Pre-culturing human adipose tissue mesenchymal stem cells under hypoxia increases their adipogenic and osteogenic differentiation potentials.

OBJECTIVES: Hypoxia is an important factor in many aspects of stem-cell biology including their viability, proliferation, differentiation and migration. We evaluated whether low oxygen level (2%) affected human adipose tissue mesenchymal stem-cell (hAT-MSC) phenotype, population growth, viability, apoptosis, necrosis and their adipogenic and osteogenic differentiation potential. MATERIALS AND METHODS: hAT-MSCs from four human donors were cultured in growth medium under either normoxic or hypoxic conditions for 7 days and were then transferred to normoxic conditions to study their differentiation potential. RESULTS: Hypoxia enhanced hAT-MSC expansion and viability, whereas expression of mesenchymal markers such as CD90, CD73 and endothelial progenitor cell marker CD34, remained unchanged. We also found that pre-culturing hAT-MSCs under hypoxia resulted in their enhanced ability to differentiate into adipocytes and osteocytes. CONCLUSIONS: This protocol could be useful for maximizing hAT-MSC potential to differentiate in vitro into the adipogenic and osteogenic lineages, for use in plastic and reconstructive surgery, and in tissue engineering strategies.

Recognition of Ii-Key/MHC class II epitope hybrids derived from proinsulin and GAD peptides by T cells in type 1 diabetes.

In order to determine whether the Ii-Key technology can enhance the presentation of specific epitopes associated with type 1 diabetes, we have designed and synthesized a series of Ii-Key/proinsulin and GAD epitope hybrid peptides. Peptides of proinsulin and GAD shown to be recognized by CD4+ T cells of type 1 diabetes patients have been selected from the literature and modified with Ii-Key. A total of 23 Caucasian type 1 diabetes subjects and 17 normal subjects as controls were included in the study. Reactive T cells were identified using an IFN-γ ELISPOT assay. We selected 5 proinsulin and 5 GAD epitopes. Regarding the activity of the proinsulin Ii-Key hybrids, 3 out of 15 patients (20%) demonstrated a positive response to one or more Ii-Key hybrid peptides compared to no responders in the control subjects. Two out of 8 patients demonstrated a positive response to one or more Ii-Key/GAD65 hybrids. Proinsulin Ii-Key hybrids and peptides were recognized only by DR3/DR4 0302+ve diabetic patients. Control subjects showed no detectable response to stimulation with Ii-Key hybrids or peptides, neither for proinsulin nor GAD65. We have now shown that the use of Ii-Key-modified MHC class II epitopes, derived from proteins associated with insulin-secreting cells, can detect the presence of specifically activated CD4+ T helper cells with greater sensitivity than unmodified epitopes in the standard ELISPOT assay. The use of these technologies may be of use in identifying patients at the earliest stages of type 1 diabetes.

Hypoxia increases Sca-1/CD44 co-expression in murine mesenchymal stem cells and enhances their adipogenic differentiation potential.

Mesenchymal stem cells (MSCs) are usually cultured under normoxic conditions (21% oxygen). However, in vivo, the physiological "niches" for MSCs have a much lower oxygen tension. Because of their plasticity, stem cells are particularly sensitive to their environments, and oxygen tension is one developmentally important stimulus in stem cell biology and plays a role in the intricate balance between cellular proliferation and commitment towards differentiation. Therefore, we investigated here the effect of hypoxia (2% oxygen) on murine adipose tissue (AT) MSC proliferation and adipogenic differentiation. AT cells were obtained from the omental fat and AT-MSCs were selected for their ability to attach to the plastic dishes, and were grown under normoxic and hypoxic conditions. Prior exposure of MSCs to hypoxia led to a significant reduction of ex vivo expansion time, with significantly increased numbers of Sca-1(+) as well as Sca-1(+)/CD44(+)double-positive cells. Under low oxygen culture conditions, the AT-MSC number markedly increased and their adipogenic differentiation potential was reduced. Notably, the hypoxia-mediated inhibition of adipogenic differentiation was reversible: AT-MSCs pre-exposed to hypoxia when switched to normoxic conditions exhibited significantly higher adipogenic differentiation capacity compared to their pre-exposed normoxic-cultured counterparts. Accordingly, the expression of adipocyte-specific genes, peroxisome proliferator activated receptor gamma (Ppargamma), lipoprotein lipase (Lpl) and fatty acid binding protein 4 (Fabp4) were significantly enhanced in hypoxia pre-exposed AT-MSCs. In conclusion, pre-culturing MSCs under hypoxic culture conditions may represent a strategy to enhance MSC production, enrichment and adipogenic differentiation.

Characterization of endothelial progenitor cells in the NOD mouse as a source for cell therapies.

BACKGROUND: Endothelial progenitor cells (EPCs) in bone marrow (BM) and peripheral blood (PB) contribute to tissue repair in various pathological conditions via the formation of new blood vessels. Previous studies indicate that diabetic patients have reduced EPC number and deregulated EPC function, although the regenerative properties of EPCs in diabetes are unknown. We wish to characterize and compare EPCs from pre-diabetic and diabetic non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D), in order to delineate the role of these cells in the pathogenesis of autoimmune diabetes. METHODS: Whole BM was obtained by flushing femurs, tibias and illiac crests from pre-diabetic and diabetic NOD mice (5-30 weeks) in which the diabetic status was confirmed by measuring blood glucose levels (> or =11.5 mmol/L); PB was collected in heparin-coated tubes and lysed after incubation with antibodies directed against EPCs. RESULTS: FACS analyses revealed a significant decrease in EPC number (CD31(+), c-Kit(+), Sca-1(+), Lin(-)) in BM from diabetic compared to pre-diabetic mice (P = 0.02). Conversely, EPC number was significantly increased in PB from diabetic compared to pre-diabetic mice (P = 0.01). CONCLUSIONS: These data suggest that at the onset of diabetes, BM-derived EPCs are stimulated to enter the systemic circulation likely in response to signals from the pancreas. Further studies are required to elucidate whether EPCs home the damaged pancreas, thus representing a prospective source of autologous cells for beta-cell regeneration therapy.

Antinuclear antibodies prevalence in Filipinos migrated to Italy.

Antinuclear antibodies (ANA) represent the main diagnostic markers for systemic autoimmune disease (AD), although their presence can be detected in blood donors. The aim of this study was to study ANA prevalence in healthy subjects of different racial groups, exposed to the same environmental factors, in order to understand the relevance of genetics or environment in determining autoimmunity. We enrolled in this study 80 healthy Filipinos (Polynesian), migrated to Italy from an average of 15 years, and 60 healthy native Italians (Caucasian) and ANA were detected in their sera (at 1:80 screening dilution) through indirect immunofluorescence assay. We found a higher prevalence of ANA positivity in Filipinos compared to Italians (23.7% vs 8.3%--P = 0.02; OR 3.43; 95% CI 1.2-9.8), above all in females and elderly, although demographic characteristics, clinical history and habits were not significantly different between the two groups. These data confirm that ANA positivity is not a rare condition and healthy non-Caucasians present a higher prevalence of autoantibodies. This could be determined by their autoimmunity-prone immune system or by the exposition to infective agents, pollution, drugs or nutrition of a western country. Future studies to evaluate the ANA prevalence in Filipinos in their own country and the follow-up of positive patients could clarify the real predisposition to AD of this population.

Length of gestation and gender are associated with HLA genotypes at risk for Type 1 diabetes (Italian DIABFIN 3).

AIM: The aim of this study, which is part of the ongoing DIABFIN project, was to correlate HLA class II genotypes, classified for their effect on susceptibility to Type 1 diabetes (T1D), with various risk factors during pregnancy and the neonatal period. METHODS: Cord blood was collected from 4349 neonates; 1.0% were at high HLA risk (HR), 9.0% at moderate HLA risk (MR), and 90.0% at low HLA risk (LR) for T1D. Information about the mother's pregnancy, type of delivery, the neonates' clinical features at birth, and family history for autoimmune diseases were collected. RESULTS: Significant correlations were found between the different HLA risk categories and length of gestation, even when adjusted for sex, weight and length at birth of the neonate, birth order and mother's age (adjusted P = 0.007). The male : female ratio tended to increase from the LR to the HR category, from 1.00 and 1.21, respectively, in the LR and MR groups, to 1.62 in the HR group (P = 0.05). CONCLUSIONS: Length of gestation is inversely correlated with HLA risk categories for T1D. The higher the HLA risk for T1D, the shorter the gestational age, especially in male neonates.

Raised C-reactive protein levels in patients with recent onset type 1 diabetes.

BACKGROUND: To investigate serum concentrations of high-sensitive C-reactive protein (CRP) and alpha(1)-acid glycoprotein (AGP) in patients with T1DM, at diagnosis and after 12 months of intensive insulin therapy (T12). METHODS: CRP and AGP were measured in 44 recent onset T1DM patients (26M/18F, mean age 14.9 +/- 9.1 years), and 44 age- and sex-matched controls, using a highly sensitive immunonephelometric assay. RESULTS: There were no significant differences in the concentrations of high-sensitive C-reactive protein (hs-CRP) and AGP between patients and controls. hs-CRP levels significantly increased in patients at T12 compared to the levels at diagnosis [0.69 (0.14-15.5) versus 0.43 (0.14-7.47) mg/L, p < 0.05; for males: 0.77 (0.14-15.5) versus 0.35 (0.14-7.47) mg/L, p < 0.05; for females the increase was not significant]. AGP levels were not different at T12 compared to diagnosis. No significant correlations were found between hs-CRP and body mass index (BMI), C-peptide, glycosylated haemoglobin, or insulin dose. A strong correlation was found between hs-CRP values at diagnosis and those at T12 (rho = 0.73, p < 0.001); indeed, patients with hs-CRP levels above the 50th percentile at diagnosis showed significantly increased hs-CRP values at T12 compared to patients with baseline hs-CRP levels under the 50th percentile [1.61 (0.18-15.5) versus 0.16 (0.14-1.92) mg/L, p < 0.0001)], and to controls [0.55 (0.14-6.50), p = 0.001]. CONCLUSIONS: This is the first report showing that, despite good metabolic control, 1 year of overt T1DM is sufficient to increase hs-CRP levels, especially in males. hs-CRP levels at diagnosis is a predictor for the values observed at 12 months, suggesting the possibility to select a subgroup of patients requiring strict follow-up for cardiovascular complications.

Lack of effect of vitamin D administration during pregnancy and early life on diabetes incidence in the non-obese diabetic mouse.

BACKGROUND AND AIMS: Several studies have suggested that vitamin D supplementation in early life may reduce the risk of developing type 1 diabetes in later life. The non-obese diabetic (NOD) mouse is a model of spontaneous type 1 diabetes currently used for testing hypothesis/compounds aimed at disease prevention. In this study, we tested the effect of vitamin D (16 IU by gavage) on diabetes incidence in NOD/Ba mice treated from conception with olive oil containing vitamin D via maternal dosing up to 10 weeks of age and followed up until 32 weeks of age. METHODS: Twelve breeding pairs were administered olive oil containing vitamin D during pregnancy, 15 days following the birth of the pups and for the next 10 weeks subsequently. The same breeding pairs were bred again after a clearance period of 15 days using a control solution to produce a control litter. This control group received a control solution for the same period of time. Diabetes incidence, degree of insulitis, and insulin content in the pancreas were investigated in the two groups. RESULTS: 12 vitamin D-treated NOD mice developed diabetes compared to 15 animals in the control group (Log rank test p = 0.899, NS). There were no significant differences between the groups in diabetes incidence, time of onset of the disease, degree of insulitis, or the insulin content in the pancreas. CONCLUSION: Vitamin D administered in utero and in the early stages of life at the dosage used does not change the incidence of diabetes or modify the disease process that leads to beta cell destruction in the NOD mouse.

The natural history of insulin content in the pancreas of female and male non-obese diabetic mouse: implications for trials of diabetes prevention in humans.

BACKGROUND: The non-obese diabetic (NOD) mouse is a well-established animal model used to study the pathogenesis of type 1 diabetes. The NOD mouse spontaneously develops an autoimmune form of the disease between 12 and 18 weeks of age, characterized by an infiltration of the endocrine pancreas by autoreactive mononuclear cells. In our colony, all animals showed signs of insulitis, but only approximately 60% of females and 15% of males developed diabetes. The aim of this study was to determine the natural history of insulin content in the pancreas of female and male NOD/Ba mice during their life span. METHODS: Pancreata were collected at two-week intervals, from 4 weeks of age to 30 weeks of age. Four animals at each age as well as 18 diabetic female NOD mice were studied. Pancreata were homogenized, supernatants collected and insulin measured by radioimmunoassay. RESULTS: Female and male non-diabetic NOD mice showed significantly higher levels of insulin in the pancreata in comparison to the diabetic female animals. Pancreata from female (n = 56) animals showed more insulin content than male pancreata (n = 56), suggesting beta-cell hyperactivity as a result of the ongoing beta-cell destruction. However this difference was only significant at an early age (4-12 weeks of age) (p < 0.04). Insulin content in diabetic female NOD pancreas declines with time, and was very low at the age of 25 to 34 weeks. This decline was not observed in male pancreata despite the presence of lymphocytic infiltration. CONCLUSION: We conclude that a reduction in pancreatic insulin content occurs slowly in the natural history of the disease and that such reduction only becomes apparent after diagnosis of hyperglycaemia. Occurrence of extensive lymphocytic infiltration in non-diabetic male mice is not accompanied by a reduction of insulin content in the pancreas. These findings have implications for designing studies in humans which aims to protect residual beta-cell function.

Reduced cumulative incidence of diabetes but not insulitis following administration of chimeric human IL-15-murine IgG2b in NOD mice.

BACKGROUND: It has been recently demonstrated that apoptosis is involved in beta-cell destruction in the NOD mouse model of diabetes. The aim of the present study was to investigate whether IL-15, a cytokine involved in the modulation of the apoptotic process, is capable of modifying the natural history of diabetes and/or insulitis in pre-diabetic NOD mice. The rationale for the use of IL-15-IgG2b recombinant cytokine is related to its long half-life (28 +/- 4 h). METHODS: At 10 weeks of age, 2 groups of 24 female mice were treated with single or multiple i.p. doses of IL-15-IgG2b respectively. As control, 2 groups of 24 age- and litter-matched female mice were injected intra-peritoneally with single or multiple doses of IgG2b immunoglobulin. RESULTS: Diabetes incidence at 33 weeks of age was lower in the group of mice treated with multiple doses than in the control group (p = 0.03). The cumulative incidence of diabetes at 33 weeks of age between single-dose treated mice and the control group was similar. No significant differences in the calculated index of insulitis were observed in all treated and control mice. CONCLUSIONS: We conclude that IL-15-IgG2b reduces the cumulative incidence of diabetes, without affecting the extent and severity of the insulitis process. Considering this and the well-defined anti-apoptotic effects of IL-15, we suggest that the reduction of diabetes incidence could be due to a down-regulation of beta-cell apoptosis.

[Partial deletion of the long arm of chromosome 17. Presentation of a clinical case]. / Delezione parziale del braccio lungo del cromosoma 17. Presentazione di un caso clinico.

A partial deletion of the long arm of chromosome 17 has been found in a liveborn infant. The clinical findings of the propositus are compared with those of a previously reported case allowing delineation of a hitherto unrecognized partial deletion (17q) syndrome.

Ring 20 chromosome phenotype.

A baby with a 46,XY,r(20) karyotype is described. The distinguishing features of this rare chromosome anomaly are analysed in the light of the suggested r(20) syndrome.