Jean-Benoît Arlet

An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia

The pathophysiology of ineffective erythropoiesis in β-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of β-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with β-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in β-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas–Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in β-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in β-thalassaemia

β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.

Ineffective Erythropoiesis in β-Thalassemia

In humans, β-thalassemia dyserythropoiesis is characterized by expansion of early erythroid precursors and erythroid progenitors and then ineffective erythropoiesis. This ineffective erythropoiesis is defined as a suboptimal production of mature erythrocytes originating from a proliferating pool of immature erythroblasts. It is characterized by (1) accelerated erythroid differentiation, (2) maturation blockade at the polychromatophilic stage, and (3) death of erythroid precursors. Despite extensive knowledge of molecular defects causing β-thalassemia, less is known about the mechanisms responsible for ineffective erythropoiesis. In this paper, we will focus on the underlying mechanisms leading to premature death of thalassemic erythroid precursors in the bone marrow.

Pulmonary fibrosis in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis: a series of 49 patients and review of the literature.

AbstractPulmonary fibrosis (PF) is an uncommon manifestation observed in patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), particularly microscopic polyangiitis (MPA). While patients with PF associated with AAV seem to have a worse prognosis, these patients have been described only in case reports or small retrospective case series. In this retrospective multicenter study, we report the main features and long-term outcomes of patients with PF associated with AAV, fulfilling the American College of Rheumatology criteria and/or Chapel Hill definitions. Forty-nine patients (30 men [61%]; median age at diagnosis of AAV, 68 [interquartile range, 58–73] years) with PF associated with AAV were identified. Forty (81.6%) patients had MPA and 9 (18.4%) had granulomatosis with polyangiitis. The diagnosis of PF preceded the onset of vasculitis in 22 (45%) patients. Usual interstitial pneumonia was the main radiologic pattern (n = 18, 43%). ANCA were mostly of antimyeloperoxidase specificity (88%). All patients were treated with glucocorticoids as induction therapy, combined with cyclophosphamide (CYC) (n = 36, 73.5%) or rituximab (RTX) (n = 1, 2%). Factors associated with mortality included occurrence of chronic respiratory insufficiency (hazard ratio [HR], 7.44; 95% confidence interval [CI], 1.6–34.5; p = 0.003), induction therapy with glucocorticoids alone (HR, 2.94; CI, 1.05–8.33; p = 0.04), and initial weigh loss (HR, 2.83; CI, 1.05–7.65; p = 0.041). The 3-year survival rate in patients treated with glucocorticoids alone or combined with an immunosuppressant (CYC or RTX) as induction therapy was 64% (95% CI, 41–99) and 94% (95% CI, 86–100), respectively (p = 0.03). After a median follow-up of 48 months [interquartile range, 14–88 mo], 18 (37%) patients died, including 11 related to respiratory insufficiency. PF is a rare manifestation of AAV with a very poor prognosis. Induction therapy with CYC might improve the outcome.

Defective nuclear localization of Hsp70 is associated with dyserythropoiesis and GATA-1 cleavage in myelodysplastic syndromes

Normal human erythroid cell maturation requests the transcription factor GATA-1 and a transient activation of caspase-3, with GATA-1 being protected from caspase-3-mediated cleavage by interaction with the chaperone heat shock protein 70 (Hsp70) in the nucleus. Erythroid cell dysplasia observed in early myelodysplastic syndromes (MDS) involves impairment of differentiation and excess of apoptosis with a burst of caspase activation. Analysis of gene expression in MDS erythroblasts obtained by ex vivo cultures demonstrates the down-regulation of a set of GATA-1 transcriptional target genes, including GYPA that encodes glycophorin A (GPA), and the up-regulation of members of the HSP70 family. GATA-1 protein expression is decreased in MDS erythroblasts, but restores in the presence of a pan-caspase inhibitor. Expression of a mutated GATA-1 that cannot be cleaved by caspase-3 rescues the transcription of GATA-1 targets, and the erythroid differentiation, but does not improve survival. Hsp70 fails to protect GATA-1 from caspases because the protein does not accumulate in the nucleus with active caspase-3. Expression of a nucleus-targeted mutant of Hsp70 protects GATA-1 and rescues MDS erythroid cell differentiation. Alteration of Hsp70 cytosolic-nuclear shuttling is a major feature of MDS that favors GATA-1 cleavage and differentiation impairment, but not apoptosis, in dysplastic erythroblasts.

Nuclear-to-cytoplasmic Relocalization of the Proliferating Cell Nuclear Antigen (PCNA) during Differentiation Involves a Chromosome Region Maintenance 1 (CRM1)-dependent Export and Is a Prerequisite for PCNA Antiapoptotic Activity in Mature Neutrophils

Background: PCNA is exclusively cytoplasmic in neutrophils and inhibits their apoptosis. Results: Nuclear export of PCNA is mediated by a CRM1-dependent nuclear export sequence (NES) and is pivotal for its antiapoptotic activity in mature neutrophils. Conclusion: Cytoplasmic relocalization of PCNA is part of the terminal differentiation of neutrophils. Significance: This process might be relevant to modulate neutrophil survival in inflammation or neutropenia. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34+ primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.

Visceral Leishmaniasis Mimicking Systemic Lupus Erythematosus

To the Editor: We read with interest the recent report by Kritikos et al on an atypical presentation of visceral leishmaniasis (VL) in a patient with rheumatoid arthritis previously treated with infliximab. We would like to emphasize another atypical presentation of this parasitic infection that may mimic systemic lupus erythematosus (SLE), and report on an erroneous diagnosis of lupusrelated hemophagocytic syndrome (HS) in a patient with VL. A 55-year-old woman originating from the south of France presented with a 2-month history of weight loss, fever, and pancytopenia. Her medical history was remarkable for a remote pulmonary tuberculosis, a Hashimoto thyroiditis, nonalcoholic steatohepatitis, and nonactive hepatitis B virus infection. Since September 2005, she was complaining of myalgia, wrist, and proximal interphalangeal arthralgia without synovitis, and Raynaud phenomenon. She denied sicca syndrome, photosensitivity, or skin rash. Physical examination was normal at that time. Laboratory findings were positive for antinuclear antibodies (ANA) (1/640, homogeneous pattern), with positive antidoublestranded DNA antibodies (antidsDNA, using Crithidia luciliae 1/10). C-reactive protein and blood count were normal except for a mild leukopenia. Rheumatoid factor was negative, but anticyclic citrullinated peptide testing was positive. Standard radiographs of both hands were unremarkable. A presumptive diagnosis of articular lupus erythematosus was considered. Nonsteroidal anti-inflammatory drugs were ineffective, and the patient was secondarily briefly treated with prednisone. She was admitted in January 2007 because of a 10 kg weight loss, spiking fever (up to 39°C), fatigue, and dry cough. Physical examination revealed moderate hepatomegaly and splenomegaly. Laboratory tests showed hemoglobin 11.2 g/dL, leukocytes 1,600/ L with neutrophils 980/ L and lymphocytes 410/ L, platelets 125,000/ L, Creactive protein 54 mg/L, ferritin 5,000 g/L, triglyceride serum level 3.7 g/L (N 1.8), elevated serum transaminase levels (4 N), estimated creatinine clearance of 53 mL/min, mild proteinuria (0.6 g/24 hours) without hematuria, and normal serum level of gammaglobulin. Blood and sputum bacterial and mycobacterial cultures were negative. Viral serologic tests including HIV and hepatitis B PCR testing were negative. ANA titer was 1/1000 (both speckled and homogenous pattern), antidsDNA title was 1/54 (by ELISA; negative by indirect immunofluorescence assay), and auto-antibodies against SSB and RNP were detected. Auto-antibodies against Sm, SSA, Scl-70, JO1, and neutrophil cytoplasm were absent. Isolated lupus anticoagulant was detected (anticardiolipin and anti2GPI antibodies were absent). Serum complement level was normal. Two bone marrow aspirations and biopsy did not show evidence of infectious agent, hemophagocytosis, or malignancy. Bone marrow culture for mycobacterium tuberculosis was negative. Serological test and culture for leishmaniasis were not performed at that time. Thoracic and abdominal CT scan confirmed hepatomegaly and splenomegaly, without lymph node enlargement. Fever persisted, pancytopenia, and hepatosplenomegaly became worse. A lupus-related HS was suspected but could not be demonstrated (negative bone marrow aspiration). Methylprednisolone pulse and then intravenous immunoglobulins (1 g/kg daily for 2 days) were ineffective. Intravenous etoposide (150 mg/ m) was then administered with a dramatic effect in 24 hours. Over the next 2 months, intravenous cyclophosphamide (0.6 g/m/ mo), hydroxychloroquine (400 mg daily), and prednisone (1 mg/kg daily) were added. Two suspected HS relapse with fever, pancytopenia, hyperferritinemia (up to 18,900 g/L), hypertriglyceridemia (up to 7.8 g/L), elevated liver enzymes, spleen and liver enlargement were treated with new etoposide pulses. In May 2007, at the time of the second suspected HS relapse, the general status was poor. ANA titer was 1/500 and antidsDNA was negative. Splenectomy and liver biopsy were performed. Histologic findings showed numerous intraand extracellular Leishmania amastigotes with signs of hemophagocytosis, both in the liver and spleen specimens (Figure 1). Testing for leishmaniasis infection was positive in blood, spleen, and liver using PCR, and culture revealed Leishmania infantum species. Retrospectively, serological testing for Leishmania antibodies and PCR test in the blood revealed that the patient had positive results since her initial admission, before intensification of immunosuppressive therapy. The patient was treated by intravenous liposomal amphotericin B (4 mg/kg/d, days 1–5 and 10 and then every week for 4 weeks). The fever disappeared within 24 hours, blood count and CRP normalized within 5 days, and detection of Leishmania by PCR was negative after 2 weeks. All immunosuppressive drugs were discontinued and prednisone rapidly tapered. Thirty months after amphotericin B therapy was completed, the patient was asymptomatic; ANA titer remained positive (1/500), without any specificity. L. infantum produces both cutaneous and VL, and is endemic in the South-East of France. The parasite invades and grows up in reticuloendothelial cells, which then rupture and release the organism into the bloodstream. The incubation period for VL varies from months to years, and during this time the parasite multiplies in the reticuloendothelial system, including the liver, the spleen, and the bone marrow. Underlying factors such as HIV coinfection, malnutrition, or treatment with corticosteroids or immunosuppressive drugs may lead to disease reactivation, which explains that VL may also occur in patients with SLE as an opportunistic infection and mimic a SLE flare as it has been previously reported in 11 patients. FIGURE 1. Spleen biopsy showing intraand extracellular Leishmania amastigotes within macrophages (RAL reagent 100).

The senescent form of Jaccoud arthropathy.

A 84-year-old woman presented with a 20 year history of mechanical pain in hands attributed to osteoarthritis. She had no significant medical history except for degenerative chronic low back pain and cervical lymph node tuberculosis. Especially, she had no history of rheumatic fever or systemic lupus. Physical examination showed a painless bilateral metacarpophalangeal (MCP) joint subluxation with ulnar deviation without synovitis (panel A) that could be passively corrected (panel B). The patient had noticed progressive bilateral hand deformity for the past 15 years. She only reported minor and intermittent pain in the fingers that occurred during activity. Standard radiographs revealed a nonerosive deforming arthropathy and evidence of digital osteoarthritis (panel C). There was evidence of small hooks at the third MCPs and minimal and questionable erosions of the second MCPs. There was no increase of acute phase reactants and no clinical or biologic signs for systemic disease (negative tests for antinuclear antibodies, rheumatoid factor, and anti-CCP antibodies) or evidence of crystal-induced arthropathy. These characteristics conformed to the arthropathy initially described by Jaccoud in 1869 as a rare complication of rheumatic fever. Currently, the more common cause of Jaccoud arthropathy in young patients is systemic lupus erythematosus. It has also been reported in various diseases including other connective tissue diseases or crystal-induced arthropathies. Jaccoud arthropathy may be misleading and suggestive of rheumatoid arthritis. The presence of erosions and hooks are exceptional in Jaccoud arthropathy and if present are not typical of rheumatoid arthritis. Our case illustrates the idiopathic or “senescent” form of the syndrome, which affects older patients and may concern 40% of patients with Jaccoud arthropathy.

Efficiency of hydroxychloroquine in the treatment of granulomatous complications in chronic granulomatous disease.

Chronic granulomatous disease (CGD) represents a group of genetic disorders in which impaired intracellular microbial killing by phagocytes leads to recurrent bacterial and fungal infections and granuloma formation. Granulomatous lesions involved gastrointestinal tract in more than one-third of all CGD patients, with various clinical presentations, and may be life threatening. Corticosteroid therapy may relieve symptoms but increase the risk of infections in such immunocompromised patients. We report here the first successful management, with hydroxychloroquine therapy alone, of a 29-year-old CGD man with severe gastric granulomatous involvement. Hydroxychloroquine appears to be a safe and effective alternative treatment of granulomas in CGD patients.

Novel players in β-thalassemia dyserythropoiesis and new therapeutic strategies.

Purpose of reviewThe review provides an overview of recent data regarding the molecular players in &bgr;-thalassemia dyserythropoiesis and the corresponding therapeutic implications. Recent findings&bgr;-thalassemia dyserythropoiesis is characterized by four steps: expansion of erythroid progenitors, accelerated erythroid differentiation until the polychromatophilic stage, maturation arrest, and apoptosis at the polychromatophilic stage. Excess &agr;-globin chains are the primary culprit in the disease, but the link between this excess and ineffective erythropoiesis has only recently been established. Important recent advances in understanding the molecular determinants involved in two critical steps of dyserythropoiesis are paving the way to new alternative targets for the treatment of this disease. SummaryGrowth differentiation factor 11 (GDF11) blockade increases the apoptosis of erythroblasts with excess &agr;-chains by upregulating Fas-ligand in late basophilic and polychromatophilic erythroblasts, thereby decreasing cell expansion (step 1). Blocking GDF11 alleviates anemia in a mouse model of &bgr;-thalassemia and also in humans, most likely by promoting cells of ‘good’ erythroblastic lineage containing an &agr;-/non-&agr;-globin chain ratio of close to 1. Maturation arrest at the polychromatophilic stage (step 3) is associated with the depletion of GATA binding protein 1 (GATA-1) from the nucleus, which results from cytoplasmic sequestration of heat shock protein 70 (HSP70) by &agr;-globin chains. Small molecules disrupting the HSP70/&agr;-globin complex in the cytoplasm or decreasing HSP70 nuclear export might increase the nuclear localization of HSP70, thereby protecting GATA-1 and alleviating anemia. Finally, increasing the serum levels of hepcidin or transferrin alleviates anemia and dyserythropoiesis by diminishing iron uptake by erythroblasts in mouse models.