Kok Onn Lee

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.

Type 2 diabetic patients with resistant hypertension should be screened for primary aldosteronism

BP control in diabetic patients is often poor. The contribution of secondary hypertension due to undiagnosed PA in hypertensive type 2 diabetic patients is not well studied. We prospectively screened 100 consecutive Asian type 2 diabetic patients with difficult-to-control or resistant hypertension for PA. PAC (pmol/L) to PRA (ng/mL/h) ratio was measured; those with PAC-to-PRA ratio >550 (corresponding PAC >415) underwent intravenous 0.9% SLT. Patients with PAC ≥140 following SLT had CT adrenals and bilateral AVS. Thirteen patients (13%) were confirmed to have PA, and all had resistant hypertension. Eight had a surgically correctable form of PA. Patients with PA had higher mean (SD) systolic [159.0 (10.6) vs. 146.0 (10.7) mmHg, p=0.001] and diastolic BP [94.6 (6.0) vs. 87.6 (5.9) mmHg, p=0.001], lower serum potassium [3.5 (0.6) vs. 4.3 (0.5) mmol/L, p=0.001], and higher PAC [679.3 (291.0) vs. 239.5 (169.4) pmol/L, p=0.001]. Identification and institution of definitive treatment for PA resulted in better BP control and in a reduction in the use of antihypertensive medications. Our findings demonstrate a high prevalence of PA in type 2 diabetic patients with resistant hypertension. Systematic screening for PA in this select group is recommended, as targeted treatment improves BP control.

Stem cell and gene therapies for diabetes mellitus

In this Perspectives article, we comment on the progress in experimental stem cell and gene therapies that might one day become a clinical reality for the treatment of patients with diabetes mellitus. Research on the ability of human embryonic stem cells to differentiate into islet cells has defined the developmental stages and transcription factors involved in this process. However, the clinical applications of human embryonic stem cells are limited by ethical concerns, as well as the potential for teratoma formation. As a consequence, alternative forms of stem cell therapies, such as induced pluripotent stem cells and bone marrow-derived mesenchymal stem cells, have become an area of intense study. Finally, gene therapy shows some promise for the generation of insulin-producing cells. Here, we discuss two of the most frequently used approaches: in vitro gene delivery into cells which are then transplanted into the recipient and direct delivery of genes in vivo.

Visfatin and its genetic variants are associated with obesity‐related morbidities and cardiometabolic risk in severely obese children

Visfatin is an adipokine, associated with obesity and possibly glucose regulation.

Characterization of Human Umbilical Cord Lining-Derived Epithelial Cells and Transplantation Potential

In this study we describe the derivation and immunological characterization of a primary epithelial cell type from the human umbilical cord membrane. These cord lining epithelial cells (CLECs) expressed and/or secreted isoforms of the nonclassical human leukocyte antigen class I (HLA-1b) glycoproteins, HLA-G and E. Conditioned media from CLECs inhibited mitogen-stimulated T-lymphocyte responses, and in a mixed leukocyte reaction (MLR) assay, cocultured CLECs inhibited allogeneic responses with a concomitant reduction in proinflammatory cytokines. Using a transwell coculture system, it was demonstrated that these immunoregulatory effects were mediated by soluble factors secreted by CLECs, in a dose-dependent manner. Functional studies using HLA-G blocking antibody showed that the effects of CLEC-secreted products could be inhibited, thus demonstrating a significant and important role for soluble HLA-G. In vivo, we show that transplanted CLECs could be maintained for extended periods in immunocompetent mice where xenorejection rapidly destroyed primary keratinocytes, a control human epithelial cell type. Additionally, CLECs delayed the rejection of keratinocytes and extended their survival when cotransplanted, indicating an ability to protect adjacent human cell types that would otherwise be rejected if transplanted alone. We also show that CLECs transduced with a modified human proinsulin gene were transplanted intraperitoneally into streptozotocin (STZ)-induced diabetic mice, resulting in significantly lower levels of serum glucose compared to control mice. This study has characterized the immunological properties of CLECs and tested a potential therapeutic application in the treatment of a type 1 diabetes mouse model.

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 LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 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.

The association of a nucleobindin 2 gene (NUCB2) variant with childhood adiposity.

Nucleobindin 2 (NUCB2) is a precursor of nesfatin-1, a hypothalamic anorectic neuropeptide. The association between variants of the NUCB2 gene and adiposity was examined. 142 severely obese Chinese children in Singapore, and 384 normal weight Chinese children from a longitudinal cohort from Da Qing, China, were studied. NUCB2 was screened using PCR and direct sequencing in 29 severely obese children and 24 non-obese children, then screened for a variant c.1012C>G (Q338E, or rs757081) in the rest of the cohort using TaqMan probe. Five variants, including c.1012C>G (Q338E) were found. Genotyping for c.1012C>G found that the GG genotype was significantly less frequent in the obese group; odds ratio for obese subjects carrying the CC and CG genotypes was 2.29 (95% CI 1.17-4.49) in the dominant model, CC genotype 2.86 (95% CI 1.41-5.81) in the additive model, and C allele 1.57 (95% CI 1.17-2.1). The findings were replicated in an independent cohort of 372 obese and 390 normal weight Chinese children, where the odds ratio of obese subjects with CC and CG genotypes was 1.69 (95% CI 1.12-2.55). Within the Da Qing cohort, subjects with the GG genotype had significantly lower BMI and percentage ideal weight for height (WFH) at 5 and 8years of age. Subjects with lower birth weights also had more pronounced difference in WFH and BMI at 5 and 10years of age between GG subjects versus CC/CG subjects. We postulate that GG genotype is protective against excessive weight gain, and factors which predispose to excessive weight gain such as higher birth weights may ameliorate the effect.

EMLA-Induced Skin Wrinkling for the Detection of Diabetic Neuropathy

Objective: To determine the usefulness of Eutectic Mixture of Local Anesthetic (EMLA)-induced stimulated skin wrinkling (SSW) to detect diabetic sensorimotor polyneuropathy (DSPN). Research Design and Methods: Two hundred and ten diabetics were prospectively recruited (mean age 58.5 ± 12.7 years) from a large tertiary center from 2009 to 2011. EMLA was applied to the tips of digits 2, 3, and 4 and the degree of wrinkling graded. Diabetic Neuropathy Symptom (DNS) score, nerve conduction studies (NCS), Semmes–Weinstein monofilament (SWMF) tests, and vibratory perception thresholds (VPTs) testing were chosen as comparative clinical standards to diagnose length-dependent DSPN. Results: Inter-rater agreement for two examiners of SSW was high, with Cohen’s weighted κ of 0.912 for the right hand, and 0.823 for the left. K measure of agreement of SSW with the NCS, DNS scores, SWMF testing, and VPT testing was 0.486, 0.243, 0.289, and 0.395 respectively. SSW was able to distinguish between normal and abnormal NCS and DNS results, with median scores of 3.333 vs. 1.667 (p < 0.0005); and 3.167 vs. 2.000 (p < 0.0005) respectively. Following receiver operating characteristic-analysis, at a cut-off point of <3 for an abnormal SSW test, sensitivity of SSW test for diagnosing DSPN using NCS as a reference standard was 81.3%, and specificity was 67.0%, on par with other testing methods. Conclusion: SSW shows comparable sensitivity to other methods for detecting DSPN. Given its low cost and easy administration, SSW can be considered a useful alternative screening method for diagnosing diabetic neuropathy.

Longitudinal study of childhood adiposity and the risk of developing components of metabolic syndrome-the Da Qing children cohort study.

Childhood adiposity is increasingly recognized as a significant predictor of cardiometabolic risks in later life. The aim of this study was to investigate factors associated with longitudinal changes in weight during childhood and the development of metabolic disease risk factors. Four hundred twenty-four children from DaQing city, China, were recruited at 5 y old and followed up for 5 y. Birth weight, television (TV) viewing time at 5 y old, blood pressure, anthropometric measurements, fasting plasma insulin (FI), and triglycerides (TG) levels were measured at 5 and 10 y old. Both birth weight and TV viewing time at 5 y old significantly correlated with percentage of ideal weight for height (WFH) at 5 y old (WFH5; p = 0.0032 and p = 0.01), but only TV time was significantly correlated with WFH at 10 y old (WFH10; p < 0.0001). Blood pressures, FI, homeostasis model assessment for insulin resistance (HOMA-IR), and TG at 10 y old were significantly greater in those children who had greater change in WFH from 5 to 10 y old (ΔWFH). We concluded that TV viewing time was the stronger determinant of later childhood adiposity. A greater ΔWFH was associated with increased cardiometabolic risk factors at 10 y old.

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.