Ikaros Is a Negative Regulator of B1 Cell Development and Function.

B1 B cells secrete most of the circulating natural antibodies and are considered key effector cells of the innate immune response. However, B1 cell-associated antibodies often cross-react with self-antigens, which leads to autoimmunity, and B1 cells have been implicated in cancer. How B1 cell activity is regulated remains unclear. We show that the Ikaros transcription factor is a major negative regulator of B1 cell development and function. Using conditional knock-out mouse models to delete Ikaros at different locations, we show that Ikaros-deficient mice exhibit specific and significant increases in splenic and bone marrow B1 cell numbers, and that the B1 progenitor cell pool is increased ∼10-fold in the bone marrow. Ikaros-null B1 cells resemble WT B1 cells at the molecular and cellular levels, but show a down-regulation of signaling components important for inhibiting proliferation and immunoglobulin production. Ikaros-null B1 cells hyper-react to TLR4 stimulation and secrete high amounts of IgM autoantibodies. These results indicate that Ikaros is required to limit B1 cell homeostasis in the adult.

Chronic bacterial infection activates autoreactive B cells and induces isotype switching and autoantigen-driven mutations.

The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity.

IgM rheumatoid factor amplifies the inflammatory response of macrophages induced by the rheumatoid arthritis-specific immune complexes containing anticitrullinated protein antibodies.

OBJECTIVES: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC. METHODS: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC. RESULTS: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1ß and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes. CONCLUSIONS: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity.

Responsiveness of the 36-item Short Form Health Survey and the Lupus Quality of Life questionnaire in SLE.

OBJECTIVES: This study aimed to estimate the responsiveness to change of a generic [the 36-item Short Form Health Survey (SF-36)] and a specific health-related quality of life questionnaire [the Lupus Quality if Life questionnaire (LupusQoL)] according to SLE patients' self-reported changes in health status. METHODS: In a cohort of 185 SLE patients, quality of life (QoL) was measured three times at 3 month intervals by the LupusQoL and SF-36 questionnaires. Anchors for responsiveness were defined by patients' global assessment of disease impact according to changes in a visual analogue scale (VAS), a 7-point Likert scale and a 0-3 scale of five patient-reported symptoms. Mean change and s.d. in worsening and improving patients according to anchors were estimated using mixed models for repeated measures. Standardized response means (SRMs) were calculated in each group. RESULTS: Patients [mean age 39.6 years (s.d. 10.5), mean Safety of Estrogen in Lupus Erythematosus National Assessment-SLEDAI score 2.6 (s.d. 3.5)] answered a total of 515 questionnaires. For the VAS and Likert global anchors, worsening patients showed a significant decrease in all LupusQoL domains except for burden to others, body image and fatigue and all SF-36 domains with low to moderate responsiveness. Improving patients had a significant increase in all LupusQoL domains except for intimate relationship and all SF-36 domains except for physical functioning and global health with low to moderate responsiveness. Regarding similar domains in the SF-36 and LupusQoL, SRMs were higher in LupusQoL domains in improving patients, while SF-36 domains had larger SRMs in worsening patients. CONCLUSION: Both the SF-36 and LupusQoL were responsive to changes in QoL in SLE patients over a 3 month interval. LupusQoL seems to be more appropriate to measure improvements in QoL.

Validity of the global anti-phospholipid syndrome score to predict thrombosis: a prospective multicentre cohort study.

OBJECTIVE: To investigate the validity of the global APS score (GAPSS) to predict thrombosis in patients with autoimmune diseases. METHODS: This prospective cohort study included consecutive patients with aPL or SLE. aPL, aPS-PT and GAPSS were determined. A Cox proportional hazards model assessed the validity of GAPSS and identified other potential independent predictors of thrombosis. RESULTS: One hundred and thirty-seven patients [43.5 (s.d. 15.4) years old; 107 women] were followed up for a mean duration of 43.1 (s.d. 20.7) months. Mean GAPSS was significantly higher in patients who experienced a thrombotic event compared with those without [10.88 (s.d. 5.06) vs 8.15 (s.d. 5.31), respectively, P = 0.038]. In univariate analysis, age [hazard ratio (HR) = 1.04 (95% CI 1.01, 1.08)] and GAPSS above 16 [HR = 6.86 (95% CI 1.90, 24.77)] were each significantly associated with thrombosis during follow-up, while history of arterial thrombosis [HR = 2.61 (95% CI 0.87, 7.82)] failed to reach significance. Among aPL assays, IgG aPS/PT--a component of the GAPSS--was significantly associated with thrombosis [HR = 2.95 (95% CI 1.02, 8.51)]. In multivariate analysis, GAPSS above 16 remained the only significant predictor of thrombosis [HR = 6.17 (95% CI 1.70, 22.40)]. CONCLUSION: This first external validation study confirmed that GAPSS can predict thrombosis in patients with aPL and associated autoimmune diseases.

Progranulin antibodies in autoimmune diseases.

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

Synovial fibroblasts promote immunoglobulin class switching by a mechanism involving BAFF.

Fibroblast-like synoviocytes (FLSs) are important actors in rheumatoid arthritis (RA) pathogenesis. The autoimmune nature of RA is attributed to autoantibody production, which confers to B cells a predominant role in RA. Several arguments support an induction of class switch recombination (CSR) in RA synovium, causing--in conjunction with somatic hypermutation--the production of potentially pathogenic IgG. To determine whether RA FLSs can directly promote CSR and to analyze the role of external factors like TLR signals and BAFF (B cell activating factor) family cytokines in this FLS-B cell crosstalk, we performed cocultures of blood B cells (from normal individuals or RA patients) with RA FLSs and analyzed CSR induction by quantification of AICDA (encoding activation-induced cytidine deaminase, AID) and switch circular transcripts expression, and IgG secretion. RA FLSs--and to a lesser extent osteoarthritis or control FLSs--promoted CSR, and TLR3 stimulation potentialized it. In addition, induction of CSR by RA FLSs was totally dependent on cell-cell contact in basal conditions, and partially dependent in the case of TLR3 stimulation. Finally, we showed that the mechanism by which RA FLSs induce CSR is mostly BAFF-dependent. Our results support the hypothesis that CSR can be induced outside the ectopic lymphoid structures in RA.

LupusQoL-FR is valid to assess quality of life in patients with systemic lupus erythematosus.

OBJECTIVE: To cross-culturally adapt the LupusQoL into French, to test its measurement properties and to further investigate its domain structure. METHODS: The cultural adaptation process according to guidelines and pre-testing resulted in the LupusQoL-FR. SLE patients completed the LupusQoL-FR at baseline, 15 days, 3 months and 6 months. Validity was studied through content and construct validity (factorial and Rasch analysis for structural validity, Spearman's correlation and Mann-Whitney tests for external validity). Cronbach's α and intra-class correlation coefficients were computed for reliability. The standardized response mean was computed to evaluate responsiveness. RESULTS: In all, 182 patients, age 39.6 (10.6) years, mostly outpatients [mean SELENA-SLEDAI 2.6 (3.5)] were recruited. Factor analysis with eight imposed factors was very close to the original LupusQoL. A screeplot with parallel analysis showed that LupusQoL domains could be aggregated in two physical and mental scales. Both eight- and two-factor structures showed a good Rasch fit, internal consistency (Cronbach's α: 0.85-0.95), and test-retest reliability (intra-class correlation coefficient 0.79-0.95). External convergent (correlation with SF-36, r=0.59-0.78) and divergent validity (according to SELENA-SLEDAI) were also satisfactory. CONCLUSION: The LupusQoL-FR is valid to assess quality of life in SLE patients. A two-factor structure of physical and mental aggregated scales is a valid alternative to the original eight-domain structure.

Control of B cells expressing naturally occurring autoantibodies.

Naturally occurring autoantibodies (NAbs) are typically polyreactive, bind with low affinity to a discrete set of autoantigens and are encoded by variable region genes in germline configuration. They differ from disease-associated autoantibodies (autoAb), which are mostly monoreactive, somatically mutated and of high affinities. Structure-function studies have shown that polyreactivity of NAbs relies on the somatically generated complementarity determining region, CDR3, of the heavy chain. This finding suggested that NAb-producing B cells were positively selected from the pre-immune B-cell repertoire. The biological significance of this selection remains, however, unclear. Data originating mainly from transgenic mice have shown that mature NAb-producing B cells are frequently ignorant toward their antigen, possibly due to their low affinity, though active tolerance mechanisms are not excluded. An important issue is whether NAb-producing B cells constitute the pool from which pathologic auto Ab emerge after autoantigen-driven maturation. We summarize results obtained in mouse models, showing that some infectious agents are able to induce an autoantigen-driven activation of certain NAb-producing B cells. However direct proof that selection by autoantigen may lead to somatic hypermutation are still lacking. Other data tend to suggest that pathologic auto Abs may derive from non-autoimmune B cells that have diversified by somatic hypermutation of their variable region genes.

Wegener's granuloma harbors B lymphocytes with specificities against a proinflammatory transmembrane protein and a tetraspanin.

Wegener's granulomatosis (WG) is a severe autoimmune disorder ranging from localized granulomatous disease to generalised anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. A previous analysis of immunoglobulin heavy chain genes derived from tissue, i.e. Wegener's granuloma indicated selection and affinity maturation towards local antigen(s). The current study focused on determining the specificity of immunoglobulins from distinct B lymphocytes out of Wegener's granuloma. Four pairs of variable region immunoglobulin light and heavy chain genes, isolated before, were recombinantly expressed using the baculovirus/insect cell system. These immunoglobulins were then analysed for their antigenic target employing a protein macroarray based upon a human fetal brain tissue cDNA expression library. The lysosomal transmembrane protein 9B, a key regulator for TNFα activation, was identified as the putative antigenic target of two immunoglobulins and a tetraspanin, which might play a role in leukocyte activation and motility, was identified as the putative antigenic target of another one. Recombinant monoclonal antibodies out of Wegener's granuloma represent a new tool aiding in elucidation of its and WG immunopathogenesis.

Autoimmunity in common variable immunodeficiency: correlation with lymphocyte phenotype in the French DEFI study.

Common variable immunodeficiency (CVID) is the most frequent clinically expressed primary immunodeficiency in adults and is characterized by primary defective immunoglobulin production. Besides recurrent infectious manifestations, up to 20% of CVID patients develop autoimmune complications. In this study, we took advantages of the French DEFI database to investigate possible correlations between peripheral lymphocyte subpopulations and autoimmune clinical expression in CVID adult patients. In order to analyse homogeneous populations of patients with precise clinical phenotypes, we first focused on patients with autoimmune cytopenia because they represent prototypic autoantibody mediated diseases. In a secondary analysis, we have tested our conclusions including all "autoimmune" CVID patients. We describe one of the largest European studies with 311 CVID patients, including 55 patients with autoimmune cytopenia and 61 patients with clinical or serologic autoimmune expression, excluding autoimmune cytopenia. We clarify previous reports and we confirm a very significant correlation between an increased proportion of CD21(low) B cells and CVID associated autoimmune cytopenia, but independently of the presence of other autoimmune disorders or of splenomegaly. Moreover, in CVID associated autoimmune cytopenia, T cells display an activated phenotype with an increase of HLA-DR and CD95 expression and a decrease in the naïve T cell numbers. Patients with other autoimmune manifestations do not harbour this "T and B cells phenotypic picture". In view of recent findings on CD21(low) B cells in CVID and RA, we suggest that both a restricted subset of B cells and a T cell help are required for a breakdown of B cell tolerance against membrane auto antigens in CVID.

Peripheral B cell abnormalities in patients with systemic lupus erythematosus in quiescent phase: decreased memory B cells and membrane CD19 expression.

B lymphocytes from patients with systemic lupus erythematosus (SLE) are hyperactive and produce autoantibodies. Several B cell phenotype characteristics such as the expansion of activated populations, and of a newly identified memory compartment have already been reported. These results are not easy to interpret because of the clinical heterogeneity of SLE, as well as the difficulties to establish homogeneous and well defined groups taking in consideration the activity of the disease and the various therapies. However, although many mediators and mechanisms can contribute to the clinical presentation and subsequent progression of individuals with SLE, several data suggest that some intrinsic B cells abnormalities may be central to the disease process. In this view, we have analysed the phenotype of B cells from 18 patients with quiescent diseases (mean SLEDAI score below 2) and from 11 healthy controls. B cell surface marker expression was determined by flow cytometry. We analysed the main B cell sub-populations. We demonstrate the persistence of plasmocyte-differentiated and -activated B cells even in quiescent patients. However, quiescent patients display a decrease in memory B cells that could reflect the control of their disease. Above all, we describe a lower membrane expression of the CD19 protein on all B cells in every patient compared to controls. This lower CD19 expression is associated with reduced CD45 levels. It is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. These findings suggest certain mechanisms of lupus development.

MyD88 negatively controls hypergammaglobulinemia with autoantibody production during bacterial infection.

A large body of evidence has convincingly shown that Toll-like receptors are necessary sensors for infections with pathogens, but their activation was also suggested to generate autoimmunity. During experimental infections, the lack of these sensors or of their signaling molecules should lead to a deficient immune response. We found out that MyD88, the major adaptor of the Toll/interleukin-1 (Toll/IL-1) receptor signaling pathway, can actually act as a negative regulator of B-cell function in some settings. MyD88-deficient mice infected by Borrelia burgdorferi developed extreme hypergammaglobulinemia compared to wild-type animals, with high levels of immunoglobulin M (IgM) autoantibodies. In vivo, cell transfer experiments and cell blocking assays showed that this phenotype was not linked to the absence of MyD88 in B cells but rather to CD4 T-cell and likely dendritic cell dysfunctions leading to a Th1-to-Th2 cytokine switch. In addition, our results suggest a relative defect in the Ig class switch recombination process, since MyD88 knockout mice developed mostly IgM antibodies. Collectively, these data emphasize the complex role of the Toll/IL-1 receptor pathway in tuning the immune response against infection and avoiding autoimmunity.

Autoantigen, innate immunity, and T cells cooperate to break B cell tolerance during bacterial infection.

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.

[Pathogenesis of the antiphospholipid syndrome]. / Physiopathologie du syndrome des antiphospholipides.

The antiphospholipid syndrome is characterized by the association of clinical events (thrombosis and/or obstetrical complications) and heterogeneous autoantibodies reacting with complexes of proteins and anionic phospholipids. Most of these recognized proteins can bind to anionic phospholipids and play a role in natural regulation of coagulation. Inhibition by these autoantibodies of the natural regulators of excessive coagulation is probably responsible for the prothrombotic state that characterizes this disease. Animal models have helped explain the mechanisms of obstetrical complications. Human antiphospholipid antibodies passively transferred to pregnant mice directly cause fetal resorption. This effect is mediated by complement fractions and neutrophil activation and is inhibited by heparin. The origin of these autoantibodies is still debated. Physiologically, the cause may be associated with exposure to anionic phospholipids on the surface of apoptotic cells. The affinity maturation process, which leads to the acquisition of somatic mutations, can then generate highly pathogenic antiphospholipid antibodies.

Phenotyping of autoreactive B cells with labeled nucleosomes in 56R transgenic mice.

The phenotypic characterization of self-reactive B cells producing autoantibodies is one of the challenges to get further insight in the physiopathology of autoimmune diseases. We took advantage of our previously developed flow cytometry method, using labeled nucleosomes, prominent autoantigens in systemic lupus erythematosus, to analyze the phenotype of self-reactive B cells in the anti-DNA B6.56R mouse model. We showed that splenic anti-nucleosome B cells express mostly kappa light chains and harbor a marginal zone phenotype. Moreover, these autoreactive B cells fail to acquire a germinal center phenotype and are less abundant in the transitional T3 compartment. In conclusion, the direct detection of autoreactive B cells helped determine their phenotypic characteristics and provided a more direct insight into the B cell tolerance process in B6.56R mice. This method constitutes an interesting new tool to study the mechanisms of B cell tolerance breakdown in B6.56R mice crossed with autoimmune prone models.

Upper and lower gastrointestinal endoscopies in patients over 85 years of age: Risk-benefit evaluation of a longitudinal cohort.

After age 85, upper and lower gastrointestinal (GI) endoscopy may be indicated in 5% to 10% of inpatients, but the risk-benefit ratio is unknown. We studied patients older than 85 years undergoing upper and lower GI endoscopy.We analyzed a retrospective cohort of inpatients older than 85 years between 2004 and 2012, all explored by upper and complete lower GI endoscopy. Initial indications, including iron deficiency anemia (IDA), other anemias, GI bleeding, weight loss, and GI symptoms, were noted, as were endoscopy or anesthesia complications, immediate endoscopic diagnosis, and the ability to modify the patients' therapeutics. Deaths and final diagnosis for initial endoscopic indication were analyzed after at least 12 months.We included 55 patients, 78% women, with a median age, reticulocyte count, hemoglobin, and ferritin levels of 87 (85-99), 56 (24-214) g/L, 8.6 (4.8-12.9) g/dL, and 56 (3-799) µg/L, respectively. IDA was the most frequent indication for endoscopy (60%; n = 33). Immediate diagnoses were found in 64% of the patients (n = 35), including 25% with GI cancers (n = 14) and 22% with gastroduodenal ulcers or erosions (n = 12). Cancer diagnosis was associated with lower reticulocyte count (45 vs. 60 G/L; P = .02). Among the 35 diagnoses, 94% (n = 33) led to modifications of the patients' therapeutics, with 29% of the patients deciding on palliative care (n = 10). No endoscopic complications lead to death. Follow-up of >12 months was available in 82% (n = 45) of the patients; among these patients, 40% (n = 27) died after an average 24 ±â€Š18 months. Cancer diagnosis was significantly associated with less ulterior red cell transfusion (0% vs. 28%; P = .02) and fewer further investigations (6.7% vs. 40%; P = .02).Upper and complete lower GI endoscopy in patients older than 85 years appears to be safe, and enables a high rate of immediate diagnosis, with significant modifications of therapeutics. GI cancers represented more than one-third of the endoscopic diagnoses.

Identification of autoreactive B cells with labeled nucleosomes.

The pathogenesis of autoimmune diseases has not been completely elucidated yet, and only a few specific treatments have been developed so far. In autoimmune diseases mediated by pathogenic autoantibodies, such as systemic lupus erythematosus, the specific detection and analysis of autoreactive B cells is crucial for a better understanding of the physiopathology. Biological characterization of these cells may help to define new therapeutic targets. Very few techniques allowing the precise detection of autoreactive B cells have been described so far. Herein we propose a new flow cytometry technique for specific detection of anti-nucleosome B cells, which secrete autoantibodies in systemic lupus erythematosus, using labeled nucleosomes. We produced different fluorochrome-labeled nucleosomes, characterized them, and finally tested them in flow cytometry. Nucleosomes labeled via the cysteines present in H3 histone specifically bind to autoreactive B cells in the anti-DNA transgenic B6.56R mice model. The present work validates the use of fluorochrome-labeled nucleosomes via cysteines to identify anti-nucleosome B cells and offers new opportunities for the description of autoreactive B cell phenotype.

Outcome of endoscopy-negative iron deficiency anemia in patients above 65: A longitudinal multicenter cohort.

After the age of 65 years, iron deficiency anemia (IDA) requires the elimination of digestive neoplasia and is explored with upper and lower gastrointestinal (GI) endoscopy. However, such explorations are negative in 14% to 37% of patients. To further evaluate this issue, we evaluated the outcomes of patients aged over 65 years with endoscopy-negative IDA.We retrospectively analyzed the outcomes of in-patients over the age of 65 years with IDA (hemoglobin <12 g/dL and ferritin <70 µg/L) who had negative complete upper and lower GI endoscopies in 7 tertiary medical hospitals. Death, the persistence of anemia, further investigations, and the final diagnosis for IDA were analyzed after at least 12 months by calling the patients' general practitioners and using hospital records.Between 2004 and 2011, 69 patients (74% women) with a median age of 78 (interquartile range (IQR) 75-82) years and hemoglobin and ferritin levels of 8.4 (IQR 6.8-9.9) g/dL and 14 (IQR 8-27) µg/L, respectively, had endoscopy-negative IDA, and 73% of these patients received daily antithrombotics. After a follow-up of 41 ±â€Š22 months, 23 (33%) of the patients were dead; 5 deaths were linked with the IDA, and 45 (65%) patients had persistent anemia, which was significantly associated with death (P = 0.007). Further investigations were performed in 45 patients; 64% of the second-look GI endoscopies led to significant changes in treatment compared with 25% for the capsule endoscopies. Conventional diagnoses of IDA were ultimately established for 19 (27%) patients and included 3 cancer patients. Among the 50 other patients, 40 (58%) had antithrombotics.In endoscopy-negative IDA over the age of 65 years, further investigations should be reserved for patients with persistent anemia, and second-look GI endoscopy should be favored. If the results of these investigations are negative, the role of antithrombotics should be considered.