Links of platelet glutamate and glutathione metabolism with attenuated positive and negative symptoms in depressed patients at clinical high risk for psychosis.

Aim of the study is to reveal clinical and biological correlations in patients with adolescent depression and attenuated psychotic symptoms. Activity of platelet enzymes involved in glutamate-, glutathione- and energy metabolism was evaluated in control group and in the patients, because these systems are suspected as related to pathogenesis of psychosis. Adolescents (78 men, 16-25 years old) hospitalized with the first acute depressive state composed two groups: with prevalence of attenuated psychotic positive or negative symptoms (Gr1 and Gr2, 48 and 30 patients, respectively). Control group comprised 20 mentally healthy men of 19-25 years old. Gr1 differed significantly from Gr2 in scores by the Scale of Prodromal Symptoms (SOPS) for positive symptoms, p < 0.001, for disorganization symptoms, p < 0.003, and for total SOPS score, p < 0.001, before the treatment started. When patients from either Gr1 or Gr2 were compared with the control group, significantly decreased baseline activities of platelet glutamate dehydrogenase (GDH), glutathione reductase (GR) and glutathione S-transferase (GST) were found (p < 0.0001). Different correlations were found between baseline enzymatic activities in Gr1 and Gr2: GDH activity correlated with GR activity in Gr1 (R = 0.37), and with GST activity in Gr2 (R = 0.70). Significant correlations were found only in Gr2 between the delta of scores by SOPS negative symptoms (SOPS-N) under treatment and baseline GDH, GST, and GR activities (R = - 0.36, R = - 0.60, and R = 0.38, respectively). The found correlations of the baseline enzymatic activity levels with the value of the decrease (delta) in SOPS-N scores under the treatment represent interest for the prediction of the pharmacotherapy efficiency.

Changes in systemic GDF15 across the adult lifespan and their impact on maximal muscle power: the Copenhagen Sarcopenia Study.

BACKGROUND: Although growth differentiation factor 15 (GDF15) is known to increase with disease and is associated with low physical performance, the role of GDF15 in normal ageing is still not fully understood. Specifically, the influence of circulating GDF15 on impairments in maximal muscle power (a major contributor to functional limitations) and the underlying components has not been investigated. METHODS: Data from 1305 healthy women and men aged 20 to 93 years from The Copenhagen Sarcopenia Study were analysed. Circulating levels of GDF15 and markers of inflammation (tumor necrosis factor-alpha, interleukin-6, and high-sensitivity C-reactive protein) were measured by ELISA (R&D Systems) and multiplex bead-based immunoassays (Bio-Rad). Relative (normalized to body mass), allometric (normalized to height squared), and specific (normalized to leg muscle mass) muscle power were assessed by the Nottingham power rig [leg extension power (LEP)] and the 30 s sit-to-stand (STS) muscle power test. Total body fat, visceral fat, and leg lean mass were assessed by dual energy X-ray absorptiometry. Leg skeletal muscle index was measured as leg lean mass normalized to body height squared. RESULTS: Systemic levels of GDF15 increased progressively as a function of age in women (1.1 ± 0.4 pg·mL-1 ·year-1 ) and men (3.3 ± 0.6 pg·mL-1 ·year-1 ) (both P < 0.05). Notably, GDF15 increased at a faster rate from the age of 65 years in women (11.5 ± 1.2 pg·mL-1 ·year-1 , P < 0.05) and 70 years in men (19.3 ± 2.3 pg·mL-1 ·year-1 , P < 0.05), resulting in higher GDF15 levels in men compared with women above the age of 65 years (P < 0.05). Independently of age and circulatory markers of inflammation, GDF15 was negatively correlated to relative STS power (P < 0.05) but not LEP, in both women and men. These findings were mainly explained by negative associations of GDF15 with specific STS power in women and men (both P < 0.05). CONCLUSIONS: A J-shaped relationship between age and systemic GDF15 was observed, with men at older age showing steeper increases and elevated GDF15 levels compared with women. Importantly, circulating GDF15 was independently and negatively associated with relative STS power, supporting the potential role of GDF15 as a sensitive biomarker of frailty in older people.

Blood flow restricted training leads to myocellular macrophage infiltration and upregulation of heat shock proteins, but no apparent muscle damage.

KEY POINTS: Muscular contractions performed using a combination of low external loads and partial restriction of limb blood flow appear to induce substantial gains in muscle strength and muscle mass. This exercise regime may initially induce muscular stress and damage; however, the effects of a period of blood flow restricted training on these parameters remain largely unknown. The present study shows that short-term, high-frequency, low-load muscle training performed with partial blood flow restriction does not induce significant muscular damage. However, signs of myocellular stress and inflammation that were observed in the early phase of training and after the training intervention, respectively, may be facilitating the previously reported gains in myogenic satellite cell content and muscle hypertrophy. The present results improve our current knowledge about the physiological effects of low-load muscular contractions performed under blood flow restriction and may provide important information of relevance for future therapeutic treatment of muscular atrophy. ABSTRACT: Previous studies indicate that low-load muscle contractions performed under local blood flow restriction (BFR) may initially induce muscle damage and stress. However, whether these factors are evoked with longitudinal BFR training remains unexplored at the myocellular level. Two distinct study protocols were conducted, covering 3 weeks (3 wk) or one week (1 wk). Subjects performed BFR exercise (100 mmHg, 20% 1RM) to concentric failure (BFRE) (3 wk/1 wk), while controls performed work-matched (LLE) (3 wk) or high-load (HLE; 70% 1RM) (1 wk) free-flow exercise. Muscle biopsies (3 wk) were obtained at baseline (Pre), 8 days into the intervention (Mid8), and 3 and 10 days after training cessation (Post3, Post10) to examine macrophage (M1/M2) content as well as heat shock protein (HSP27/70) and tenascin-C expression. Blood samples (1 wk) were collected before and after (0.1-24 h) the first and last training session to examine markers of muscle damage (creatine kinase), oxidative stress (total antibody capacity, glutathione) and inflammation (monocyte chemotactic protein-1, interleukin-6, tumour necrosis factor α). M1-macrophage content increased 108-165% with BFRE and LLE at Post3 (P < 0.05), while M2-macrophages increased (163%) with BFRE only (P < 0.01). Membrane and intracellular HSP27 expression increased 60-132% at Mid8 with BFRE (P < 0.05-0.01). No or only minor changes were observed in circulating markers of muscle damage, oxidative stress and inflammation. The amplitude, timing and localization of the above changes indicate that only limited muscle damage was evoked with BFRE. This study is the first to show that a period of high-frequency, low-load BFR training does not appear to induce general myocellular damage. However, signs of tissue inflammation and focal myocellular membrane stress and/or reorganization were observed that may be involved in the adaptation processes evoked by BFR muscle exercise.

Separate developmental programs for HLA-A and -B cell surface expression during differentiation from embryonic stem cells to lymphocytes, adipocytes and osteoblasts.

A major problem of allogeneic stem cell therapy is immunologically mediated graft rejection. HLA class I A, B, and Cw antigens are crucial factors, but little is known of their respective expression on stem cells and their progenies. We have recently shown that locus-specific expression (HLA-A, but not -B) is seen on some multipotent stem cells, and this raises the question how this is in other stem cells and how it changes during differentiation. In this study, we have used flow cytometry to investigate the cell surface expression of HLA-A and -B on human embryonic stem cells (hESC), human hematopoietic stem cells (hHSC), human mesenchymal stem cells (hMSC) and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA-B though at lower levels. IFNγ induced HLA-A to very high levels on both hESC and hMSC and HLA-B on hMSC. Even on hESC, a low expression of HLA-B was achieved. Differentiation of hMSC to osteoblasts downregulated HLA-A expression (P = 0.017). Interestingly HLA class I on T lymphocytes differed between different compartments. Mature bone marrow CD4(+) and CD8(+) T cells expressed similar HLA-A and -B levels as hHSC, while in the peripheral blood they expressed significantly more HLA-B7 (P = 0.0007 and P = 0.004 for CD4(+) and CD8(+) T cells, respectively). Thus different HLA loci are differentially regulated during differentiation of stem cells.

Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells.

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.

Teratoma formation by human embryonic stem cells is site dependent and enhanced by the presence of Matrigel.

When implanted into immunodeficient mice, human embryonic stem cells (hESCs) give rise to teratoma, tumor-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data are available regarding the effects of implantation site and the methods employed for implantation on the success rate of teratoma formation. In this study, the rate of teratoma formation in immunodeficient mice was site dependent: subcutaneous (25-100%), intratesticular (60%), intramuscular (12.5%), and under the kidney capsule (100%). Co-injecting the hESCs with Matrigel increased subcutaneous teratoma formation efficiency from 25-40% to 80-100%. We did not observe site-specific differences in the teratoma composition at the histological level. However, subcutaneous teratomas were quite distinct, easy to remove, and caused minimal discomfort to the mice. Also, subcutaneous teratomas displayed larger proportion of solid tissues as opposed to cyst formation that dominated the teratomas formed at the other sites. Interestingly, a chromosomally abnormal hESCs with trisomy 20 formed teratomas where the ratio of differentiated to undifferentiated tissues was significantly decreased suggesting defective pluripotency of the cells. In conclusion, subcutaneous implantation of hESCs in presence of Matrigel appears to be the most efficient, reproducible, and the easiest approach for teratoma formation by hESCs. Also, teratoma formation can be employed to study the development defects exhibited by the chromosomally abnormal hESC lines.

The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly.

The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.