diseases, we isolated the RF producing B cells from the peripheral blood and from the synovial fluid of a patient suffering from rheumatoid arthritis [RA]. We previously demonstrated that a significant fraction of these RF were derived from three V kappa III genes known to encode most of the monoclonal RF light chain variable regions. To get more insight into the actual repertoire of RF-V kappa genes during RA, we analyzed the nucleotide sequences of RF light chain variable regions of other V kappa families. Using two sets of polymerase chain reactions in order to amplify the cDNA derived from RF producing cells from the same patient KRA, we isolated only three different rearranged V kappa-J kappa complexes: slkv5, slkv7 and bkv42, all derived from V kappa I germ-line genes not previously known to be associated with RF activity; this suggests that the repertoire of VL genes coding for RF during RA is more diverse than the one involved in the generation of paraprotein RF during monoclonal lymphoid proliferations, although there remains a possible bias in favor of the V kappa III family. Moreover, each of these genes is somatically mutated with a pattern suggesting a selective pressure of the antigen. Particularly interesting is the additional proline residue at the V kappa-J kappa junction of bkv42, an unorthodox feature that we found previously in more than 50% of RF V kappa III-J kappa gene complexes. Finally, the homogeneity of some non conservative mutations suggests the existence of a restricted set of pathogenic epitopes driving the production of RF during RA.

Antiphospholipid antibodies and preeclampsia: a case-control study.

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.

CD5 negative IGM rheumatoid factor B cells in B-chronic lymphocytic leukemia and benign mixed cryoglobulinemia.

IgM-RF B cell precursors are abnormally overrepresented in "well differentiated" lymphoid monoclonal proliferations while data on less mature lymphoid malignancies are still awaited. This nevertheless suggests that RF activity plays a role in the transforming process perhaps by inducing constant stimulation of the precursor B cells. Despite the preferential use of similar VH and VL genes with little or no somatic hypermutations in both malignant B-cell CLL and nonmalignant mixed cryoglobulinemia, these proliferations do differ in CD5 membrane expression and in their clinical evolution. One possibility could be that CD5 glycoprotein is lost during maturation of the lymphocyte into a secreting cell as suggested by data on Waldenström's disease and the LES-CLL and by in vitro studies. Alternatively, CD5 expression could play an additional direct role in malignant transformation as suggested by recent data on the CD5 receptor ligand. Further data on the proliferating cells in both situations as well as on the genetic control of CD5 expression in B cells and its physiology should shed additional light on the mechanisms of B-cell malignancy.

Molecular analysis of rearranged VH genes during B cell chronic lymphocytic leukemia: intraclonal stability is frequent but not constant.

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.

Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope.

The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

[Early surgical treatment of a septal perforation complicating a posterior infarct. Value of the diaphragmatic left ventricular approach]. / Cure chirurgicale précoce d'une perforation septale compliquant un infarctus postérieur. Intérêt de l'abord ventriculaire gauche diaphragmatique.

The authors report the case of a 53 year old patient who required operation on the 5th day after postero-inferior myocardical infarction for a poorly tolerated perforation of the ventricular septum. In discussing this case, they recall that the results for surgical repair of septal perforations complicating myocardial infarction are poorer when the infarction is posterior than when it is anterior. They suggest that this difference in prognosis is in large part due to the customary use in postero-inferior infarcts, of the right transventricular approach, which does not allow the infarct to be resected at the same time as the septum is closed. They finish by recommending the systematic use of a diaphragmatic approach to the left ventricle, including resection of the infarct, for all cases of septal perforations with posterior infarction in which surgery is necessary.

[The immune system: new therapeutic targets]. / Système immunitaire: nouvelles cibles thérapeutiques.

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.