Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO2, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised.

The efficacy and safety of the new drug, BGP-15, were compared with placebo in insulin-resistant patients in a 28-day dose-ranging study. Forty-seven nondiabetic patients with impaired glucose tolerance were randomly assigned to 4 weeks of treatment with 200 or 400 mg of BGP-15 or placebo. Insulin resistance was determined by hyperinsulinemic euglycemic clamp technique and homeostasis model assessment method, and beta-cell function was measured by intravenous glucose tolerance test. Each BGP-15 dose significantly increased whole body insulin sensitivity (M-1, p=0.032), total body glucose utilization (M-2, p=0.035), muscle tissue glucose utilization (M-3, p=0.040), and fat-free body mass glucose utilization (M-4, p=0.038) compared to baseline and placebo. No adverse drug effects were observed during treatment. BGP-15 at 200 or 400 mg significantly improved insulin sensitivity in insulin-resistant, nondiabetic patients during treatment compared to placebo and was safe and well-tolerated. This was the first clinical study demonstrating the insulin-sensitizing effect of a molecule, which is considered as a co-inducer of heat shock proteins.

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Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.

Weight gain and dysfunction of glucose and lipid metabolism are well-known side effects of atypical antipsychotic drugs (AAPD). Here, we address the question whether a heat-shock protein (HSP) co-inducer, insulin sensitizer drug candidate, BGP-15, can prevent AAPD-induced glucose, lipid, and weight changes. We also examined how an AAPD alters HSP expression and whether BGP-15 alters that expression. Four different experiments are reported on the AAPD BGP-15 interventions in a human trial of healthy men, a rodent animal model, and an in vitro adipocyte cell culture system. Olanzapine caused rapid insulin resistance in healthy volunteers and was associated with decreased level of HSP72 in peripheral mononuclear blood cells. Both changes were restored by the administration of BGP-15. In Wistar rats, weight gain and insulin resistance induced by clozapine were abolished by BGP-15. In 3T3L1 adipocytes, clozapine increased intracellular fat accumulation, and BGP-15 inhibited this process. Taken together, our results indicate that BGP-15 inhibits multiple metabolic side effects of atypical antipsychotics, and this effect is likely to be related to its HSP co-inducing ability.

Background: In this study, the effect of antigen-presenting cells (APC), peptide concentration, and CD28 costimulation on calcium signaling, induced by antigen-specific T-cell activation, was studied by flow cytometry. Methods: We used two experimental approaches, which differed in their time scale and in the duration of the T cell-APC interaction, to measure the increase of intracellular free calcium levels ([Ca 2ϩ ] i ) in activated T cells: (1) Fluo-3-loaded T cells were activated by cocentrifugation with peptide-loaded APC and the kinetics of fluorescence intensity changes was monitored continuously and (2) peptide-loaded APC and T cells were mixed, cocultured, and the fluorescence intensity was measured at various time intervals. Results: The calcium signal of T cells was dependent on the APC as demonstrated by the ratio of cells exhibiting high versus low fluorescence intensity and by the magnitude of the calcium signal in the activated population. Short-term interaction of T cells with less potent APC or

There is no clinically available cancer immunotherapy that exploits Langerhans cells (LCs), the epidermal precursors of dendritic cells (DCs) that are the natural agent of antigen delivery. We developed a DNA formulation with a polymer and obtained synthetic 'pathogen-like' nanoparticles that preferentially targeted LCs in epidermal cultures. These nanoparticles applied topically under a patch-elicited robust immune responses in human subjects. To demonstrate the mechanism of action of this novel vaccination strategy in live animals, we assembled a high-resolution two-photon laser scanning-microscope. Nanoparticles applied on the native skin poorly penetrated and poorly induced LC motility. The combination of nanoparticle administration and skin treatment was essential both for efficient loading the vaccine into the epidermis and for potent activation of the LCs to migrate into the lymph nodes. LCs in the epidermis picked up nanoparticles and accumulated them in the nuclear region demonstrating an effective nuclear DNA delivery in vivo. Tissue distribution studies revealed that the majority of the DNA was targeted to the lymph nodes. Preclinical toxicity of the LC-targeting DNA vaccine was limited to mild and transient local erythema caused by the skin treatment. This novel, clinically proven LC-targeting DNA vaccine platform technology broadens the options on DC-targeting vaccines to generate therapeutic immunity against cancer.

Atypical antipsychotic drugs (AAPD) are widely used to treat severe psychiatric disorders, have well documented metabolic side effects such as disturbances in glucose metabolism, insulin resistance and weight gain. It has been shown that BGP-15, a hydroxylamine derivative with insulin sensitizing activity can prevent AAPD provoked fat accumulation in adipocyte cultures, and insulin resistance in animal experiments and in healthy volunteers. The aim of this study was to compare the preventive effect of BGP-15 with conventional oral antidiabetics on metabolic side effects of AAPDs. We found that BGP-15 that does not belong to either conventional insulin sensitizers or oral antidiabetics, is able to counteract insulin resistance and weight gain provoked by antipsychotic agents in rats while rosiglitazone and metformin were not effective in the applied doses. Our results confirm that BGP-15 is a promising new drug candidate to control the metabolic side effects of atypical antipsychotics. Data indicate that this rat model is suitable to analyze the metabolic side effects of AAPDs and the protective mechanism of BGP-15.