Response of blood glucose and GLP-1 to different food temperature in normal subject and patients with type 2 diabetes.

BACKGROUND: Eating behavior is a major factor in type 2 diabetes. We investigated the different responses of glucose-regulating hormones to cold and hot glucose solutions in normal subjects and patients with type 2 diabetes. METHODS: In this crossover, self-controlled study, normal subjects (N = 19) and patients with type 2 diabetes (N = 22) were recruited and randomly assigned to a hot (50 °C) or a cold (8 °C) oral glucose-tolerance test (OGTT). The subsequent day, they were switched to the OGTT at the other temperature. Blood glucose, insulin, GIP, glucagon-like peptide-1 (GLP-1), and cortisol were measured at 0, 5, 10, 30, 60, and 120 min during each OGTT. After the hot OGTT, all subjects ingested hot (>42 °C) food and water for that day, and ingested food and water at room temperature (≤24 °C) for the day after cold OGTT. All participants had continuous glucose monitoring (CGM) throughout the study. RESULTS: Compared to cold OGTT, blood glucose was significantly higher with hot OGTT in both groups (both P < 0.05). However, insulin and GLP-1 levels were significantly higher in hot OGTT in normal subjects only (both P < 0.05). The GIP and cortisol responses did not differ with temperature in both groups. CGM showed that normal subjects had significantly higher 24-h mean glucose (MBG) (6.11 ± 0.13 vs. 5.84 ± 0.11 mmol/L, P = 0.021), and standard deviation of MBG with hot meals (0.59 ± 0.06 vs. 0.48 ± 0.05 mmol/L, P = 0.043), T2DM patients had higher MBG only (8.46 ± 0.38 vs. 8.88 ± 0.39 mmol/L, P = 0.022). CONCLUSIONS: Food temperature is an important factor in glucose absorption and GLP-1 response. These food temperatures elicited differences are lost in type 2 diabetes.

First use of gene therapy to treat growth hormone resistant dwarfism in a mouse model.

The only treatment tested for growth hormone receptor (GHR) defective Laron Syndrome (LS) is injections of recombinant insulin-like-growth factor 1 (rhIGF1). The response is suboptimal and associated with progressive obesity. In this study, we treated 4-5-week-old Laron dwarf mice (GHR-/-) with an adeno-associated virus expressing murine GHR (AAV-GHR) injection at a dose of 4 × 1010 vector genome per mouse. Serum growth hormone (GH) levels decreased, and GH-responsive IGF1, IGF binding protein 3 (IGFBP3) and acid labile subunit (ALS) increased. There was a significant but limited increase in body weight and length, similar to the response to rhIGF1 treatment in LS patients. All the major organs increased in weight except the brain. Our study is the first to use gene therapy to treat GH-receptor deficiency. We propose that gene therapy with AAV-GHR may eventually be useful for the treatment of human LS.

Association of decreased sperm motility and increased seminal plasma IGF-I, IGF-II, IGFBP-2, and PSA levels in infertile men.

PURPOSE: Previous studies have suggested the involvement of serum insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in the regulation of the female reproductive system. Little is known of these peptides in the seminal plasma (SP) of men and their potential effects on fertility. We assessed SP levels of these peptides in infertile men with low sperm motility (asthenozoospermic; AZ) and low sperm counts (oligozoospermic; OZ), its effects on in vivo sperm motility, and whether there is a correlation with aging. METHODS: Twenty-eight infertile men (AZ; n = 18 and OZ; n = 10) and 20 fertile normozoospermic (NZ) men were studied. Seminal plasma IGF-I, IGF-II, IGFBP-2, IGFBP-3, and prostate-specific antigen (PSA) levels were measured, and spermatozoa mRNA transcript patterns were examined. RESULTS: Asthenozoospermic men had higher SP IGF-I, IGF-II, IGFBP-2, and PSA levels than NZ and OZ men, whereas SP IGFBP-3 levels were similar between the three groups. Sperm count positively correlated with SP IGF-I, IGF-II, and IGFBP-2; sperm motility negatively correlated with SP IGF-II and IGFBP-2; and age correlated positively with SP IGF-II. The expression of IGF-I and IGF-II mRNA and mRNA receptors was detectable, but no variations in transcript levels were noted. CONCLUSION: Decreased sperm motility, but not sperm count, in infertile AZ men is associated with increased SP IGF-I, IGF-II, IGFBP-2, and PSA levels. Changes in SP IGFs and their interactions with IGFBPs and IGF receptors, and PSA levels suggest a role of these SP peptides in modulating sperm motility and possibly prostate disease development in aging men.

Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice.

We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3' albumin enhancer (3'iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 109 vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 1010 vg/mouse. The further inclusion of an 860 base-pairs 3'iALB enhancer component in the 3' untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production.

Efficacy and safety of pulsed intravenous methylprednisolone in early active thyroid eye disease.

Introduction: The mainstay of therapy for active inflammatory phase of thyroid eye disease (TED) is immunosuppression. Patients in our centre with early active TED are treated with pulsed intravenous methylprednisolone (IVMP). Two different protocols are offered in our centre: High dose (1g/day for 3 days, monthly for 6 months), or EUGOGO protocol (500 mg weekly for six weeks, followed by 250 mg weekly for the next 6 weeks). Methods: A prospective cohort study of patients undergoing the two IVMP protocols was performed from January 2009 to May 2015. Main outcome measures were improvement of Clinical Activity Score (CAS) and International Thyroid Eye Disease (ITEDS) - VISA Inflammatory Index. Results: We had a total of 63 patients. Mean age was 43.1 ± 13.1years, females comprised 49.2% (n = 31), and 31 (49.2%) had a positive smoking history. Following IVMP, 65.0% (n = 41) had good response, 31.7% (n = 20) partial, and 3.3% (n = 2) poor. There were significant differences (p < 0.001) in CAS and ITEDS scores between pre-IVMP and post-IVMP visits, for both protocols. A higher proportion of patients receiving the modified EUGOGO protocol (58.3%) had persistent activity and required additional immunosuppression compared to those who underwent the high dose protocol (33.3%). Mild side effects were experienced by 5 (7.9%) and 3 (4.8%) patients at 3 and 6 months, respectively. There were no severe side effects, cardiovascular events or liver failure. Conclusion: With adequate screening and follow-up, six repeated cycles of high dose pulsed IVMP is an effective treatment for TED and can significantly reduce the morbidity associated with this debilitating condition. None of the 51 patients from the high dose protocol met with any serious side effects.

Development of a liver-specific Tet-off AAV8 vector for improved safety of insulin gene therapy for diabetes.

BACKGROUND: Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno-associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)-induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long-acting insulin therapy. METHODS: We have developed a single AAV8 vector with a Tet-Off regulatable system as a safety mechanism to turn off insulin secretion should hypoglycaemia develop in vector-treated diabetic mice. We first transfected HepG2 cells or freshly isolated rat hepatocytes in vitro with the Tet-Off system (pAAV-Tetoffbidir -Alb-luc) regulating a luciferase reporter gene. We subsequently incorporated a furin-cleavable codon-optimised human proinsulin cDNA into pAAV-Tetoffbidir backbone to form the doxycycline inducible pAAV-Tetoffbidir -Alb-hINSco. RESULTS: Using STZ-induced diabetic mice, we were able to switch off insulin secretion repeatedly with doxycycline administration, and showed full restoration of insulin secretion on withdrawing doxycycline. CONCLUSIONS: The present study provides proof of concept that, under circumstances when inappropriate basal insulin secretion is a safety concern, insulin secretion from AAV8 gene therapy can be turned off reversibly with doxycycline.

Immunosuppression overcomes insulin- and vector-specific immune responses that limit efficacy of AAV2/8-mediated insulin gene therapy in NOD mice.

We report the restoration of euglycaemia in chemically induced diabetic C57BL/6 mice and spontaneously diabetic Non Obese Diabetic (NOD) mice by intravenous systemic administration of a single-stranded adeno-associated virus (ssAAV2/8) codon optimised (co) vector encoding furin cleavable human proinsulin under a liver-specific promoter. There were no immunological barriers to efficacy of insulin gene therapy in chemically induced C57BL/6 mice, which enjoyed long-lasting correction of hyperglycaemia after therapy, up to 250 days. Euglycaemia was also restored in spontaneously diabetic NOD mice, although these mice required a 7-10-fold higher dose of vector to achieve similar efficacy as the C57BL/6 mice and the immunodeficient NODscid mice. We detected CD8+ T cell reactivity to insulin and mild inflammatory infiltration in the livers of gene therapy recipient NOD mice, neither of which were observed in the treated C57BL/6 mice. Efficacy of the gene therapy in NOD mice was partially improved by targeting the immune system with anti-CD4 antibody treatment, while transfer of NOD mouse AAV2/8-reactive serum to recipients prevented successful restoration of euglycaemia in AAV2/8-HLP-hINSco-treated NODscid mice. Our data indicate that both immune cells and antibodies form a barrier to successful restoration of euglycaemia in autoimmune diabetic recipient mice with insulin gene therapy, but that this barrier can be overcome by increasing the dose of vector and by suppressing immune responses.

The characteristics of blood glucose fluctuations in patients with fulminant type 1 diabetes mellitus in the stable stage

ABSTRACT Objective: The aim was to characterize blood glucose fluctuations in patients with fulminant type 1 diabetes (FT1DM) at the stable stage using continuous blood glucose monitoring systems (CGMSs). Subjects and methods: Ten patients with FT1DM and 20 patients with classic type 1 diabetes mellitus (T1DM) (the control group) were monitored using CGMSs for 72 hours. Results: The CGMS data showed that the mean blood glucose (MBG), the standard deviation of the blood glucose (SDBG), the mean amplitude glycemic excursions (MAGE), the blood glucose areas and the percentages of blood glucose levels below 13.9 mmol/L were similar between the two groups. However, the percentage of blood glucose levels below 3.9 mmol/L was significantly higher in the FT1DM group compared to the T1DM group (p < 0.05). The minimum (Min) blood glucose level in the FT1DM group was significantly lower than that of the T1DM group (p < 0.05). Patients with FT1DM had severe dysfunction of the islet beta cells and alpha cells compared to patients with T1DM, as indicated by lower C-peptide values and higher glucagon/C-peptide values. Conclusion: In conclusion, patients with FT1DM at the stable stage were more prone to hypoglycemic episodes as recorded by CGMSs, and they had a greater association with severe dysfunction of both the beta and alpha islet cells compared to patients with T1DM.

Comparison of Diabetic and Non-diabetic Human Leukocytic Responses to Different Capsule Types of Klebsiella pneumoniae Responsible for Causing Pyogenic Liver Abscess.

The major risk factor for Klebsiella liver abscess (KLA) is type 2 diabetes mellitus (DM), but the immunological mechanisms involved in the increased susceptibility are poorly defined. We investigated the responses of neutrophils and peripheral blood mononuclear cells (PBMCs) to hypervirulent Klebsiella pneumoniae (hvKP), the causative agent of KLA. DNA and myeloperoxidase levels were elevated in the plasma of KLA patients compared to uninfected individuals indicating neutrophil activation, but diabetic status had no effect on these neutrophil extracellular trap (NET) biomarkers in both subject groups. Clinical hvKP isolates universally stimulated KLA patient neutrophils to produce NETs ex vivo, regardless of host diabetic status. Ability of representative capsule types (K1, K2, and non-K1/K2 strains) to survive intra- and extra-cellular killing by type 2 DM and healthy neutrophils was subsequently examined. Key findings were: (1) type 2 DM and healthy neutrophils exhibited comparable total, phagocytic, and NETs killing against hvKP, (2) phagocytic and NETs killing were equally effective against hvKP, and (3) hypermucoviscous K1 and K2 strains were more resistant to total, phagocytic, and NETs killing compared to the non-mucoviscous, non-K1/K2 strain. The cytokine response and intracellular killing ability of type 2 DM as well as healthy PBMCs upon encounter with the different capsule types was also examined. Notably, the IL-12-IFNγ axis and its downstream chemokines MIG, IP-10, and RANTES were produced at slightly lower levels by type 2 DM PBMCs than healthy PBMCs in response to representative K1 and non-K1/K2 strains. Furthermore, type 2 DM PBMCs have a mild defect in its ability to control hvKP replication relative to healthy PBMCs. In summary, our work demonstrates that type 2 DM does not overtly impact neutrophil intra- and extra-cellular killing of hvKP, but may influence cytokine/chemokine production and intracellular killing by PBMCs.

Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING.

The anti-proliferative agent hexamethylene bisacetamide (HMBA) belongs to a class of hybrid bipolar compounds developed more than 30 y ago for their ability to induce terminal differentiation of transformed cells. Recently, HMBA has also been shown to trigger HIV transcription from latently infected cells, via a CDK9/HMBA inducible protein-1 dependent process. However, the effect of HMBA on the immune response has not been explored. We observed that pretreatment of human peripheral blood mononuclear cells with HMBA led to a markedly increased production of IL-12 and IFN-γ, but not of TNF-α, IL-6, and IL-8 upon subsequent infection with Burkholderia pseudomallei and Salmonella enterica HMBA treatment was also associated with better intracellular bacterial control. HMBA significantly improved IL-12p70 production from CD14+ monocytes during infection partly via the induction of type I IFN in these cells, which primed an increased transcription of the p35 subunit of IL-12p70 during infection. HMBA also increased early type I IFN transcription in human monocytic and epithelial cell lines, but this was surprisingly independent of its previously reported effects on positive transcription elongation factor b and HMBA inducible protein-1. Instead, the effect of HMBA was downstream of a calcium influx, and required the pattern recognition receptor and adaptor STING but not cGAS. Our work therefore links the STING-IRF3 axis to enhanced IL-12 production and intracellular bacterial control in primary monocytes. This raises the possibility that HMBA or related small molecules may be explored as therapeutic adjuvants to improve disease outcomes during intracellular bacterial infections.

Use of cabergoline for the management of persistent Cushing's disease in pregnancy.

Cushing's disease (CD) is rare during pregnancy and is associated with significant maternal and fetal complications. It is important to control hypercortisolism during pregnancy, either surgically or medically, for a successful maternal and fetal outcome. We report a patient with recurrent CD who was treated with low-dose cabergoline (CAB) for persistent hypercortisolism throughout pregnancy. A 36-year-old woman was diagnosed with CD at the age of 23. She underwent trans-sphenoidal surgery with initial complete remission. However, 4 years after surgery, CD recurred and she underwent Gamma Knife radiosurgery (GKRS). Following GKRS, her cortisol levels remained elevated despite no evidence of visible tumour on pituitary MRI. Medical treatment was commenced with ketoconazole and cyproheptadine. This was changed to CAB as she was keen for pregnancy. She conceived spontaneously and was on CAB throughout pregnancy. She delivered a healthy male neonate, weighing 3195 g at 40 weeks of gestation.

Correction of Murine Diabetic Hyperglycaemia With A Single Systemic Administration of An AAV2/8 Vector Containing A Novel Codon Optimized Human Insulin Gene.

We report the correction of hyperglycemia of STZ induced diabetic mice using one intravenous systemic administration of a single stranded serotype 8 pseudotyped adeno-associated virus (ssAAV2/8) vector encoding the human proinsulin gene under a constitutive liver specific promoter. In vivo dose titration experiments were carried out and we identified an optimal range that achieved maintenance of euglycaemia or a mild diabetic condition for at least 9 months and ongoing to beyond 1 year for some animals, accompanied by human C-peptide secretion and weight gain. Further DNA codon optimization of the insulin gene construct resulted in approximately 3-10 times more human C-peptide secreted in the blood of codon optimized treated animals thereby reducing the number of vector particles required to achieve the same extent of reduction in blood glucose levels as the non-codon optimized vector. The constitutive secretion of insulin achieved with a single administration of the vector could be of therapeutic value for some diabetic patients.

Liraglutide treatment causes upregulation of adiponectin and downregulation of resistin in Chinese type 2 diabetes.

AIMS: To assess the effect of 16 weeks of liraglutide administration on the plasma levels of adiponectin and resistin in Chinese patients diagnosed with type 2 diabetes mellitus (T2DM). METHODS: Forty-four subjects were recruited and randomly assigned to once-a-day dosage of either liraglutide, or glimepiride (4 mg) in a double-blinded double-dummy active-controlled study. All treatments were administered in combination with metformin (1 g, twice daily). The efficacy of liraglutide was estimated by measuring and comparing the levels of HbA1c, adiponectin and resistin in the plasma before and after the 16-week treatment. RESULTS: The plasma level of adiponectin was significantly increased (0.74±0.19 ng/ml, p<0.05) and resistin was significantly lowered (-1.34±0.34 pg/ml, p<0.05) in a dose-dependent manner in the liraglutide group when compared with the glimepiride group (-0.44±0.09 ng/ml of adiponectin and 0.14±0.41 pg/ml of resistin). In contrast, we found no differences in the decrease in HbA1c between the two groups (8.3±1.2% to 7.2±1.1% in NGSP units vs. 8.3±1.0% to 7.3±1.2% in NGSP units; 67±13 mmol/mol to 55±12 mmol/mol vs. 67±11 mmol/mol to 56±13 mmol/mol in IFCC units). CONCLUSIONS: In Chinese T2DM patients, liraglutide treatment led to increased adiponectin and decreased resistin levels compared to glimepiride-treated subjects, while inducing similar glycemic changes.

Thyroid eye disease: a Southeast Asian experience.

AIM: To study the demographics, comorbidities, clinical manifestations and treatment methods of thyroid eye disease (TED) in Singapore. METHODS: In this retrospective case series, we analysed the case records of all patients with TED who presented at our multidisciplinary Thyroid Eye Clinic from November 2002 to October 2012. RESULTS: There were a total of 174 patients-111 female patients (63.8%) and 63 male patients (36.2%). The majority of the patients were ethnically Chinese (80.5%), followed by Malay (10.3%) and Indian (6.3%). The mean age was 40.2 years (SD±15.5, range 0.3-87.0). The commonest sign on ophthalmic examination was eyelid retraction (62.1%), followed by proptosis (61.0%) and lid lag (57.5). Acquired epiblepharon and corneal erosions were noted in 11.5% and 29.3% respectively. Eight patients (4.6%) had dysthyroid optic neuropathy. The mean exophthalmometry reading was 18.8 mm (SD±3.32, range 10.0-28.0). Mild, moderate and severe disease was noted in 71.3%, 20.7% and 8.0% respectively. Thyroid dysfunction was managed with anti-thyroid medication only (40.2%), ß blockers (19.5%), thyroxine replacement (14.4%), radioactive iodine (14.4%) and block-replace regime (9.8%). Clinically significant active orbitopathy was managed with intravenous corticosteroids (12.1%). Surgical procedures consisted of thyroidectomy (10.3%), eyelid surgery (8.6%), orbital decompression (7.5%), epiblepharon correction (2.3%) and strabismus surgery (0.6%). CONCLUSIONS: Corneal erosion secondary to acquired epiblepharon is a common sign in East Asian patients with TED, thus increased awareness among physicians should be encouraged. Mean exophthalmometry values and frequencies of upper eyelid retraction and oedema are lower in East Asian patients compared with Caucasian patients. Among Singapore's multi-ethnic population, Malay patients with TED had the highest exophthalmometry reading.

Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy In Vivo.

Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection. Although murine models of diabetes have been extensively used in gene and cell therapy research, few studies have shown efficacy in large preclinical animal models. Here we report optimized conditions for ex vivo expansion and characterization of porcine bone marrow stromal cells and their permissive expression of a transfected insulin gene. Our data show that these cells resemble human bone marrow stromal cells in surface antigen expression, are homogeneous, and can be reproducibly isolated from outbred Yorkshire-Landrace pigs. Porcine bone marrow stromal cells were efficiently expanded in vitro to >10(10) cells from 20 ml of bone marrow and remained karyotypically normal during expansion. These cells were electroporated with an insulin expression plasmid vector with high efficiency and viability, and secreted human insulin and C-peptide indicating appropriate processing of proinsulin. We showed that autologous insulin-secreting bone marrow stromal cells implanted and engrafted in the liver of a streptozotocin-diabetic pig that modeled type 1 diabetes resulted in partial, but significant, improvement in hyperglycemia that could not be ascribed to regeneration of endogenous ß-cells. Glucose-stimulated insulin secretion in vivo from implanted cells in the treated pig was documented by a rise in serum human C-peptide levels during intravenous glucose tolerance tests. Compared to a sham-treated control pig, this resulted in significantly reduced fasting hyperglycemia, a slower rise in serum fructosamine, and prevented weight loss. Taken together, this study suggests that bone marrow stromal cells merit further development as autologous cell therapy for diabetes.

Effect of oral N-acetyl cysteine supplementation in type 2 diabetic patients on intracellular glutathione content and innate immune responses to Burkholderia pseudomallei.

Type 2 diabetic patients have increased susceptibility to melioidosis, an infectious disease caused by Burkholderia pseudomallei. We had previously shown that peripheral blood mononuclear cells (PBMCs) from diabetic patients with poor glycemic control had a defective IL-12 and IFNγ response to B. pseudomallei infection, resulting in poor intracellular bacterial control. The impaired IL-12 response was due to glutathione (GSH) deficiency characterized by a low reduced to oxidized glutathione ratio (GSH ratio) and could be restored by the addition of reduced GSH to the infected cells. Our goal is to determine whether N-acetyl cysteine (NAC, a GSH pro-drug) supplementation in diabetic patients could improve their immune control of B. pseudomallei. Type 2 diabetic patients with poor glycemic control were given oral supplementation of NAC for six weeks at 1200 mg daily. Their PBMCs and subsets of immune cells showed a significant increase in free GSH concentration. However, the GSH ratio, IL-12 and IFNγ production, and intracellular bacterial killing upon ex-vivo infection did not improve. Thus, oral NAC supplementation in diabetic patients is sufficient to increase intracellular GSH content in blood cells. However, modulating the free GSH content is not sufficient to improve infection outcome as it is the GSH ratio that regulates the IL-12 response in monocytes.

Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies.

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1(125-133), LMP2A(426-434) or EBNA1(562-570) in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1(562-570) in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease.

Endocrine emergencies in pregnancy.

Endocrine emergencies in pregnancy are rare and are more likely to occur in the absence of good obstetric care. Serious thyroid and diabetes related events in pregnancy are more common because of their higher prevalence in the normal population. Pituitary complications in pregnancy are now relatively rare. A high index of suspicion is needed for early diagnosis, and medical treatment is directed primarily at maintaining maternal hemodynamic stability. A close liaison between an endocrinologist, maternal-fetal specialist and intensivist is critical in optimising both maternal and fetal outcomes.

Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria.

Individuals with type 2 diabetes are at increased risk of acquiring melioidosis, a disease caused by Burkholderia pseudomallei infection. Although up to half of melioidosis patients have underlying diabetes, the mechanisms involved in this increased susceptibility are unknown. We found that B. pseudomallei-infected PBMCs from diabetic patients were impaired in IL-12p70 production, which resulted in decreased IFN-γ induction and poor bacterial killing. The defect was specific to the IL-12-IFN-γ axis. Defective IL-12 production was also observed during Mycobacterium tuberculosis infection, in which diabetes is likewise known to be a strong risk factor. In contrast, IL-12 production in diabetic cells was not affected upon Salmonella enterica infection or in response to TLR2, -3, -4, and -5 ligands. Poor IL-12 production correlated with a deficiency in intracellular reduced glutathione (GSH) concentrations in diabetic patients. Addition of GSH or N-acetylcysteine to PBMCs selectively restored IL-12 and IFN-γ production and improved bacterial killing. Furthermore, the depletion of GSH in mice led to increased susceptibility to melioidosis, reduced production of IL-12p70, and poorer disease outcome. Our data thus establish a link between GSH deficiency in diabetes and increased susceptibility to melioidosis that may open up new therapeutic avenues to protect diabetic patients against some intracellular bacterial pathogens.

Des-aspartate-angiotensin-I and angiotensin IV improve glucose tolerance and insulin signalling in diet-induced hyperglycaemic mice.

Although clinical studies suggested that blockade of the renin-angiotensin system may prevent diabetes, the mechanism is uncertain. As a follow-up to an earlier study, we investigated how des-aspartate-angiotensin-1 (DAA-1) and its metabolite, angiotensin IV (Ang-IV) improved glucose tolerance in diet-induced hyperglycaemic mice. Male C57BL/6J mice were fed a high-fat-high-sucrose (HFD) or normal (ND) diet for 52 weeks. HFD animals were orally administered either DAA-I (600nmol/kg/day), Ang-IV (400nmol/kg/day) or distilled water. Body weight, blood glucose and insulin were measured fortnightly. Inflammatory and insulin signalling transducers that are implicated in hyperglycaemia were analyzed in skeletal muscles at 52 weeks. HFD animals developed hyperglycemia, hyperinsulinemia and obesity. DAA-I and Ang-IV improved glucose tolerance but had no effect on hyperinsulinemia and obesity. Skeletal muscles of HFD animals showed increased level of ROS, gp91 of NADPH oxidase, pJNK and AT(1)R-JAK-2-IRS-1 complex. Both DAA-I and Ang-IV attenuated these increases. Insulin-induced activation of IR, IRS-1, IRS-1-PI3K coupling, phosphorylation of Akt, and GLUT4 translocation were attenuated in skeletal muscles of HFD animals. The attenuation was significantly ameliorated in DAA-I-treated HFD animals. In corresponding Ang-IV treated animals, insulin induced IRAP and PI3K interaction, activation of pAkt and GLUT4 translocation, but no corresponding activation of IR, IRS-1 and IRS-1-PI3K coupling were observed. DAA-I and Ang-IV improved glucose tolerance, insulin signalling, and para-inflammatory processes linked to hyperglycaemia. DAA-I acts via the angiotensin AT(1) receptor and activates the insulin pathway. Ang-IV acts via IRAP, which couples PI3K and activates the later part of the insulin pathway.