In utero exposure to Azathioprine in autoimmune disease. Where do we stand?

Azathioprine (AZA), an oral immunosuppressant, is safe during pregnancy. Some reports suggested different impairments in the offspring of mothers with autoimmune diseases (AI) exposed in utero to AZA. These observations are available from retrospective studies or case reports. However, data with respect to the long-term safety in the antenatally exposed child are still lacking. The aim of this study is to summarize the current knowledge in this field and to focus on the need for a prospective study on this population. We performed a PubMed search using several search terms. The actual data show that although the risk of congenital anomalies in offspring, as well as the infertility risk, are similar to those found in general population, there is a higher incidence of prematurity, of lower weight at birth and an intra-uterine delay of development. There is also an increased risk of materno- fetal infections, especially cytomegalovirus infection. Some authors raise the interrogations about neurocognitive impairment. Even though the adverse outcomes might well be a consequence of maternal illness and disease activity, interest has been raised about a contribution of this drug. However, the interferences between the external agent (in utero exposure to AZA), with the host (child genetic susceptibility, immune system anomalies, emotional status), environment (public health, social context, availability of health care), economic, social, and behavioral conditions, cultural patterns, are complex and represent confounding factors. In conclusion, it is necessary to perform studies on the medium and long-term outcome of children born by mothers with autoimmune diseases, treated with AZA, in order to show the safety of AZA exposure. Only large-scale population studies with long-term follow-up will allow to formally conclude in this field. TAKE HOME MESSAGES.

In situ detection of PR3-ANCA+ B cells and alterations in the variable region of immunoglobulin genes support a role of inflamed tissue in the emergence of auto-reactivity in granulomatosis with polyangiitis.

Circulating anti-neutrophilic cytoplasmic autoantibodies targeting proteinase 3 (PR3-ANCA) are a diagnostic and pathogenic hallmark of granulomatosis with polyangiitis (GPA). It is, however, incompletely understood if inflamed tissue supports presence or emergence of PR3-ANCA+ B cells. In search of such cells in inflamed tissue of GPA, immunofluorescence staining for IgG and a common PR3-ANCA idiotype (5/7 Id) was undertaken. Few 5/7 Id+/IgG+ B cells were detected in respiratory and kidney tissue of GPA. To gain more insight into surrogate markers possibly indicative of an anti-PR3-response, a meta-analysis comprising IGVH and IGVL genes derived from respiratory tract tissue of GPA (231 clones) was performed. Next generation sequencing-based IGHV genes derived from peripheral blood of healthy donors (244.353 clones) and previously published IGLV genes (148 clones) served as controls. Additionally, Ig genes of three murine and five known human monoclonal anti-PR3 antibodies were analyzed. Primary and probably secondary rearrangements led to altered VDJ usage and an extended complementarity determining region 3 (CDR3) of IGHV clones from GPA tissue. Selection against amino acid exchanges was prominent in the framework region of IGHV clones from GPA tissue. The comparison of V(D)J rearrangements and deduced amino acid sequences of the CDR3 yielded no identities and few similarities between clones derived from respiratory tissue of GPA and anti-PR3 antibodies, arguing against a presence of B cells that carry PR3-ANCA-prone Ig genes among the clones. In line with the scarcity of 5/7 Id+ B lymphocytes in GPA tissue, the results suggest that with respect to a local anti-PR3 response, methods detecting rare clones are required.

Plasma cells within granulomatous inflammation display signs pointing to autoreactivity and destruction in granulomatosis with polyangiitis.

INTRODUCTION: Plasma cells residing in inflamed tissues produce antibodies in chronic inflammatory and systemic autoimmune diseases. This study examined if plasma cells, located within inflamed nasal tissue in granulomatosis with polyangiitis (GPA), express features potentially associated with the autoimmune and destructive character of this disease. METHODS: Ig gene mutation patterns of individual tissue-derived plasma cells from GPA (n = 5) were analyzed, by using laser-assisted microdissection followed by semi-nested polymerase chain reaction (PCR). Signs of B-lymphocyte maturation (ectopic lymphoid structures, ELS) and survival (a proliferation-inducing ligand, APRIL; B-cell maturation antigen, BCMA; transmembrane-activator and calcium modulator and cyclophilin interactor, TACI; receptor activator of nuclear factor κB ligand, RANKL) were examined in nasal tissues or serum, respectively, by using immunohistochemistry/fluorescence and enzyme-linked immunosorbent assay, ELISA. RESULTS: Plasma-cell derived Ig genes (light- and heavy-chain pairs, n = 4; heavy chains, n = 33) resembled mutation patterns seen in other autoimmune diseases, predominantly displaying selection against replacement mutations within the framework region of Ig genes (10 of 15), which is responsible for structural integrity. Ectopic lymphoid structures were similar between GPA and a disease control (that is, unspecific chronic rhinosinusitis. However, histomorphologic features distinguishing GPA from rhinosinusitis (that is, neutrophilic microabscess and granuloma) expressed considerable amounts of membrane-associated and secreted APRIL, respectively. The latter was co-localized with CD138 and found in close proximity to cells expressing IgG, TACI, and BCMA. Interestingly, plasma cells strongly expressed receptor activator of nuclear factor κB ligand (RANKL), apart from fibroblast-like cells. CONCLUSIONS: Plasma cells within granulomatous inflammation appear to display features that might be required for autoreactivity and, possibly, RANKL-mediated destruction in GPA.

Human mesenchymal stem cells provide protection against radiation-induced liver injury by antioxidative process, vasculature protection, hepatocyte differentiation, and trophic effects.

To evaluate the potential therapeutic effect of the infusion of hMSCs for the correction of liver injuries, we performed total body radiation exposure of NOD/SCID mice. After irradiation, mir-27b level decreases in liver, increasing the directional migration of hMSCs by upregulating SDF1 α . A significant increase in plasmatic transaminases levels, apoptosis process in the liver vascular system, and in oxidative stress were observed. hMSC injection induced a decrease in transaminases levels and oxidative stress, a disappearance of apoptotic cells, and an increase in Nrf2, SOD gene expression, which might reduce ROS production in the injured liver. Engrafted hMSCs expressed cytokeratin CK18 and CK19 and AFP genes indicating possible hepatocyte differentiation. The presence of hMSCs expressing VEGF and Ang-1 in the perivascular region, associated with an increased expression of VEGFr1, r2 in the liver, can confer a role of secreting cells to hMSCs in order to maintain the endothelial function. To explain the benefits to the liver of hMSC engraftment, we find that hMSCs secreted NGF, HGF, and anti-inflammatory molecules IL-10, IL1-RA contributing to prevention of apoptosis, increasing cell proliferation in the liver which might correct liver dysfunction. MSCs are potent candidates to repair and protect healthy tissues against radiation damages.

New insights for pelvic radiation disease treatment: Multipotent stromal cell is a promise mainstay treatment for the restoration of abdominopelvic severe chronic damages induced by radiotherapy.

Radiotherapy may induce irreversible damage on healthy tissues surrounding the tumor. It has been reported that the majority of patients receiving pelvic radiation therapy show early or late tissue reactions of graded severity as radiotherapy affects not only the targeted tumor cells but also the surrounding healthy tissues. The late adverse effects of pelvic radiotherapy concern 5% to 10% of them, which could be life threatening. However, a clear medical consensus concerning the clinical management of such healthy tissue sequelae does not exist. Although no pharmacologic interventions have yet been proven to efficiently mitigate radiotherapy severe side effects, few preclinical researches show the potential of combined and sequential pharmacological treatments to prevent the onset of tissue damage. Our group has demonstrated in preclinical animal models that systemic mesenchymal stromal cell (MSC) injection is a promising approach for the medical management of gastrointestinal disorder after irradiation. We have shown that MSCs migrate to damaged tissues and restore gut functions after irradiation. We carefully studied side effects of stem cell injection for further application in patients. We have shown that clinical status of four patients suffering from severe pelvic side effects resulting from an over-dosage was improved following MSC injection in a compationnal situation.

Fifteen years of preclinical and clinical experiences about biotherapy treatment of lesions induced by accidental irradiation and radiotherapy.

High dose radiation exposures involving medical treatments or accidental irradiation may lead to extended damage to the irradiated tissue. Alleviation or even eradication of irradiation induced adverse events is therefore crucial. Because developments in cell therapy have brought some hope for the treatment of tissues damages induced by irradiation, the Institute for Radiation and Nuclear Safety contributed to establish the clinical guidelines for the management of accidentally irradiated victims and to provide the best supportive care to patients all over the world. In the past 15 years, we contributed to develop and test cell therapy for protection against radiation side effects in several animal models, and we proposed mechanisms to explain the benefit brought by this new therapeutic approach. We established the proof of concept that mesenchymal stem cells (MSCs) migrate to damaged tissues in the nonobese diabetic/severe combined immunodeficiency immunotolerant mice model and in non-human primate after radiation exposure. We showed that the intravenous injection of MSCs sustains hematopoiesis after total body irradiation, improves wound healing after radiodermatitis and protects gut function from irradiation damages. Thanks to a tight collaboration with clinicians from several French hospitals, we report successful treatments of therapeutic/accidental radiation damages in several victims with MSC infusions for hematopoiesis correction, radio-induced burns, gastrointestinal disorders and protection homeostatic functions of gut management after radio-therapy.

Gastro-intestinal autoimmunity: preclinical experiences and successful therapy of fistulizing bowel diseases and gut Graft versus host disease by mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are multipotent adult stem cells with the potential to regenerate tissue damage and inhibit inflammation and fibrosis in parallel. As they are non-immunogenic, MSC can be safely auto- and allotransplanted and consequently represent a therapeutic option for refractory connective tissue diseases and fistulizing colitis like Crohn's disease. Actually, there are more than 200 registered clinical trial sites for evaluating MSC therapy, 22 are on autoimmune diseases and 27 are actually recruiting bowel disease' patients. More than 1,500 patients with bowel diseases like Crohn's disease were treated in clinical trials by local as well as systemic MSC therapy. Phase I and II trials on fistula documented the feasibility and safety of MSC therapy, and a significant superiority compared to fibrin glue in fistulizing bowel diseases was demonstrated. Autologous as well as allogeneic use of Bone marrow as well as of adipose tissue-derived MSC are feasible. In refractory Graft versus host disease, especially in refractory gut Graft versus host diseases, encouraging results were reported using MSC. Systemic MSC therapy of refractory irradiation-induced colitis was safe and effective on pain, diarrhea, hemorrhage, inflammation and fistulization accompanied by modulation of the lymphocyte subsets toward an increase in T regulatory cells and a decrease in activated effector T cells. Mesenchymal stem cells represent a safe therapy for patients with refractory inflammatory bowel diseases.

Use of mesenchymal stem cells (MSC) in chronic inflammatory fistulizing and fibrotic diseases: a comprehensive review.

Mesenchymal stem cells (MSC), multipotent adult stem cells, feature the potential to regenerate tissue damage and, in parallel, inhibit inflammation and fibrosis. MSC can be safely transplanted in autologous and allogeneic ways as they are non-immunogenic, and consequently represent a therapeutic option for refractory connective tissue diseases, fibrosing diseases like scleroderma and fistulizing colitis like in Crohn's disease. Actually, there are more than 200 registered clinical trial sites for evaluating MSC therapy, and 22 are on autoimmune diseases. In irradiation-induced colitis, MSC accelerate functional recovery of the intestine and dampen the systemic inflammatory response. In order to provide rescue therapy for accidentally over-irradiated prostate cancer patients who underwent radiotherapy, allogeneic bone marrow-derived MSC from family donors were intravenously infused to three patients with refractory and fistulizing colitis resembling fistulizing Crohn's disease. Systemic MSC therapy of refractory irradiation-induced colitis was safe and effective on pain, diarrhoea, hemorrhage, inflammation and fistulization accompanied by modulation of the lymphocyte subsets towards an increase of T regulatory cells and a decrease of activated effector T cells. The current data indicate that MSC represent a promising alternative strategy in the treatment of various immune-mediated diseases. Encouraging results have already been obtained from clinical trials in Crohn's disease and SLE as well as from case series in systemic sclerosis. MSC represent a safe therapeutic measure for patients who suffer from chronic and fistulizing colitis. These findings are instructional for the management of refractory inflammatory bowel diseases that are characterized by similar clinical and immunopathological features.

New autoimmune diseases after cord blood transplantation: a retrospective study of EUROCORD and the Autoimmune Disease Working Party of the European Group for Blood and Marrow Transplantation.

To describe the incidence, risk factors, and treatment of autoimmune diseases (ADs) occurring after cord blood transplantation (CBT), we analyzed both CBT recipients reported to EUROCORD who had developed at least 1 new AD and those who had not. Fifty-two of 726 reported patients developed at least 1 AD within 212 days (range, 27-4267) after CBT. Cumulative incidence of ADs after CBT was 5.0% +/- 1% at 1 year and 6.6% +/- 1% at 5 years. Patients developing ADs were younger and had more nonmalignant diseases (P < .001). ADs target hematopoietic (autoimmune hemolytic anemia, n = 20; Evans syndrome, n = 9; autoimmune thrombocytopenia, n = 11; and immune neutropenia, n = 1) and other tissues (thyroiditis, n = 3; psoriasis, n = 2; Graves disease, n 1; membranous glomerulonephritis, n = 2; rheumatoid arthritis, n = 1; ulcerative colitis, n = 1; and systemic lupus erythematosus, n = 1). Four patients developed 2 ADs (3 cases of immune thrombocytopenia followed by autoimmune hemolytic anemia and 1 Evans syndrome with rheumatoid arthritis). By multivariate analysis, the main risk factor for developing an AD was nonmalignant disease as an indication for CBT (P = .0001). Hematologic ADs were most often treated with steroids, rituximab, and cyclosporine. With a median follow-up of 26 months (range, 2-91), 6 of 52 patients died as a consequence of ADs. We conclude that CBT may be followed by potentially life-threatening, mainly hematologic ADs.

Secondary autoimmune diseases occurring after HSCT for an autoimmune disease: a retrospective study of the EBMT Autoimmune Disease Working Party.

To specify the incidence and risk factors for secondary autoimmune diseases (ADs) after HSCT for a primary AD, we retrospectively analyzed AD patients treated by HSCT reported to EBMT from 1995 to 2009 with at least 1 secondary AD (cases) and those without (controls). After autologous HSCT, 29 of 347 patients developed at least 1 secondary AD within 21.9 (0.6-49) months and after allogeneic HSCT, 3 of 16 patients. The observed secondary ADs included: autoimmune hemolytic anemia (n = 3), acquired hemophilia (n = 3), autoimmune thrombocytopenia (n = 3), antiphospholipid syndrome (n = 2), thyroiditis (n = 12), blocking thyroid-stimulating hormone receptor antibody (n = 1), Graves disease (n = 2), myasthenia gravis (n = 1), rheumatoid arthritis (n = 2), sarcoidosis (n = 2), vasculitis (n = 1), psoriasis (n = 1), and psoriatic arthritis (n = 1). After autologous HSCT for primary AD, the cumulative incidence of secondary AD was 9.8% ± 2% at 5 years. Lupus erythematosus as primary AD, and antithymocyte globulin use plus CD34(+) graft selection were important risk factors for secondary AD by multivariate analysis. With a median follow-up of 6.2 (0.54-11) years after autologous HSCT, 26 of 29 patients with secondary AD were alive, 2 died during their secondary AD (antiphospholipid syndrome, hemophilia), and 1 death was HSCT-related. This European multicenter study underlines the need for careful management and follow-up for secondary AD after HSCT.

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.

Rituximab in patients with rheumatoid arthritis and vasculitis-associated cutaneous ulcers.

OBJECTIVES: To test the efficacy of treatment with rituximab in refractory rheumatoid vasculitis in patients with rheumatoid arthritis (RA). METHODS: Retrospective study of four female patients with histologically proven RA associated vasculitic cutaneous ulcers. All patients developed the lesions on long term treatment with methotrexate or leflunomide, and three of them with tumour necrosis factor alpha (TNF) blockers. All patients were refractory to prednisolone in the dosage between 0.5 and 1 mg/kg body weight for at least 4 weeks prior to rituximab. Rituximab were administered in two intravenous applications in the interval of 14 days accompanied by continued treatment with methotrexate or leflunomide and prednisolone. RESULTS: Three out of four patients achieved a rapid clinical remission of the lesions within 4 to 6 weeks after rituximab therapy continuing at least for four months with a successful corticoid reduction till prednisolone 10 mg a day. One patient showed no remission of the skin lesions accompanied by increasing levels for ESR and CRP. CONCLUSIONS: Rituximab treatment seems to be very effective in several cases of vasculitis-associated cutaneous ulcers in RA patients. However, the effectiveness of rituximab in cases with this indication remains to be shown in larger number of patients.

Efficacy of antibiotic therapy for SAPHO syndrome is lost after its discontinuation: an interventional study.

INTRODUCTION: The acronym SAPHO was introduced in 1987 to unify the various descriptions of a seronegative arthritis associated with skin manifestations and to show synovitis, acne, pustulosis, hyperostosis, and osteitis with and without sterile multifocal osteomyelitis. The etiology of SAPHO syndrome is unknown, but an association with infection by semipathogenic bacteria like Propionibacterium acnes has been suggested. We conducted an interventional study of SAPHO patients receiving antibiotics. METHODS: Thirty-seven patients met the clinical criteria of SAPHO syndrome, 21 of them underwent a needle biopsy of the osteitis lesion, and 14 of them showed positive bacteriological cultures for P. acnes. Thirty patients (14 bacteriological positive and 16 without biopsy) were treated with antibiotics for 16 weeks. The activity of skin disease and osteitis were assessed by a physician using a scoring model (from 0 to 6). In addition, patients completed a Health Assessment Score (HAS, from 0 to 6). The erythrocyte sedimentation rate was determined and a MRI (of the osteitis lesion, radiologic activity score from 0 to 2) was performed in week 1 (W1), week 16 (W16), and week 28 (W28, 12 weeks after antibiotics). RESULTS: Twenty-seven patients continued the medication (azithromycin, n = 25, 500 mg twice a week; clindamycin, n = 1, 300 mg daily; or doxycycline, n = 1, 100 mg daily) for 16 weeks. After W16 the scores for MRI (1.5 to 1.1, P = 0.01), skin activity (3.2 to 1.2, P = 0.01), osteitis activity (4.0 to 2.1, P = 0.02), and HAS (3.3 to 2.1, P = 0.01) decreased significantly. However, this was followed by increasing values for MRI scores (1.2 to 1.4, P = 0.08), skin activity (1.2 to 1.7, P = 0.11), osteitis activity (1.9 to 2.7, P = 0.01), and HAS (2.2 to 3.3, P = 0.02) from W16 to W28. The comparison of the scores in W1 and W28 in these 12 patients showed no significant differences. CONCLUSIONS: For the period of application, the antibiotic therapy seems to have controlled the disease. After antibiotic discontinuation, however, disease relapse was observed. SAPHO syndrome thus groups with other chronic inflammatory arthropathies with a need for permanent therapy.

Expansion of circulating NKG2D+ effector memory T-cells and expression of NKG2D-ligand MIC in granulomaous lesions in Wegener's granulomatosis.

Expansion of circulating CD28- T-cells reminiscent of effector memory T-cells (T(EM)) has been reported in Wegener's granulomatosis (WG) recently. To investigate the role of T(EM) in WG, we analyzed the expression of the activating NK-receptor NKG2D and its ligand MIC on circulating T(EM) and in granulomatous lesions, respectively. NKG2D was anomalously expressed and preferentially detected on circulating CD4+CD28- T(EM) in WG. Compared to healthy controls, T(EM) display a more activated phenotype potentially favoring unbalanced proinflammatory responses in WG. Cluster-like formations of "Wegener's autoantigen" PR3 were surrounded by NKG2D+ and NKG2D-ligand MIC+ cells in WG-granulomata, but not in disease controls. Further, IL-15 - known to drive T(EM) differentiation and proliferation--was also expressed in WG-granulomata. Thus, through acquisition of NK-like "innate" properties, IL-15 stimulated NKG2D+ T(EM) could interact with MIC+ cells within WG-granulomata, thereby sustaining inflammation and autoimmunity and promoting self-perpetuating pathology in WG.

Human proteinase 3 (PR3) and its binding molecules: implications for inflammatory and PR3-related autoimmune responses.

Human proteinase 3 (PR3) is a multifunctional serine protease, mainly located in the azurophilic granules and on the cell surface of polymorphonuclear leukocytes (PMN). Cumulated data indicate that PR3, which is the main target autoantigen of antineutrophilic cytoplasmic antibodies (ANCA), interacts with several surface receptors and participates in the local inflammatory response. Herein, we summarize the efforts made to elucidate ANCA-binding epitopes of PR3, extended by data derived from the use of a random peptide library. The inserts for 107 peptides were obtained by panning of a random peptide library with PR3-ANCA(+) immunoglobulins. Analysis of the amino acid sequences of the inserted peptides derived from isolated positive clones suggested that they do not belong to linear epitopes of PR03 and possess a high proportion of positively charged amino acids. Furthermore, this article focuses on immune functions of PR3 with respect to PR3 modulation of cell activation via cleavage of protease-activated receptor-2 (PAR-2) and as binding protein for the proinflammatory cytokine IL-32alpha. Altogether, there are a number of (auto)molecules that bind to PR3, some of them even competitive and each binding interaction seems to have specific implications.

Alterations in the phenotype of CMV-specific and total CD8+ T-cell populations in Wegener's granulomatosis.

Wegener's granulomatosis (WG) is an autoimmune disease of as yet unknown etiology. To date it has remained obscure what causes WG or determines disease progression. Case reports suggest that viral infections such as cytomegalovirus (CMV) reactivation may contribute to disease flares. In this study we found a skewing of the phenotype of CMV-specific CD8+tet(ramer)+ T-cells in WG. A marked proportion of these cells displayed a late differentiated "effector memory" T-cell phenotype with decreased expression of CD28 and CD62L, and heterogeneous CD27 expression, features which were also seen in CD8+tet- T-cells in WG, but not in controls. Our results might reflect profound generalized changes in the CD8+ T-cell compartment also affecting virus-specific T-cell responses in WG.

From immunoglobulin gene fingerprinting to motif-specific hybridization: advances in the analysis of B lymphoid clonality in rheumatic diseases.

In rheumatic diseases, autoantibody-producing cells of interest are often hidden in a polyclonal B-lymphocyte population. Immunoglobulin gene fingerprinting is a useful approach to screen for expanding clones and to detect recirculation between different locations. The gene fingerprinting approach and the Southern blot technique have been amalgamated, using electrophoretic transfer of a PCR product from an acrylamide gel onto a nylon membrane followed by hybridization with specific oligonucleotide probes. In contrast to conventional fingerprinting, the authenticity of immunoglobulin genes can be confirmed, individual genes can be detected and handling radionucleotides can be avoided. Also, the membrane may be reused for further investigations.