Peripheral autoreactive CD8 T-cell frequencies are too variable to be a reliable predictor of disease progression of human type 1 diabetes.

OBJECTIVES: The detection of a peripheral immune cell signature that specifically reflects autoimmunity in type 1 diabetes would enable the prediction and staging of disease on an individual basis. However, defining such a signature is technically challenging. Reliable interpretation of immune cell-related biomarkers depends on their inherent variability and, to understand this variability, longitudinal analyses are required. METHODS: We performed a longitudinal observational study in which 40 individuals with elevated genetic risk of type 1 diabetes and persistent islet autoantibodies provided a blood sample every 4-6 weeks for > 1 year. RESULTS: Peripheral immune cell composition (T cells, NK cells and monocytes) was assessed using well-validated flow cytometry panels and demonstrated that, while non-antigen-specific immune cell subsets were stable over time, autoantigen-reactive T-cell frequencies were highly variable in and between individuals. Neither the frequency nor phenotype of non-antigen-specific subsets or autoreactive CD8+ T cells associated with clinical onset of T1D. CONCLUSION: The findings from the Type 1 Diabetes Longitudinal BIomarker Trial underscore the inherent challenge of evaluating changes in peripheral immune cell populations as surrogates of organ-specific disease activity. The variability of peripheral antigen-specific T cells precludes their use as a prognostic marker and clearly demonstrates that a reliable prognostic cell signature remains elusive.

POSSIBLE ROLE OF NUTRITION IN PREVENTION OF SARCOPENIA AND FALLS.

Objective: In this review, we analyze the foundation of sarcopenia as a potentially modifiable risk factor for falls, and we try to formulate practical strategies for nutritional interventions aimed at reducing the risk for sarcopenia and falls in our elderly patients. Methods: An extensive literature search was performed using the PubMed and the Google Scholar databases. Results: Falls are a common and costly source of injury and death in elderly adults. A large proportion of injurious falls are due to a trip or slip, suggesting that muscular factors are major determinants of both fall risk and the risk for fall-related injury. Conclusion: An increasing body of evidence links sarcopenia, the loss of muscle strength and mass that occurs with advancing age, with an increased risk for falls. Nutritional factors, as well as exercise, can help with both prevention and treatment of sarcopenia and may reduce the risk of falls in the elderly. Abbreviations: 25-OHD = 25-hydroxyvitamin D; EAA = essential amino acid; IGF-1 = insulin-like growth factor 1; IU = international units; MPS = muscle protein synthesis; PUFA = polyunsaturated fatty acid.

Biochemical and Clinical Features of Insulinoma in a Patient with Turner Syndrome.

Turner syndrome (TS), i.e., mosaic or nonmosaic states with only one normal X chromosome in females, is characterized by a wide spectrum of somatic, hormonal, and metabolic features. Here we report an unusual case of recurrent hypoglycemia in a 53-year-old woman with TS. Biochemical work-up following a 72h fast revealed detectable, inappropriate for low glucose insulin levels and elevated proinsulin and beta-hydroxybutyrate (BOHB) levels. MR and multiphase CT showed a solid 2.5 cm pancreatic tail mass with absent uptake in the 111In-pentetreotide (Octreoscan) scan. Subsequent hepatic vein blood sampling after intra-arterial calcium stimulation showed sharp increase in insulin and modest increase in proinsulin levels. The patient underwent excision of the mass with resolution of symptoms. Histopathologic examination confirmed the neuroendocrine etiology of the tumor. This is, to our knowledge, the third report of TS and concomitant insulinoma. Impaired counterregulatory response to hypoglycemia in patients with TS may result in symptomatic hypoglycemia with only mild insulin elevation and elevated proinsulin in setting of hypoglycemia may be the only indication of insulinoma in these patients. BOHB levels should not be used for ruling out EHH in patients with TS.

Development and Standardization of an Improved Type 1 Diabetes Genetic Risk Score for Use in Newborn Screening and Incident Diagnosis.

OBJECTIVE: Previously generated genetic risk scores (GRSs) for type 1 diabetes (T1D) have not captured all known information at non-HLA loci or, particularly, at HLA risk loci. We aimed to more completely incorporate HLA alleles, their interactions, and recently discovered non-HLA loci into an improved T1D GRS (termed the "T1D GRS2") to better discriminate diabetes subtypes and to predict T1D in newborn screening studies. RESEARCH DESIGN AND METHODS: In 6,481 case and 9,247 control subjects from the Type 1 Diabetes Genetics Consortium, we analyzed variants associated with T1D both in the HLA region and across the genome. We modeled interactions between variants marking strongly associated HLA haplotypes and generated odds ratios to create the improved GRS, the T1D GRS2. We validated our findings in UK Biobank. We assessed the impact of the T1D GRS2 in newborn screening and diabetes classification and sought to provide a framework for comparison with previous scores. RESULTS: The T1D GRS2 used 67 single nucleotide polymorphisms (SNPs) and accounted for interactions between 18 HLA DR-DQ haplotype combinations. The T1D GRS2 was highly discriminative for all T1D (area under the curve [AUC] 0.92; P < 0.0001 vs. older scores) and even more discriminative for early-onset T1D (AUC 0.96). In simulated newborn screening, the T1D GRS2 was nearly twice as efficient as HLA genotyping alone and 50% better than current genetic scores in general population T1D prediction. CONCLUSIONS: An improved T1D GRS, the T1D GRS2, is highly useful for classifying adult incident diabetes type and improving newborn screening. Given the cost-effectiveness of SNP genotyping, this approach has great clinical and research potential in T1D.

Alpha cells, the main source of IL-1ß in human pancreas.

Interleukin-1ß (IL-1ß) is known to trigger beta cell dysfunction in vitro and could potentially play a role during the pathogenesis of type 1 diabetes and type 2 diabetes. However, several clinical trials attempting to block IL-1ß function have had minimal success. We therefore re-investigated local expression of IL-1ß in human diabetic and non-diabetic pancreata. We obtained pancreatic tissue sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) including non-diabetic (n = 9), non-diabetic auto-antibody positive (AAb+, n = 5), type 1 diabetes (n = 6), and type 2 diabetes (n = 6) donors. Islets were systematically investigated for the presence of IL-1ß mRNA by in situ hybridization and IL-1ß protein by indirect immunofluorescence. We found that intra-islet IL-1ß was produced at comparable level in both non-diabetic and diabetic donors. Interestingly, the main source for IL-1ß was alpha cells but not beta cells. Our findings call into question the role of IL-1ß in the diabetic pancreas as it has been proposed in previous literature. Additionally, our results regarding the localization of IL-1ß should lead to further investigation into the role of IL-1ß in the physiology of pancreatic alpha cells.

Pancreatic Tissue Transplanted in TheraCyte Encapsulation Devices Is Protected and Prevents Hyperglycemia in a Mouse Model of Immune-Mediated Diabetes.

Type 1 diabetes (T1D) is characterized by destruction of glucose-responsive insulin-producing pancreatic ß-cells and exhibits immune infiltration of pancreatic islets, where CD8 lymphocytes are most prominent. Curative transplantation of pancreatic islets is seriously hampered by the persistence of autoreactive immune cells that require high doses of immunosuppressive drugs. An elegant approach to confer graft protection while obviating the need for immunosuppression is the use of encapsulation devices that allow for the transfer of oxygen and nutrients, yet prevent immune cells from making direct contact with the islet grafts. Here we demonstrate that macroencapsulation devices (TheraCyte) loaded with neonatal pancreatic tissue and transplanted into RIP-LCMV.GP mice prevented disease onset in a model of virus-induced diabetes mellitus. Histological analyses revealed that insulin-producing cells survived within the device in animal models of diabetes. Our results demonstrate that these encapsulation devices can protect from an immune-mediated attack and can contain a sufficient amount of insulin-producing cells to prevent overt hyperglycemia.

Potential viral pathogenic mechanism in human type 1 diabetes.

In type 1 diabetes, as a result of as yet unknown triggering events, auto-aggressive CD8(+) T cells, together with a significant number of other inflammatory cells, including CD8(+) T lymphocytes with unknown specificity, infiltrate the pancreas, leading to insulitis and destruction of the insulin-producing beta cells. Type 1 diabetes is a multifactorial disease caused by an interactive combination of genetic and environmental factors. Viruses are major environmental candidates with known potential effects on specific key points in the pathogenesis of type 1 diabetes and recent findings seem to confirm this presumption. However, we still lack well-grounded mechanistic explanations for how exactly viruses may influence type 1 diabetes aetiology. In this review we provide a summary of experimentally defined viral mechanisms potentially involved in the ontology of type 1 diabetes and discuss some novel hypotheses of how viruses may affect the initiation and natural history of the disease.

Transient B-cell depletion with anti-CD20 in combination with proinsulin DNA vaccine or oral insulin: immunologic effects and efficacy in NOD mice.

A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks) was also ineffective, while proinsulin DNA (weekly for up to 12 weeks) showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04). In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.

Viruses and Type 1 diabetes: a dynamic labile equilibrium.

Type 1 diabetes (T1D) results from the specific immune-mediated destruction of the insulin-producing ß-cells of the pancreas. In genetically susceptible individuals, a still undetermined initiating 'hit' triggers a cascade of events that eventually leads to autoreactive CD8 T cells infiltrating the pancreatic islets and, subsequently, destroying them. There is increasing evidence that viruses, especially enteroviruses, are major environmental candidates; however, despite decades of investigation, we still lack certainty with regard to the causation of T1D. Moreover, studies in animal models of diabetes suggest a protective role of certain enteroviral infections upon diabetes contraction, making the quest for viral involvement in T1D even more difficult. Analyzing the foundation and the results of the most current work in the field, this article gives a brief overview of current knowledge, as well as providing an outlook for future directions.

A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis.

Hematogenous dissemination of melanoma is a life-threatening complication of this malignant tumor. Here, we identified junctional adhesion molecule-C (JAM-C) as a novel player in melanoma metastasis to the lung. JAM-C expression was identified in human and murine melanoma cell lines, in human malignant melanoma, as well as in metastatic melanoma including melanoma lung metastasis. JAM-C expressed on both murine B16 melanoma cells as well as on endothelial cells promoted the transendothelial migration of the melanoma cells. We generated mice with inactivation of JAM-C. JAM-C(-/-) mice as well as endothelial-specific JAM-C-deficient mice displayed significantly decreased B16 melanoma cell metastasis to the lung, whereas treatment of mice with soluble JAM-C prevented melanoma lung metastasis. Together, JAM-C represents a novel therapeutic target for melanoma metastasis.

Vascular endothelial growth factor (VEGF)-induced up-regulation of CCN1 in osteoblasts mediates proangiogenic activities in endothelial cells and promotes fracture healing.

Angiogenesis is indispensable during fracture repair, and vascular endothelial growth factor (VEGF) is critical in this process. CCN1 (CYR61) is an extracellular matrix signaling molecule that has been implicated in neovascularization through its interactions with several endothelial integrin receptors. CCN1 has been shown to be up-regulated during the reparative phase of fracture healing; however, the role of CCN1 therein remains unclear. Here, the regulation of CCN1 expression in osteoblasts and the functional consequences thereof were studied. Stimulation of osteoblasts with VEGF resulted in a dose- and time-dependent up-regulation of CCN1 mRNA and protein. An up-regulation of both cell surface-associated CCN1 as well as extracellular matrix-associated CCN1 in osteoblasts was found. The supernatant of VEGF-prestimulated osteoblasts was chemotactic for endothelial cells, increasing their migration and stimulated capillary-like sprout formation. These effects could be attributed to the presence of CCN1 in the osteoblast supernatant as they were prevented by an antibody against CCN1 or by small interfering RNA-mediated knockdown of osteoblast CCN1. Moreover, the supernatant of VEGF-prestimulated osteoblasts induced angiogenesis in Matrigel plugs in vivo in a CCN1-dependent manner. In addition, blockade of CCN1 prevented bone fracture healing in mice. Taken together, the present work demonstrates a potential paracrine loop consisting of the VEGF-mediated up-regulation of CCN1 in osteoblasts that attracts endothelial cells and promotes angiogenesis. Such a loop could be operative during fracture healing.

Inhibition of breast cancer cell adhesion and bone metastasis by the extracellular adherence protein of Staphylococcus aureus.

Bone metastasis is a common sequelae of breast cancer and the interaction of alpha v beta3-integrin with osteopontin (OPN) found in the extracellular matrix of mineralized tissues is implicated in this process. The integrin-dependent proadhesive and promigratory functions of OPN are particularly attributed to the 40 kD N-terminal fragment that derives upon matrix metalloproteinase (MMP) cleavage. Based on the broad repertoire of interactions between Staphylococcus aureus extracellular adherence protein (Eap) and host components, we here characterized Eap to specifically interact with recombinant full-length OPN and the 40 kD N-terminal MMP cleavage fragment, but not with the 32 kD or the 25 kD C-terminal fragments of OPN. Eap thereby prevented the OPN/alpha v beta3-integrin interaction, as well as the alpha v beta3-integrin-dependent adhesion of MDA-MB-231 breast cancer cells to full-length OPN or to the 40 kD fragment and the migration of these cells towards OPN. Furthermore, Eap treatment markedly impaired the development of osseous metastasis of human MDA-MB-231 cells in vivo. Taken together, Eap may represent an attractive novel treatment for the prevention of breast cancer bone metastasis.

Isolated pancreatic islets in three-dimensional matrices are responsive to stimulators and inhibitors of angiogenesis.

The formation of a new microvasculature is essential for the long-term survival and function of the islet graft. In this study we examined endothelium of isolated pancreatic islets by stimulation with growth factors, different culture conditions, and genetic modification. We also inspected the effect of immunosuppressives used in human transplantation on angiogenesis. Isolated islets were embedded in a three-dimensional fibrin or Matrigel matrix. The effect of hyperglycemia, hypoxia, and the addition of VEGF and bFGF was investigated. We exposed islets from transgenic mice expressing the VEGF gene (RIP1VEGF-A) to high glucose (16.7 mmol/L) medium and tested the immunosuppressive agents rapamycin (100 ng/ml) and FK506 (100 ng/ml). To quantify angiogenesis the percentage of sprouting islets was determined. New endothelial capillary-like structures protruded from isolated pancreatic islets. Addition of VEGF to the islets and transgenic RIP-VEGF islets showed a two- to threefold increase of sprouting islets compared to control. Hypoxic culture conditions stimulated angiogenesis, resulting in a twofold increase of capillary sprouting. Rapamycin and FK506 proved to be potent inhibitors of angiogenesis in this system, because a decrease of sprouting islets of more than 20% by both agents was observed. Isolated pancreatic islets are capable of forming new capillary structures and are susceptible to pro- and antiangiogenic stimuli.

Suppression of experimental autoimmune encephalomyelitis by extracellular adherence protein of Staphylococcus aureus.

Multiple sclerosis (MS) is a devastating inflammatory disorder of the central nervous system (CNS). A major hallmark of MS is the infiltration of T cells reactive against myelin components. T cell infiltration is mediated by the interaction of integrins of the beta1 and beta2 family expressed by lymphocytes with their endothelial counter-receptors, vascular cell adhesion molecule 1 and intercellular adhesion molecule (ICAM)-1, respectively. We have reported previously that extracellular adherence protein (Eap) of Staphylococcus aureus exerts antiinflammatory activities by interacting with ICAM-1 and blocking beta2-integrin-dependent neutrophil recruitment. Here, we report that Eap inhibits experimental autoimmune encephalomyelitis (EAE) in mice. In vitro, Eap reduced adhesion of peripheral blood T cells to immobilized ICAM-1 as well as their adhesion and transmigration of TNF-activated human endothelium under static and shear flow conditions. These inhibitory effects were corroborated in two mouse models of inflammation. In a delayed-type hypersensitivity model, both T cell infiltration and the corresponding tissue edema were significantly reduced by Eap. In addition, Eap administration prevented the development of EAE and markedly decreased infiltration of inflammatory cells into the CNS. Strikingly, intervention with Eap after the onset of EAE suppressed the disease. Collectively, our findings indicate that Eap represents an attractive treatment for autoimmune neuroinflammatory disorders such as MS.

The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

Vascular endothelial growth factor increases functional beta-cell mass by improvement of angiogenesis of isolated human and murine pancreatic islets.

BACKGROUND: Blood flow is impaired in islet transplants, but there is conflicting evidence on improving the outcome by promoting vascularization. We previously reported that islet endothelial cells (EC) possess significant angiogenic capacity. METHODS: To further address this issue, we studied human islets in culture under hypoxic conditions. Moreover, we used a transgene mouse model with human vascular endothelial growth factor (VEGF) production in beta-cells under the control of the rat insulin promoter (RIP) to stimulate islet EC proliferation. RESULTS: Subsequent to a hypoxic stimulus, islets responded with specific expression patterns of VEGF and fibroblast growth factor; however, this was not sufficient to prevent the decay of islet EC. VEGF release of RIP-VEGF transgenic islets was controlled by glucose and resulted in the formation of sprouts. When transplanted to the kidney capsule of diabetic mice, RIP-VEGF islets significantly enhanced microvascular density and functional blood flow to the graft compared with controls. CONCLUSIONS: Optimized angiogenesis of islet transplants resulted in greater availability of insulin caused by beta-cell proliferation and a significantly higher percentage (90% versus 20%) of mice cured from diabetes.

Polymers for induction of revascularization in the rat fascial flap: application of vascular endothelial growth factor and pancreatic islet cells.

One of the major obstacles in transplanting avascular tissue or metabolically active cells for ischemic diseases is the loss of transplanted cells due to lack of oxygen and nutrients in the early posttransplantation period. Biodegradable polymeric tissue engineering scaffolds and hydrogels have a potential to incorporate cells or cellular organoids such as islets of Langerhans and growth factors. In this study, we tested the efficiency of two types of polymeric materials to carry recombinant human vascular endothelial growth factor (rhVEGF) or pancreatic tumor cell lines, namely Ins-1 and AR42J, for the induction of new vessels. Chitosan hydrogel fibers with micropores were prepared and molded into a cylinder construct (5 mm phi; 8 mm height). Macroporous PLGA scaffolds with a pore size of 250-400 microm were prepared and cut into cylinders (6 mm phi; 3 mm height). Both chitosan and PLGA constructs were loaded with rhVEGF (3 microg) or seeded with the cell lines (5 x 10(5) cells and 3 x 10(5) cells/construct, respectively, for AR42J and INS-1 cells), and transplanted into the fascial flaps of Wistar rats. At distinct time points up to 4 weeks postimplantation, polymers were explanted, fixed, and vessel density was counted on sections stained with anti-Factor-VIII antibody. Additionally, the kinetics of rhVEGF release from PLGA microspheres (phi of 50-80 microm) was determined using VEGF Elisa. Endogenous VEGF release from pancreatic rat cell lines was also determined. Light microscopy study was performed on H&E-stained paraffin sections of the islet-polymer samples. The vascular density of rhVEGF-loaded chitosan constructs was increased fourfold 2 weeks after subcutaneous transplantation compared with rhVEGF-unloaded controls (465 +/- 144 vs. 104 +/- 80 vessels per mm2, p < 0.05). Protein leakage occurred, but was not observed after 2 weeks. Higher insulin content was detected in rat islet grafts transplanted following VEGF application. More than 50% of total rhVEGF was released on the first day of in vitro culture of PLGA microspheres. rhVEGF secretion had another, but smaller, peak on the third day followed by a constant release. By comparison, endogeneous VEGF secretion of pancreatic tumor cells was measured within a 3-day culture period. Biodegradable polymer scaffolds and hydrogels may have potential use as solid supports to induce angiogenesis for pancreatic cell transplantation.