Helen Koutala

Rituximab therapy reduces activated B cells in both the peripheral blood and bone marrow of patients with rheumatoid arthritis: depletion of memory B cells correlates with clinical response

IntroductionBone marrow (BM) is an immunologically privileged site where activated autoantibody-producing B cells may survive for prolonged periods. We investigated the effect of rituximab (anti-CD20 mAb) in peripheral blood (PB) and BM B-cell and T-cell populations in active rheumatoid arthritis (RA) patients.MethodsActive RA patients received rituximab (1,000 mg) on days 1 and 15. PB (n = 11) and BM (n = 8) aspirates were collected at baseline and at 3 months. We assessed B-cell and T-cell populations using triple-color flow cytometry.ResultsRituximab therapy decreased PB (from a mean 2% to 0.9%, P = 0.022) but not BM (from 4.6% to 3.8%, P = 0.273) CD19+ B cells, associated with a significant reduction in the activated CD19+HLA-DR+ subset both in PB (from 55% to 19%, P = 0.007) and in BM (from 68% to 19%, P = 0.007). Response to rituximab was preceded by a significant decrease in PB and BM CD19+CD27+ memory B cells (P = 0.022). These effects were specific to rituximab since anti-TNF therapy did not reduce total or activated B cells. Rituximab therapy did not alter the number of activated CD4+HLA-DR+ and CD4+CD25+ T cells.ConclusionsRituximab therapy preferentially depletes activated CD19+HLA-DR+ B cells in the PB and BM of active RA patients. Clinical response to rituximab is associated with depletion of CD19+CD27+ memory B cells in PB and BM of RA patients.

Investigation of bone marrow mesenchymal stem cells (BM MSCs) involvement in idiopathic pulmonary fibrosis (IPF)

BACKGROUND Experimental data have provided evidence that progenitor cells of bone marrow (BM) origin may play a role in the fibrogenic process of the lung. OBJECTIVE To probe the possible involvement of BM mesenchymal stem cells (MSCs) in the pathophysiology of Idiopathic Pulmonary Fibrosis (IPF) by investigating the molecular profile of these cells. DESIGN BM MSCs were studied in 10 IPF patients and 10 healthy controls. MSCs were identified by their immunophenotypic characteristics and their potential to differentiate towards adipocytes/osteocytes/chondrocytes. We evaluated the mRNA expression of genes involved in the lung injury of IPF, namely the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor beta-1 (TGF-beta1) and the axis stromal-cell-derived factor-1 (SDF-1)/CXCR4 in BM MSCs using quantitative RT-PCR. RESULTS The BM MSCs of IPF patients displayed normal immunophenotypic characteristics and differentiation potential. No statistically significant difference was found between patients and controls in VEGF and FGF mRNA expression. TGF-beta1 was not expressed in either patients or controls. A significant increase in SDF-1-TR1 and CXCR4 mRNA expression was detected in IPF patients (1.6 x 10(25) +/- 1.2 x 10(25) and 3.1 x 10(7) +/- 3.1 x 10(7), respectively) compared to controls (0.32 x 10(25) +/- 0.07 x 10(25) and 1.67 x 10(7) +/- 0.30 x 10(7), respectively) (p = 0.001 and p = 0.001, respectively) whereas SDF-1 levels in MSC supernatants were similar in patients and controls. CONCLUSIONS The increased CXCR4 expression by patient MSCs suggests that the BM is probably implicated in the pathophysiology of IPF by mobilizing MSCs in response to or preceding lung injury. The potential role of BM MSCs in IPF is another interesting field for further investigation.

Investigation of Toll-like receptors in the pathogenesis of fibrotic and granulomatous disorders: a bronchoalveolar lavage study

Background and aimToll-like receptors (TLRs), a key component of innate immunity, have recently been implicated in the pathogenesis of interstitial lung diseases (ILDs). As the involvement of TLRs has not yet been fully elucidated, the aim of the current study was to examine the expression of various TLRs in the bronchoalveolar lavage fluid (BALF) of patients with ILDs.Patients and MethodsWe studied prospectively three groups of patients: (1) one group of 35 patients with fibrotic disorders, 16 with idiopathic pulmonary fibrosis (IPF) and 19 with fibrotic interstitial pneumonias associated with collagen tissue disorders (CTD-IPs); (2) one group of 14 patients with pulmonary sarcoidosis; and (3) 11 normal subjects. We evaluated TLR expression with flow cytometry and mRNA expression with real-time PCR.ResultsAn overexpression of TLR-3 mRNA was found in fibrotic disorders (CTD-IPs/IPF) in comparison with sarcoidosis (mean ± SD, 1.104 ± 1.087 versus 0.038 ± 0.03; P = 0.04). Additionally, TLR-3 mRNA was increased in CTD-IPs in comparison with IPF (P = 0.001), sarcoidosis (P = 0.002) and controls (P = 0.05). An upregulation in TLR-7 and -9 mRNA expression was detected in IPF (P = 0.05) and sarcoidosis (P = 0.05), respectively, when compared to controls. A higher percentage of TLR-9-expressing cells was found in BALF of CTD-IPs when compared to IPF (mean ± SD, 36.7 ± 7.06 versus 14.85 ± 3.82; P = 0.025).ConclusionWe observed distinct profiles of TLR expression in fibrotic and granulomatous disorders. It is likely that they could play a key role in the pathogenesis of these diseases and represent future therapeutic targets.

T-cell receptor Vβ repertoire analysis in patients with chronic idiopathic neutropenia demonstrates the presence of aberrant T-cell expansions.

Chronic idiopathic neutropenia (CIN) is a bone marrow (BM) disorder characterized by presence of activated T-lymphocytes in peripheral blood (PB) and BM. We investigated the pattern of T-cell responses in CIN by analyzing the T-cell receptor β-chain variable (Vβ) gene repertoire. Compared to controls, CIN patients displayed different patterns of Vβ gene usage in PB CD3(+), CD4(+) and CD8(+) cells. The frequency of Vβ skewing and the number of expanded Vβ families per subject were higher in patients compared to controls in all cell subpopulations. Skewing was more profound within the CD8(+) cells. The number of Vβ expansions per patient was higher in BM compared to PB. The majority of patients displayed a skewed oligoclonal/monoclonal pattern within the PB and/or BM CD8(+) cells and a polyclonal profile within the CD4(+) cells. We concluded that aberrant T-cell expansions are invariably detected in CIN patients and may have a role in the disease pathogenesis.

Increased Levels of the High Mobility Group Box 1 Protein Sustain the Inflammatory Bone Marrow Microenvironment in Patients with Chronic Idiopathic Neutropenia via Activation of Toll-Like Receptor 4

PurposeChronic idiopathic neutropenia (CIN) is a granulocytic disorder characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells and an inflammatory BM microenvironment. The aim of this study was to investigate the possible involvement of Toll-like receptors (TLRs) in the production of pro-inflammatory mediators in CIN BM.MethodsWe evaluated the expression of TLRs in patient BM cell subsets and adherent cells of long-term BM cultures (LTBMCs) using flow cytometry. We also examined the activation of TLR-mediated signaling using real-time PCR arrays and explored for potential endogenous TLR-specific ligands in CIN BM.ResultsCIN patients (n = 30) displayed significantly increased expression of surface TLR4 in monocytes of BM and LTBMC adherent cells compared to controls (n = 27). The TLR signaling gene array study in purified BM CD14+ cells showed that numerous TLR-related genes displayed at least two-fold increase in patients compared to controls. Among the over-expressed genes were genes related to the MyD88-dependent and MyD88-independent pathway suggesting a TLR4-mediated signaling. BM plasma from CIN patients induced the production of pro-inflammatory mediators including interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and IL-8 by autologous BM monocytes, and this effect was abrogated by a specific TLR4 inhibitor. The levels of the high mobility group box 1 protein (HMGB1), representing a TLR4 ligand, were significantly increased in patient LTBMC supernatants compared to controls.ConclusionThese data demonstrate a significant role of BM monocytes in the pathophysiology of CIN through the production of pro-inflammatory cytokines in a TLR4-mediated mechanism under the influence of endogenous ligands such as HMGB1.

An open label, single dose study to evaluate the safety, efficacy, and effects on CD25 expression of ciclosporin in patients with active rheumatoid arthritis despite treatment with methotrexate and infliximab

Objective: To explore the safety, efficacy, and lymphocyte activation of a triple therapeutic regimen with infliximab, methotrexate (MTX), and ciclosporin A (CsA) by an open label, pilot study. Patients and methods: 19 patients (mean age 52.9 years) with active rheumatoid arthritis (mean DAS28 7.3) after a mean of 16.8 infliximab infusions and dose adjustments of both infliximab and MTX were enrolled. CsA was added to a stable therapeutic regimen. Disease activity was evaluated by the DAS28. Lymphocyte activation was evaluated by assessing CD25 expression on peripheral blood mononuclear cells (PBMCs). Primary end points were safety and efficacy according to the EULAR response criteria at 24 weeks. Results: Eight patients (42%) discontinued treatment: adverse events (3), inefficacy (2) or non-compliance (2). One patient had a stroke and died. 5/11 (45%) patients who completed 24 weeks’ treatment were moderate responders. CD25 expression, both on unstimulated and phytohaemagglutinin stimulated PBMCs in five patients assessed, was reduced (mean (SD) values from 37 (34)% to 15 (10)% and from 50 (15)% to 29 (20)%, respectively). Conclusion: In this group of patients with refractory, highly active disease, addition of CsA reduced lymphocyte activation, and resulted in a modest response and a high rate of discontinuation. In such patients, other new approaches need to be explored.

High Frequency of Thyroid Disorders in Patients Presenting With Neutropenia to an Outpatient Hematology Clinic STROBE-Compliant Article

Abstract Granulopoiesis abnormalities have been described in association with thyroid disorders (TD). However, data regarding systematic evaluation of adult neutropenia and concurrent or prior TD are scarce. To investigate the frequency of TD among patients presenting with neutropenia, and the immunophenotypic and immunologic profile of neutropenic patients with concomitant thyroidopathy. Two hundred eighteen consecutive neutropenic patients were prospectively evaluated in our outpatient Hematology Clinic, with a detailed laboratory screen, including thyroid function tests, antineutrophil antibodies, blood lymphocytes immunophenotyping, and detection of T-cell clonality by PCR. Among 218 patients with neutropenia, 95 (43.6%) had TD, 65 chronic immunologic neutropenia, 20 clonal proliferation of T-large granular lymphocytes (T-LGL), 5 autoimmune disorders, and 33 other diagnoses. TD-patients had an increased frequency of recurrent infections compared with other patients (P = 0.045). The following correlations were found: negative correlation between FT3 and absolute neutrophil count (ANC) (r 2 = −0.274, P = 0.007), negative correlation between TPO-Abs/TG-Abs and C4 (r 2 = −0.16, P = 0.045; r 2 = −0.266, P = 0.001), and CD4+ counts were inversely correlated to T4 and positively to TSH (r 2 = −0.274, P = 0.024; r 2 = 0.16, P = 0.045). In addition, TD-patients had significantly higher percentages of CD4+ lymphocytes (P = 0.003). Among TD-patients, 23.4% had Hashimoto thyroiditis (HT), 4.1%, Graves disease (GD), 8.2% nontoxic multinodular goiter (NTMG), 5% subclinical hypothyroidism, and 2.8% had undergone total thyroidectomy associated with nodules (TTM). Thirteen TD-patients displayed T-LGL. Patients with autoimmune thyroidopathy had an increased frequency of concomitant autoimmune manifestations (P = 0.03). Significant differences between the different thyroidopathies included: HT-patients had higher percentages of B-lymphocytes, while the opposite was evident for the TTM-subgroup (P = 0.009, 0.02); GD-patients showed an increase of the proportion of NK cells and a decrease in the percentage of TCR&ggr;&dgr;+ lymphocytes (P = 0.001, 0.045); and NTMG-patients had significantly higher ANC (P = 0.004) compared to other thyroidopathies. Antineutrophil antibodies were found in 37.2% of TD-patients tested. Anti-TPO titers were significantly higher in patients with positive antineutrophil antibodies (P = 0.04). The frequency of TD among neutropenic patients may be higher than previously reported. The existence of antineutrophil antibodies, as well as the different distribution of lymphocyte subsets among patients with different TD, suggests both humoral and cellular mechanisms in the pathophysiology of thyroid disease-associated neutropenia.

Lymphopenia in patients with chronic idiopathic neutropenia is associated with decreased number of T‐lymphocytes containing T‐cell receptor excision circles

Objectives:  Chronic idiopathic neutropenia (CIN) is a disorder of granulopoiesis characterized by the presence of activated T‐lymphocytes that induce/sustain apoptosis of bone marrow (BM) granulocytic progenitors. T‐cell lymphopenia is commonly found in CIN. The aim of the study is to probe the mechanisms underlying T‐cell lymphopenia in CIN.

Minor populations of paroxysmal nocturnal hemoglobinuria‐type cells in patients with chronic idiopathic neutropenia

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells under the influence of pro‐inflammatory mediators and oligoclonal/monoclonal T‐lymphocytes. Because patients with immune‐mediated BM failure display frequently paroxysmal nocturnal hemoglobinuria (PNH)‐type cells in the peripheral blood (PB), we investigated the possible existence of PNH‐type cells in 91 patients with CIN using flow cytometry. The patients displayed increased proportions of PNH‐type glycophorin A+/CD59dim and glycophorin A+/CD59− red blood cells (RBCs), FLAER−/CD24− granulocytes, and FLAER−/CD14− monocytes, compared to controls (n = 55). A positive correlation was found between the proportions of PNH‐type RBCs, granulocytes, and monocytes and an inverse correlation between the number of PB neutrophils and the proportions of PNH‐type cell populations. The number of patients, displaying percentages of PNH‐type cells above the highest percentage observed in the control group, was significantly increased among patients with skewed compared to those with normal T‐cell receptor repertoire suggesting that T‐cell‐mediated immune processes underlie the emergence of PNH‐type cells in CIN. Our findings suggest that patients with CIN display PNH‐type cells in the PB at a high frequency corroborating the hypothesis that CIN belongs to the immune‐mediated BM failure syndromes.

Immunoglobulin and B-cell disturbances in patients with chronic idiopathic neutropenia

Chronic idiopathic neutropenia (CIN) is a granulocytic disorder associated with presence of activated, myelosuppressive T-lymphocytes. In the present study we have evaluated constituents of humoral immunity in CIN patients (n=48) compared to healthy controls (n=52). CIN patients displayed lower serum IgG levels due to a reduction in IgG1, IgG3, IgG4 but not IgG2, lower IgA and increased IgM levels compared to controls. The proportion of CD19+ cells did not differ between patients and controls; however the proportion of the naïve IgD+/CD27- B-cells was increased and the proportion of class-switched memory IgD-/CD27+ B-cells was decreased in the patients. The percentage of CD40+ B-cells did not differ between patients and controls and no aberrations in the CD40-meadiated signal transduction pathway or in CD40-gene polymorphisms were identified. These data provide further evidence that immune disturbances are associated with the pathophysiology of CIN and point out for the first time the implication of the B-cell system.