We conclude that chronic uncontrolled HIV-infection is associated with elevated levels of MDSC, which potentially contribute to the impaired T-cell responses characteristic for the progressive disease stage.

BackgroundInformation regarding the variability of metabolite levels over time in an individual is required to estimate the reproducibility of metabolite measurements. In intervention studies, it is critical to appropriately judge changes that are elicited by any kind of intervention. The pre-analytic phase (collection, transport and sample processing) is a particularly important component of data quality in multi-center studies.MethodsReliability of metabolites (within-and between-person variance, intraclass correlation coefficient) and stability (shipment simulation at different temperatures, use of gel-barrier collection tubes, freeze-thaw cycles) were analyzed in fasting serum and plasma samples of 22 healthy human subjects using a targeted LC-MS approach.ResultsReliability of metabolite measurements was higher in serum compared to plasma samples and was good in most saturated short-and medium-chain acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids and hexose. The majority of metabolites were stable for 24 h on cool packs and at room temperature in non-centrifuged tubes. Plasma and serum metabolite stability showed good coherence. Serum metabolite concentrations were mostly unaffected by tube type and one or two freeze-thaw cycles.ConclusionA single time point measurement is assumed to be sufficient for a targeted metabolomics analysis of most metabolites. For shipment, samples should ideally be separated and frozen immediately after collection, as some amino acids and biogenic amines become unstable within 3 h on cool packs. Serum gel-barrier tubes can be used safely for this process as they have no effect on concentration in most metabolites. Shipment of non-centrifuged samples on cool packs is a cost-efficient alternative for most metabolites.

Several recent studies have suggested that the adult bone marrow harbors cells that can differentiate into tissues from all three germ layers. Other reports have contradicted these findings or attributed them to cell fusion. In this study, we investigated whether bone marrow؊derived cells contribute to the renewal of adult pancreatic endocrine cells, in particular insulinproducing ␤-cells, in vivo. To address this issue, we studied mice transplanted with green fluorescent protein (GFP)؊positive, sex-mismatched bone marrow. We also extended our studies to pancreatic injury models (partial pancreatectomy and streptozotocin administration). All animals showed stable full donor chimerism in the peripheral blood and microscopic analysis at 4 -6 weeks and 3 months after transplantation, indicating that the GFP ؉ and Y chromosome؊positive donor bone marrow contributed substantially to blood, lymphatic, and interstitial cells in the pancreas. However, after examining >100,000 ␤-cells, we found only 2 ␤-cells positive for GFP, both of which were in control animals without pancreatic injury. Thus our study results did not support the concept that bone marrow contributes significantly to adult pancreatic ␤-cell renewal.

No abstract

CD8 T-cell responses are thought to be crucial for control of viremia in human immunodeficiency virus (HIV) infection but ultimately fail to control viremia in most infected persons. Studies in acute infection have demonstrated strong CD8-mediated selection pressure and evolution of mutations conferring escape from recognition, but the ability of CD8 T-cell responses that persist in late-stage infection to recognize viruses present in vivo has not been determined. Therefore, we studied 24 subjects with advanced HIV disease (median viral load ‫؍‬ 142,000 copies/ml; median CD4 count ‫؍‬ 71/l) and determined HIV-1-specific CD8 T-cell responses to all expressed viral proteins using overlapping peptides by gamma interferon Elispot assay. Chronic-stage virus was sequenced to evaluate autologous sequences within Gag epitopes, and functional avidity of detected responses was determined. In these subjects, the median number of epitopic regions targeted was 13 (range, 2 to 39) and the median cumulative magnitude of CD8 T-cell responses was 5,760 spot-forming cells/10 6 peripheral blood mononuclear cells (range, 185 to 24,700). On average six (range, one to 8) proteins were targeted. For 89% of evaluated CD8 T-cell responses, the autologous viral sequence was predicted to be well recognized by these responses and the majority of analyzed optimal epitopes were recognized with medium to high functional avidity by the contemporary CD8 T cells. Withdrawal of antigen by highly active antiretroviral therapy led to a significant decline both in breadth (P ‫؍‬ 0.032) and magnitude (P ‫؍‬ 0.0098) of these CD8 T-cell responses, providing further evidence that these responses had been driven by recognition of autologous virus. These results indicate that strong, broadly directed, and high-avidity gamma-interferonpositive CD8 T-cells directed at autologous virus persist in late disease stages, and the absence of mutations within viral epitopes indicates a lack of strong selection pressure mediated by these responses. These data imply functional impairment of CD8 T-cell responses in late-stage infection that may not be reflected by gamma interferon-based screening techniques.

The gut microbiota has been linked to metabolic diseases. However, information on the microbiome of young adults at risk for type 2 diabetes (T2D) is lacking. The aim of this cross-sectional analysis was to investigate whether insulin resistant women with previous gestational diabetes (pGDM), a high risk group for T2D, differ in their stool microbiota from women after a normoglycemic pregnancy (controls). Bacterial communities were analyzed by high-throughput 16S rRNA gene sequencing using fecal samples from 42 pGDM and 35 control subjects 3–16 months after delivery. Clinical characterization included a 5-point OGTT, anthropometrics, clinical chemistry markers and a food frequency questionnaire. Women with a Prevotellaceae-dominated intestinal microbiome were overrepresented in the pGDM group (p < 0.0001). Additionally, the relative abundance of the phylum Firmicutes was significantly lower in women pGDM (median 48.5 vs. 56.8%; p = 0.013). Taxa richness (alpha diversity) was similar between the two groups and with correction for multiple testing we observed no significant differences on lower taxonomic levels. These results suggest that distinctive features of the intestinal microbiota are already present in young adults at risk for T2D and that further investigations of a potential pathophysiological role of gut bacteria in early T2D development are warranted.

Presently, routine screening misses many cases of prediabetes and early type 2 diabetes (T2D). Therefore, better biomarkers are needed for a simple and early detection of abnormalities of glucose metabolism and prediction of future T2D. Possible candidates for this include plasma or serum amino acids because glucose and amino acid metabolism are closely connected. This review presents the available evidence of this connectivity and discusses its clinical implications. First, we examine the underlying physiological, pre-analytical, and analytical issues. Then, we summarize results of human studies that evaluate amino acid levels as markers for insulin resistance, prediabetes, and future incident T2D. Finally, we illustrate the interconnection of amino acid levels and metabolic syndrome with our own data from a deeply phenotyped human cohort. We also discuss how amino acids may contribute to the pathophysiology of T2D. We conclude that elevated branched-chain amino acids and reduced glycine are currently the most robust and consistent amino acid markers for prediabetes, insulin resistance, and future T2D. Yet, we are cautious regarding the clinical potential even of these parameters because their discriminatory power is insufficient and their levels depend not only on glycemia, but also on other components of the metabolic syndrome. The identification of more precise intermediates of amino acid metabolism or combinations with other biomarkers will, therefore, be necessary to obtain in order to develop laboratory tests that can improve T2D screening.

No abstract