Human mesenchymal stem cell-derived microvesicles modulate T cell response to islet antigen glutamic acid decarboxylase in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Mesenchymal stem cells (MSCs) have been shown to abrogate in vitro the proinflammatory response in type 1 diabetes. The mechanism involves paracrine factors, which may include microvesicles (MVs). We evaluated whether MVs derived from heterologous bone-marrow MSCs exert an immunomodulatory effect on T cell responses against GAD (glutamic acid decarboxylase) antigen in type 1 diabetes. METHODS: MVs were purified from heterologous human MSCs by differential centrifugation. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with type 1 diabetes at disease onset, and responses to GAD65 stimulation were assessed by IFN-γ enzyme-linked immunosorbent spot analysis. Levels of cytokines and prostaglandin E2 (PGE2) were measured in the supernatant fraction, and T helper 17 (Th17) and regulatory T cell analysis was performed. RESULTS: MVs were internalised by PBMCs, as assessed by confocal microscopy and flow cytometry analyses. MVs significantly decreased IFN-γ spots and levels in GAD65-stimulated PBMCs, and significantly increased transforming growth factor-ß (TGF-ß), IL-10, IL-6 and PGE2 levels. Furthermore, MVs decreased the number of Th17 cells and the levels of IL-17, and increased FoxP3(+) regulatory T cells in GAD65-stimulated PBMCs. CONCLUSIONS/INTERPRETATION: These results provide evidence that MSC-derived MVs can inhibit in vitro a proinflammatory response to an islet antigenic stimulus in type 1 diabetes. The action of MVs involves PGE2 and TGF-ß signalling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T cells.

Venous lower-limb evaluation in patients with acute pulmonary embolism.

OBJECTIVES: Compressive ultrasonography (CUS) of the lower limbs is the first choice for identifying deep venous thrombosis (DVT) in patients with symptomatic pulmonary embolism (PE). The aim of this study was to uncover clinical characteristics and CUS findings in patients with proven PE and their correlations with PE extent. METHODS: A total of 524 consecutive cases of proven symptomatic PE diagnosed between January 1996 and December 2006 were reviewed. RESULTS: Mean age was 71.06 ± 14.43 SD years; 244 patients (46.6%) were men. DVT signs or symptoms were present in 30.9% of patients and were associated with the femoral site (P = 0.029). CUS was performed in 383 patients (73.1%) and DVT was found in 75.5%. In 94.1% of patients DVT was proximal (popliteal and/or femoral), which would have been then identified by simplified CUS. CUS was performed significantly more often in presence of signs or symptoms of DVT (P < 0.001), less often in presence of medical illnesses (P = 0.040), age ≥75 years (P = 0.001) and death in hospital (P < 0.001). Signs or symptoms of DVT were predictors of positive CUS (P < 0.001), presence of medical illnesses (P = 0.020), central venous catheter (P = 0.035), death in hospital (P = 0.032) were predictors of negative CUS findings. Neither clinical findings nor CUS were associated with PE extent. CONCLUSIONS: In patients with proven symptomatic PE, signs or symptoms of DVT are present only in 1/3 of cases and are significantly more frequent when DVT is extended to the femoral vein. Simplified CUS of the lower limbs has a high sensitivity in finding proximal DVT. CUS is not able to predict PE extent.

Stretch reduces nephrin expression via an angiotensin II-AT(1)-dependent mechanism in human podocytes: effect of rosiglitazone.

Increased glomerular permeability to proteins is a characteristic feature of diabetic nephropathy (DN). The slit diaphragm is the major restriction site to protein filtration, and the loss of nephrin, a key component of the slit diaphragm, has been demonstrated in both human and experimental DN. Both systemic and glomerular hypertension are believed to be important in the pathogenesis of DN. Human immortalized podocytes were subjected to repeated stretch-relaxation cycles by mechanical deformation with the use of a stress unit (10% elongation, 60 cycles/min) in the presence or absence of candesartan (1 microM), PD-123319 (1 microM), and rosiglitazone (0.1 microM). Nephrin mRNA and protein expression were assessed using quantitative real-time PCR, immunoblotting, and immunofluorescence, and the protein expression of AT(1) receptor and angiotensin II secretion were evaluated. Exposure to stretch induced a significant approximately 50% decrease in both nephrin mRNA and protein expression. This effect was mediated by an angiotensin II-AT(1) mechanism. Indeed, podocyte stretching induced both angiotensin II secretion and AT(1) receptor overexpression, podocyte exposure to angiotensin II reduced nephrin protein expression, and both the AT-1 receptor antagonist candesartan and a specific anti-angiotensin II antibody completely abolished stretch-induced nephrin downregulation. Similar to candesartan, the peroxisome proliferator-activated receptor (PPAR)-gamma agonist, rosiglitazone, also inhibited stretch-induced nephrin downregulation, suggesting interference with stretch-induced activation of the angiotensin II-AT(1) receptor system. Accordingly, rosiglitazone did not alter stretch-induced angiotensin II secretion, but it prevented AT(1) upregulation in response to stretch. These results suggest a role for hemodynamic stress in loss of nephrin expression and allude to a role of PPAR-gamma agonists in the prevention of this loss.

Association of cytomegalovirus infections with recurrence of humoral and cellular autoimmunity to islet autoantigens and of type 1 diabetes in a pancreas transplanted patient.

Association of type 1 diabetes and cytomegalovirus (CMV) is suspected and CMV infections have also been linked to increased risk of new onset post-transplantation diabetes. We monitored response to islet autoantigens, pancreatic endocrine function, and CMV infections in one type 1 diabetic patient receiving pancreas allograft. Time course analyses of levels of islet autoantibodies (Abs), IFN-gamma ELISPOT response, analysis of T cell function, levels of C peptide together with CMV pp65 antigenaemia and viraemia and graft biopsy histopathology were performed in comparison with a cohort of diabetic recipients. Evidence of autoimmune activation to GAD and IA2, modification of CD4(+) CD25hi T cells, loss of pancreatic function, concomitantly with multiple CMV infections and allograft rejection with peri-insulitis is provided. The parallel between metabolic outcome, initiation and progression of autoreactivity to islet autoantigens and early CMV infections after transplantation, suggests that persistent CMV infections may be relevant to the pathogenesis of type 1 diabetes.

Human pancreatic islet endothelial cells express coxsackievirus and adenovirus receptor and are activated by coxsackie B virus infection.

Enteroviruses, such as the coxsackievirus (CV) group, have been linked to the induction of inflammatory and autoimmune diseases. Virus tropism and tissue access are modulated by endothelial cells. To examine the susceptibility of microvascular endothelial cells (MECs) derived from pancreatic islets to infection with CV group B (CVB), purified cultured human islet MECs were infected with CVB-4 strain, and the immunological phenotype of the infected cells was analyzed. CVB-4 persistently infected the islet MECs, which expressed the CV receptors human coxsackievirus and adenovirus receptor (HCAR) and decay accelerating factor (DAF) and maintained EC characteristics, without overt cytopathic effects. CVB-4 infection transiently up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1 and increased production of the proinflammatory cytokines IL-1beta and IL-6, and chemokines IL-8 and lymphotactin, as well as IFN-alpha. Mononuclear cell adhesion to CVB infected monolayers was increased, compared to uninfected monolayers. Moreover, infection up-regulated the viral receptors HCAR and DAF and coreceptor alpha(v)beta3 integrin on islet MECs, while down-regulating expression of HCAR on human aortic endothelial cells, indicating potential tissue-specific influence on the pathological outcome of infection. These results provide evidence that islet MECs are natural targets and reservoirs for persistent CVB infection resulting in acute endothelial cell activation by virus, which may contribute to selective recruitment of subsets of leukocytes during inflammatory immune responses, such as insulitis in type 1 diabetes.

Hyperinsulinemia and insulin resistance are independently associated with plasma lipids, uric acid and blood pressure in non-diabetic subjects. The GISIR database.

BACKGROUND AND AIMS: We evaluated whether hyperinsulinemia and/or insulin resistance are independently associated with plasma lipids, uric acid and blood pressure in non-diabetic subjects. METHODS AND RESULTS: A database of non-diabetic Italian subjects has recently been set up using data from hyperinsulinemic euglycemic clamp studies carried out using the standard technique (40 mU per min per square meter of body surface area). In this database we evaluated the relationships between fasting plasma insulin (FPI), glucose metabolized during clamp (M) and plasma levels of triglycerides (TG), high density lipoprotein cholesterol (HDL-C), uric acid (UA) as well as blood pressure (BP) in non-diabetic subjects with fasting plasma glucose <6.1 mmol/l. Parallel analyses were conducted in all subjects in the database (n=1093) and in those with all variables available (n=309). In the univariate analysis both FPI and M were significantly correlated with TG, HDL-C, UA and BP (systolic, diastolic and mean). Multivariate regression analyses including center, sex, age, body mass index (BMI), FPI and M as independent variables showed that: (1) TG and UA were positively correlated with FPI and negatively correlated with M; (2) HDL-C was negatively correlated with FPI and positively correlated with M; and (3) BP was negatively correlated with both FPI and M. Analyses of covariance showed that, after adjusting for center, sex, age and BMI, subjects with isolated hyperinsulinemia or isolated insulin resistance had higher TG and UA and lower HDL-C. Subjects with isolated insulin resistance had also higher BP whereas subjects with isolated hyperinsulinemia had lower BP. Subjects with both defects had a worse profile. CONCLUSIONS: Hyperinsulinemia and insulin resistance might contribute with distinct and independent mechanisms to the development of several metabolic and hemodynamic disorders often clustering in the same individual. In particular, hypertriglyceridemia, low HDL-cholesterol and hyperuricemia seem to be related to both hyperinsulinemia and insulin resistance, whereas hypertension seems to be related only to insulin resistance.

Platelet-activating factor synthesis and response on pancreatic islet endothelial cells: relevance for islet transplantation.

BACKGROUND: Recent data suggest that donor intraislet endothelial cells may survive islet transplantation and participate to the events that influence islet engraftment. However, the mechanisms that regulate islet endothelial behavior in this setting are poorly known. METHODS: We obtained immortalized human (hIECs) and mouse (mIECs) islet endothelial cells by transfection with SV40-T-large antigen and studied the synthesis and response to Platelet-activating factor (PAF), a multipotent phospholipid that acts as endothelial mediator of both inflammation and angiogenesis. RESULTS: HIECs showed typical endothelial markers such as expression of vWF, CD31, and CD105, uptake of acetylated-LDL and binding to ULE-A lectin. Moreover, they expressed nestin, the PAF-receptor and possess surface fenestrations and in vitro angiogenic ability of forming tubular structures on Matrigel. Likewise, mIECs showed expression of vWF, CD31, nestin, PAF-receptor and CD105, and uptake of acetylated-LDL. HIECs and mIECs rapidly produced PAF under stimulation with thrombin in a dose-dependent way. Exogenous PAF or thrombin-induced PAF synthesis increased leukocyte adhesion to hIECS and mIECs and cell motility of both endothelial cell lines. Moreover, PAF or thrombin-induced PAF synthesis accelerated in vitro formation of vessel-like tubular structures when hIECs are seeded on Matrigel. Notably, gene-microarray analysis detected up-regulation of beta3 integrin gene on hIECs stimulated with PAF, that was confirmed at the protein level. CONCLUSIONS: Based on the novel development of immortalized islet endothelium, these results suggest that PAF may have a dual role that links inflammation to angiogenesis in the early events of islet transplantation.

Low incidence of end-stage renal disease and chronic renal failure in type 2 diabetes: a 10-year prospective study.

OBJECTIVE: Data on the incidence of end-stage renal disease (ESRD) and chronic renal failure from population-based studies in Caucasian type 2 diabetic patients are lacking. To provide such data, a population-based cohort of type 2 diabetic patients was identified in Casale Monferrato, Italy, and prospectively examined from 1991 to 2001. RESEARCH DESIGN AND METHODS: During the follow-up period, patients were regularly examined with centralized measurements of plasma creatinine and HbA(1c). Independent predictors of progression to renal events were identified with multivariate Cox proportional hazards modeling, with sex, age, and individual follow-up time as confounders. RESULTS: We followed 1,408 of 1,540 (91.4%) patients (average follow-up time 6.7 years, range 0.011-11.1); 10 new cases of ESRD and 72 of chronic renal failure (plasma values of creatinine >or=2.0 mg/dl) were identified, giving incidence rates/1,000 person-years of 1.04 (95% CI 0.56-1.94) and 7.63 (6.06-9.61), respectively. Cumulative risks for chronic renal failure adjusted for competing mortality were 6.1 and 9.3% after 20 and 30 years from diagnosis of diabetes, respectively. Incidence rates and cumulative risks of chronic renal failure defined by plasma creatinine values >1.5 mg/dl increased to 13.1/1,000 person-years, 8.6 and 14.8%, respectively. In Cox regression analysis, predictors of progression (after adjustment for confounders) were hypertension (P = 0.078), diastolic blood pressure (P = 0.034), BMI (P = 0.03), and albumin excretion rate (AER) (P < 0.0001). CONCLUSIONS: We provide evidence that the individual risk of ESRD and chronic renal failure is low. AER and diastolic blood pressure are independent predictors of progression. These findings underline the relevance of primary prevention to reduce the number of diabetic patients with ESRD.

Prevalence of Q-T interval dispersion in type 1 diabetes and its relation with cardiac ischemia : the EURODIAB IDDM Complications Study Group.

OBJECTIVE: The interlead variation in duration of the Q-T interval on the surface electrocardiogram (Q-T interval dispersion [QTd]) has been shown to predict mortality in type 2 diabetic patients. We evaluated the prevalence of QTd prolongation in the EURODIAB population and its relation to corrected Q-T interval (QTc), sex, age, duration of diabetes, blood glucose control, and complications. RESEARCH DESIGN AND METHODS; A total of 3,042 type 1 diabetic patients were studied. QTc was calculated according to the Bazett's formula; QTc > 0.44 s was considered abnormally prolonged. QTd was calculated using the difference between the maximum and the minimum QTc in any thoracic lead. QTd >0.080 s was considered abnormally prolonged. RESULTS: The prevalence of an increased QTd was 7%. A significant relation was observed between QTd prolongation and diastolic blood pressure (P < 0.05). A higher prevalence of QTd prolongation was observed in patients with ischemic heart disease (P = 0.004), whereas no relationship was observed with retinopathy, albumin excretion rate, or measures of somatic and autonomic neuropathy. QTc and QTd were significantly related (P = 0.001); however, a proportion of patients with normal QTd showed a prolonged QTc (>0.44 s). CONCLUSIONS: In patients with type 1 diabetes, QTd is associated with ischemic heart disease and diastolic blood pressure but not neuropathy. Although QTd is statistically related to duration of QTc, increased QTd and increased QTc identify different patients, and their predictive value deserves prospective evaluation.

Autonomic function and autoantibodies to autonomic nervous tissues at follow-up in a cohort of young patients with type 1 diabetes. Effects of serum from diabetic patients on human adrenergic cells.

Recent studies have linked autoimmunity to nervous tissue structures and diabetic autonomic neuropathy. To evaluate prospectively the early stage of type 1 diabetes and the natural history of this association, we monitored the autonomic function and the presence of autoantibodies (Ab) to sympathetic and parasympathetic nervous structures in a cohort of 92 diabetic adolescent patients, recruited and followed-up after 40+/-3 months. The presence of circulating Ab and their ability to activate complement was also assessed using a human adrenergic neuroblastoma cell line, and the effect of patients' sera on these cells was evaluated by different methods assessing cytotoxic effects and apoptosis. Thirty-nine percent of the Ab-positive patients had one abnormal test (p=0.07 vs. Ab-negative patients). Serum from four patients positive for anti-cervical ganglia Ab showed positive staining of neuroblastoma cells and displayed ability to activate complement. Serum from two adolescent patients with anti-cervical ganglia and anti-neuroblastoma cells Ab, induced cytotoxic effects and damaged the plasma membrane of the neuroblastoma cells. Moreover, sera from two adult patients with overt autonomic neuropathy, used as positive controls, induced apoptosis of these cells, assessed by TUNEL. Our data indicate that symptomatic autonomic neuropathy is not characteristic of young diabetic patients, but that autoantibodies to autonomic structures are present and persist in the first 20 years of disease, possibly associated with subtle autonomic dysfunction and cytotoxic effect on sympathetic cells.

Retinal heat shock protein 25 in early experimental diabetes.

Diabetic retinopathy is the leading cause of blindness in adults, and oxidative stress has been pathogenically associated with retinal neurodegeneration. Cellular stresses induce expression of heat shock proteins (HSPs) and this results in cytoprotection. Our aim was to assess retinal expression of HSP25 in early experimental diabetes. Mice were rendered diabetic by streptozotocin injection. Ten weeks after diabetes onset retinal HSP25 expression were studied by real-time PCR, immunoblotting and immunohistochemistry (IHC). Expression of nitrotyrosine and Cu/Zn superoxide dismutase (SOD), was assessed by IHC and apoptosis by TUNEL. Retinal HSP25 mRNA and protein expression was significantly increased in diabetic as compared to non-diabetic animals and localised predominantly within the retinal ganglion cells (RGC) layer. This was paralleled overexpression of nitrotyrosine and SOD and enhanced apoptosis. In early experimental diabetes, HSP25 is overexpressed in the RGC layer in parallel with markers of oxidative stress and apoptosis.

Serum heat shock protein 27 levels in patients with hepatocellular carcinoma.

Levels of serum heat shock protein 27 (sHsp27) have been studied in numerous cancer types, but their potential relevance in patients with hepatocellular carcinoma (HCC) is undetermined. Our aim was to compare sHsp27 levels in patients with HCC and HCC-free controls. Specifically, we recruited 71 patients with HCC (80 % with early tumour), 80 patients with chronic liver disease (59 with liver cirrhosis and 21 with chronic active hepatitis) and 42 healthy subjects. sHsp27 was measured by immunoenzymatic assay. Results showed that sHsp27 levels were significantly (p < 0.001) higher in patients with HCC than in the other groups, particularly in those with hepatitis C virus (HCV)-related disease. In HCC patients, sHsp27 levels were not associated with prognostic risk factors, such as size/multiplicity of nodules and stage. In logistic regression analysis, performed in patients with liver disease, log-sHsp27 was associated with a significant age-adjusted 2.5-fold increased odds ratio of HCC and with a significant 4.4-fold higher odds ratio of HCC in the subgroup with HCV-related liver disease. In receiver operating characteristic curve analysis, sensitivity and specificity of the best sHsp27 cut-off value (456.5 pg/ml) for differentiating patients with HCC from those with HCC-free chronic liver disease were 70 and 73 %, respectively. In conclusion, sHsp27 levels are enhanced in patients with HCC and may represent a candidate biomarker of HCC.

Urinary exosomal microRNAs in incipient diabetic nephropathy.

MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication.

Severe hypoglycemia and cardiovascular disease incidence in type 1 diabetes: the EURODIAB Prospective Complications Study.

OBJECTIVE: Frequent episodes of severe hypoglycemia may increase the risk of cardiovascular disease (CVD) in people with diabetes. Our aim was to study the relationship between severe hypoglycemic episodes and CVD incidence in subjects with type 1 diabetes, and further, to assess if markers of inflammation/endothelial injury were enhanced in individuals who experienced hypoglycemic episodes. RESEARCH DESIGN AND METHODS: The prospective study included 2,181 type 1 diabetic patients from the EURODIAB Prospective Complications Study. At baseline, frequency of self-reported severe hypoglycemia, defined as episodes serious enough to require the help of another person, was assessed based on responses to a patient questionnaire. Both fatal/nonfatal CVD was assessed 7.3 years after baseline examination. At the follow-up visit, data on both severe and nonsevere hypoglycemic episodes in the previous year were collected through a questionnaire and markers of inflammation/stress response/endothelial injury measured by enzyme-linked immunosorbent assays in the 531 subjects of the nested case-control study, including 363 case subjects with one or more complications of diabetes and 168 control subjects with no evidence of any complication. RESULTS: During the follow-up period, 176 patients had incident CVD. Logistic regression analysis showed that severe hypoglycemia at the baseline examination was not associated with incidence of CVD (adjusted odds ratios [95% CI]: one to two episodes, 0.87 [0.55-1.37]; three or more episodes, 1.09 [0.68-1.75]). Furthermore, follow-up serum levels of markers of endothelial damage/inflammation were not cross-sectionally associated with the frequency of hypoglycemic episodes. CONCLUSIONS: Taken together our data do not support the hypothesis that in type 1 diabetes, severe hypoglycemia increases the risk of CVD.

NH2-terminal probrain natriuretic peptide is associated with diabetes complications in the EURODIAB Prospective Complications Study: the role of tumor necrosis factor-α.

OBJECTIVE: Circulating levels of NH(2)-terminal probrain natriuretic peptide (NT-proBNP), a marker of acute heart failure, are associated with increased risk of cardiovascular disease (CVD) in the general population. However, there is little information on the potential role of NT-proBNP as a biomarker of vascular complications in type 1 diabetic patients. We investigated whether serum NT-proBNP levels were associated with micro- and macrovascular disease in type 1 diabetic subjects. RESEARCH DESIGN AND METHODS: A cross-sectional nested case-control study from the EURODIAB Prospective Complications Study of 507 type 1 diabetic patients was performed. Case subjects (n = 345) were defined as those with one or more complications of diabetes; control subjects (n = 162) were those with no evidence of any complication. We measured NT-proBNP levels by a two-site sandwich electrochemiluminescence immunoassay and investigated their associations with complications. RESULTS: Mean NT-proBNP levels were significantly higher in case than in control subjects. In logistic regression analyses, NT-proBNP values >26.46 pg/mL were independently associated with a 2.56-fold increased risk of all complications. Odds ratios of CVD (3.95 [95% CI 1.26-12.35]), nephropathy (4.38 [1.30-14.76]), and distal symmetrical polyneuropathy (4.32 [1.41-13.23]) were significantly increased in patients with NT-proBNP values in the highest quartile (>84.71 pg/mL), independently of renal function and known risk factors. These associations were no longer significant after inclusion of TNF-α into the model. CONCLUSIONS: In this large cohort of type 1 diabetic subjects, we found an association between NT-proBNP and diabetic micro- and macrovascular complications. Our results suggest that the inflammatory cytokine TNF-α may be involved in this association.

Targeting the MCP-1/CCR2 System in diabetic kidney disease.

Diabetic nephropathy is the leading cause of end-stage renal failure in the Western World and accounts for significant morbidity and mortality in patients with diabetes. Although hyperglycaemia and hypertension are established key determinants in the development of the complication, recent studies suggest that a low-grade inflammatory response may also play a role. Monocyte Chemoattractant Protein 1 (MCP-1), a potent chemokine produced by renal cells, has emerged as a very important player in this process. Specifically, it has been shown that MCP-1 is overexpressed in the kidneys from diabetic animals. Furthermore, there is amelioration of both functional and structural abnormalities in MCP-1- knockout mice in the setting of concomitant diabetes. Over recent years the cellular mechanisms linking MCP-1 to kidney injury have been increasingly delineated and, in particular, it has become evident that MCP-1 contributes to the kidney damage not only by inducing mononuclear cell recruitment, but also by direct activation of resident renal cells. The present review focuses on the most significant advances in understanding the role of MCP-1 in diabetic kidney disease and future potential therapeutic implications.

Human mesenchymal stem cells modulate cellular immune response to islet antigen glutamic acid decarboxylase in type 1 diabetes.

CONTEXT: Mesenchymal stem cells (MSCs) exert an immunosuppressive effect on the immune system. However, studies on the immunomodulatory potential of MSCs in type 1 diabetes are lacking. OBJECTIVE: We aimed to evaluate whether human MSCs may inhibit in vitro pancreatic islet antigen-specific T cell activation in type 1 diabetes. DESIGN: Human MSCs were isolated and characterized. Peripheral blood mononuclear cells (PBMCs) were obtained from nine type 1 diabetic patients at disease onset and 13 healthy control subjects. IFN-gamma, IL-10, and IL-4 enzyme-linked immunospot responses of lymphocytes incubated with glutamic acid decarboxylase 65 (GAD65) were investigated in PBMC cultures and PBMC/MSC cocultures. Levels of prostaglandin E2 (PGE2), IFN-gamma, IL-4, and IL-10 in supernatants were measured by ELISA. PGE2 inhibition experiments with NS-398 and indomethacin were also performed. RESULTS: Five diabetic patients were identified with a positive PBMC IFN-gamma response to GAD65 and negative IL-10 and IL-4 response. PBMC/MSC cocultures resulted in a significant decrease in the number of spots and in detection of IL-4-secreting cells. PGE2 inhibitors abrogated the immune-suppressive effect, indicating an involvement of PGE2 production, and the constitutive production of PGE2 by MSCs was enhanced in PBMC/MSC coculture. Moreover, in GAD-responder patients, GAD-stimulated PBMC/MSC cocultures significantly decreased secretion of IFN-gamma and IL-10 and increased secretion of IL-4. CONCLUSIONS: These results provide evidence that human MSCs abrogate in vitro a proinflammatory T helper type 1 response to an islet antigenic stimulus in type 1 diabetes. MSCs induce IL-4-producing cells, suggesting a possible switch to an antiinflammatory T helper type 2 signaling of T cells.