Chiara Bocelli-Tyndall

Clinical risk assessment of organ manifestations in systemic sclerosis: a report from the EULAR Scleroderma Trials And Research group database

Background: Systemic sclerosis (SSc) is a multisystem autoimmune disease, which is classified into a diffuse cutaneous (dcSSc) and a limited cutaneous (lcSSc) subset according to the skin involvement. In order to better understand the vascular, immunological and fibrotic processes of SSc and to guide its treatment, the EULAR Scleroderma Trials And Research (EUSTAR) group was formed in June 2004. Aims and methods: EUSTAR collects prospectively the Minimal Essential Data Set (MEDS) on all sequential patients fulfilling the American College of Rheumatology diagnostic criteria in participating centres. We aimed to characterise demographic, clinical and laboratory characteristics of disease presentation in SSc and analysed EUSTAR baseline visits. Results: In April 2006, a total of 3656 patients (1349 with dcSSc and 2101 with lcSSc) were enrolled in 102 centres and 30 countries. 1330 individuals had autoantibodies against Scl70 and 1106 against anticentromere antibodies. 87% of patients were women. On multivariate analysis, scleroderma subsets (dcSSc vs lcSSc), antibody status and age at onset of Raynaud’s phenomenon, but not gender, were found to be independently associated with the prevalence of organ manifestations. Autoantibody status in this analysis was more closely associated with clinical manifestations than were SSc subsets. Conclusion: dcSSc and lcSSc subsets are associated with particular organ manifestations, but in this analysis the clinical distinction seemed to be superseded by an antibody-based classification in predicting some scleroderma complications. The EUSTAR MEDS database facilitates the analysis of clinical patterns in SSc, and contributes to the standardised assessment and monitoring of SSc internationally.

Autologous hematopoietic stem cell transplantation for autoimmune diseases

Summary:Experimental data and early phase I/II studies suggest that high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (HSCT) can arrest progression of severe autoimmune diseases. We have evaluated the toxicity and disease response in 473 patients with severe autoimmune disease treated with autologous HSCT between 1995 and 2003, from 110 centers participating in the European Group for Blood and Marrow Transplantation (EBMT) autoimmune disease working party database. Survival, transplant-related mortality, treatment response and disease progression were assessed. In all, 420 patients (89%; 86±4% at 3 years, median follow-up 20 months) were alive, 53 (11%) had died from transplant-related mortality (N=31; 7±3% at 3 years) or disease progression (N=22; 9±4% at 3 years). Of 370 patients, 299 evaluable for response (81%) showed a treatment response, which was sustained in 213 (71% of responders). Response was associated with disease (P<0.001), was better in patients who received cyclophosphamide during mobilization (relative risk (RR)3.28 (1.57–6.83)) and was worse with increasing age (>40 years, RR0.29 (0.11–0.82)). Disease progression was associated with disease (P<0.001) and conditioning intensity (high intensity, RR1; intermediate intensity, RR1.81 (0.96–3.42)); low intensity, RR2.34 (1.074–5.11)). These data from the collective EBMT experience support the hypothesis that autologous HSCT can alter disease progression in severe autoimmune disease.

Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005

Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines – an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.

Phenotypical and functional characteristics of in vitro expanded bone marrow mesenchymal stem cells from patients with systemic sclerosis

Background: Mesenchymal stem cells (MSCs) have a potential immunomodulatory role in autoimmune disease; however, the qualitative properties and haematopoietic support capacity of MSCs derived from patients with autoimmune disease is unclear. Objectives: To further characterise phenotypically and functionally bone marrow (BM)-derived MSCs from patients with systemic sclerosis (SSc). Methods: Key parameters of BM-derived MSC function and phenotype were assessed in 12 patients with SSc and compared with 13 healthy normal controls. The parameters included the ability to: form colony-forming unit fibroblasts (CFU-F), differentiate along the adipogenic and osteogenic lineages, express cell surface antigens defining the MSCs population, support normal haematopoiesis and suppress in vitro lymphocyte proliferation induced by either anti-CD3∊ plus anti-CD28 monoclonal antibodies or the mixed lymphocyte reaction. Results: SSc MSCs were shown to have a similar characteristic phenotype, capacities to form CFU-F and to differentiate along adipogenic and osteogenic lineages as those of healthy donor MSCs. The ability of SSc MSCs to support long-term haematopoiesis was also identical to that of controls. Both healthy donor and SSc BM MSCs reduced the proliferation of autologous and allogeneic peripheral blood mononuclear cells in a cell number dependent fashion. Conclusions: These results show that BM-derived MSCs from patients with SSc under the described culture conditions exhibit the same phenotypic, proliferative, differentiation potential and immunosuppressive properties as their healthy counterparts and could therefore be considered in an autologous setting. Further studies are needed to ensure the quality and safety of large-scale expansion of patient MSCs prior to their potential use in clinical trials.

Allogeneic hematopoietic SCT for patients with autoimmune diseases

Allogeneic hematopoietic SCT (HSCT) has been used as treatment for single patients with autoimmune diseases (AD). To summarise currently available information, we analyzed all patients who underwent allogeneic HSCT for AD and who reported to the European Group for Blood and Marrow Transplantation (EBMT) database. Thirty-five patients receiving 38 allogeneic transplantations for various hematological and non-hematological AD were identified. Four patients had had an allogeneic HSCT for a conventional hematological indication in the past. Fifty-five per cent of the transplantation procedures led to a complete clinical response of the refractory AD and 23% to at least a partial response. The median duration of response at the last follow-up was 70.7 (15.2–130) months. Three patients relapsed at a median of 12.3 months after HSCT. Treatment-related mortality at 2 years was 22.1% (95% CI: 7.3–36.9%). Two deaths were caused by progression of AD. The probability of survival at 2 years was 70%. No single factor predicting the outcome could be identified. The retrospective nature of this study and the heterogeneous, partly incomplete data are its limitations. However, allogeneic HSCT can induce remission in patients suffering from refractory AD. These data provide the basis for carefully conducted prospective trials.

Fibroblast growth factor 2 and platelet-derived growth factor, but not platelet lysate, induce proliferation-dependent, functional class II major histocompatibility complex antigen in human mesenchymal stem cells

OBJECTIVE To document the specificity and the mechanism of induction of a novel class II major histocompatibility complex (MHC) antigen by mitogenic growth factors in human mesenchymal stem cells (MSCs) expanded in vitro for translational applications. METHODS Expression of class II MHC molecules was measured in human MSCs and differentiated cells expanded in the presence of fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), human platelet lysate, or interferon-γ (IFNγ). The roles of cell proliferation and growth factor-induced signaling pathways were investigated as well as the class II MHC assembly machinery and functional capacity. RESULTS FGF-2 and, to a lesser extent, PDGF-BB induced in adult human MSCs the expression of HLA-DR (normally induced by inflammatory cytokines), which was able to stimulate CD4+ T cells via superantigen binding. In contrast to IFNγ, FGF induced HLA-DR expression only in human MSCs proliferating under its mitogenic effect and not in mouse MSCs or in differentiated human cells. Although it induced cell proliferation, human platelet lysate did not cause HLA-DR expression in human MSCs. HLA-DR expression occurred following FGF-specific binding to its receptor(s), mainly FGF receptor 1, without inducing IFNγ or tumor necrosis factor α expression. Both MAPK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt controlled cell proliferation and HLA-DR expression, but only MAPK/ERK-1/2 controlled the induction of the class II MHC transcription activator protein CIITA, the major determinant of HLA-DR transcription. CONCLUSION The induction of functional HLA-DR in proliferating progenitor MSCs is a property of human MSCs that have been expanded with mitogenic growth factors. This has potential biologic significance in the regulation and/or protection of progenitor cell subpopulations under sustained mitogenic proliferation and needs to be taken into account when expanding MSCs for use in in vivo applications.

Consensus statement concerning cardiotoxicity occurring during haematopoietic stem cell transplantation in the treatment of autoimmune diseases, with special reference to systemic sclerosis and multiple sclerosis

Summary:Autologous haematopoietic stem cell transplantation is now a feasible and effective treatment for selected patients with severe autoimmune diseases. Worldwide, over 650 patients have been transplanted in the context of phase I and II clinical trials. The results are encouraging enough to begin randomised phase III trials. However, as predicted, significant transplant-related morbidity and mortality have been observed. This is primarily due to complications related to either the stage of the disease at transplant or due to infections. The number of deaths related to cardiac toxicity is low. However, caution is required when cyclophosphamide or anthracyclines such as mitoxantrone are used in patients with a possible underlying heart damage, for example, systemic sclerosis patients. In November 2002, a meeting was held in Florence, bringing together a number of experts in various fields, including rheumatology, cardiology, neurology, pharmacology and transplantation medicine. The object of the meeting was to analyse existing data, both published or available, in the European Group for Blood and Marrow Transplantation autoimmune disease database, and to propose a safe approach to such patients. A full cardiological assessment before and during the transplant emerged as the major recommendation.

Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction cells from human adipose tissue

Fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 are key supplements for the culture of stromal vascular fraction (SVF) cells from adipose tissue, both for typical monolayer (2D) expansion and for streamlined generation of osteogenic-vasculogenic grafts in 3D perfusion culture. The present study investigates whether factors present in human platelet lysate (PL) could substitute for FBS and FGF-2 in 2D and 3D culture models of SVF cells from human lipoaspirates. SVF cells were grown in medium supplemented with 10% FBS+FGF-2 or with 5% PL. In 2D cultures, PL initially supported SVF cell proliferation, but resulted in growth arrest shortly after the first passage. Freshly isolated SVF cells cultured with both media under perfusion for 5 days within 3D ceramic scaffolds induced bone formation after subcutaneous implantation in nude mice. However, blood vessels of donor origin were generated only using FBS+FGF-2-cultured cells. This was unexpected, because the proportion of CD34+/CD31+ endothelial lineage cells was significantly higher with PL than that of FBS+FGF-2 (33% vs. 3%, respectively). These results support the use of PL as a substitute of FBS+FGF-2 for short-term culture of human SVF cells, and indicate that more specific serum-free formulations are required to maintain a functionally vasculogenic fraction of SVF cells expanded under 3D perfusion.

Human bone marrow mesenchymal stem cells and chondrocytes promote and/or suppress the in vitro proliferation of lymphocytes stimulated by interleukins 2, 7 and 15

Objectives: To investigate whether human bone marrow-derived mesenchymal stem cells (BM-MSCs) and articular chondrocytes (ACs) affect the in vitro proliferation of T lymphocytes and peripheral blood mononuclear cells (PBMCs) driven by the homeostatic interleukin (IL)2, IL7 and IL15 cytokines binding to the common cytokine receptor γ-chain (γc) in the absence of T cell receptor (TCR) triggering. Methods: PBMCs, total T cells and T cell subsets (CD4+ and CD8+) were stimulated with IL2, IL7 or IL15 and exposed to cultured BM-MSCs and ACs at varying cell:cell ratio either in contact or in transwell conditions. Lymphocyte proliferation was measured by 3H-thymidine uptake or by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE)-labelled lymphocytes. Results: MSCs and ACs enhanced and inhibited lymphocyte proliferation depending on the extent of lymphocyte baseline proliferation and on the MSC/AC to lymphocyte ratio. Enhancement was significant on poorly proliferating lymphocytes and mostly at lower MSC/AC to lymphocyte ratio. Suppression occurred only on actively proliferating lymphocytes and at high MSC/AC to lymphocyte ratio. Neither enhancement nor inhibition required cell–cell contact. Conclusions: There is a dichotomous effect of MSCs/ACs on lymphocytes proliferating in response to the homeostatic IL2, IL7 and IL15 cytokines likely to be encountered in homeostatic and autoimmune inflammatory conditions. The effect is determined by baseline lymphocyte proliferation, cell:cell ratio and is dependent on soluble factor(s). This should be taken into account when planning cellular therapy for autoimmune disease (AD) using stromal-derived cells such as MSCs.

Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor‐associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow‐derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial‐to‐mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT‐related genes, downregulation of E‐cadherin and acquisition of mesenchymal morphology. These effects required cell‐to‐cell contact and were mediated by surface‐bound TGF‐β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC‐conditioned CRC cells were larger and characterized by higher vessel density, decreased E‐cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC‐conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT‐related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.