Routing Nanomolar Protein Cargoes to Lipid Raft-Mediated/Caveolar Endocytosis through a Ganglioside GM1-Specific Recognition Tag

There is a pressing need to develop ways to deliver therapeutic macromolecules to their intracellular targets. Certain viral and bacterial proteins are readily internalized in functional form through lipid raft-mediated/caveolar endocytosis, but mimicking this process with protein cargoes at therapeutically relevant concentrations is a great challenge. Targeting ganglioside GM1 in the caveolar pits triggers endocytosis. A pentapeptide sequence WYKYW is presented, which specifically captures the glycan moiety of GM1 (K-D = 24 nm). The WYKYW-tag facilitates the GM1-dependent endocytosis of proteins in which the cargo-loaded caveosomes do not fuse with lysosomes. A structurally intact immunoglobulin G complex (580 kDa) is successfully delivered into live HeLa cells at extracellular concentrations ranging from 20 to 160 nm, and escape of the cargo proteins to the cytosol is observed. The short peptidic WYKYW-tag is an advantageous endocytosis routing sequence for lipid raft-mediated/caveolar cell delivery of therapeutic macromolecules, especially for cancer cells that overexpress GM1.Peer reviewe

Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury

Background: Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. Methods: In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNF alpha and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. Results: Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNF alpha in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNF alpha compared to wild-type cells under inflammatory conditions. Conclusions; Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.Peer reviewe

EQ-5D in Central and Eastern Europe : 2000-2015

Objective: Cost per quality-adjusted life year data are required for reimbursement decisions in many Central and Eastern European (CEE) countries. EQ-5D is by far the most commonly used instrument to generate utility values in CEE. This study aims to systematically review the literature on EQ-5D from eight CEE countries. Methods: An electronic database search was performed up to July 1, 2015 to identify original EQ-5D studies from the countries of interest. We analysed the use of EQ-5D with respect to clinical areas, methodological rigor, population norms and value sets. Results: We identified 143 studies providing 152 country-specific results with a total sample size of 81,619: Austria (n=11), Bulgaria (n=6), Czech Republic (n=18), Hungary (n=47), Poland (n=51), Romania (n=2), Slovakia (n=3) and Slovenia (n=14). Cardiovascular (20%), neurologic (16%), musculoskeletal (15%) and endocrine/nutritional/metabolic diseases (14%) were the most frequently studied clinical areas. Overall 112 (78%) of the studies reported EQ VAS results and 86 (60%) EQ-5D index scores, of which 27 (31%) did not specify the applied tariff. Hungary, Poland and Slovenia have population norms. Poland and Slovenia also have a national value set. Conclusions: Increasing use of EQ-5D is observed throughout CEE. The spread of health technology assessment activities in countries seems to be reflected in the number of EQ-5D studies. However, improvement in informed use and methodological quality of reporting is needed. In jurisdictions where no national value set is available, in order to ensure comparability we recommend to apply the most frequently used UK tariff. Regional collaboration between CEE countries should be strengthened

Impact of Endocrine Disorders on IVF Outcomes: Results from a Large, Single-Centre, Prospective Study

Endocrine disorders negatively influence the ovarian function, and increasing incidence of endocrine diseases with age may have further negative effects on pregnancy rate. Prospective cohort study of 231 consecutively enrolled patients underwent IVF treatment. In patients with known endocrine disorders, the laboratory parameters were corrected before IVF treatment. One hundred sixty one patients (69.7%) had at least one known and treated endocrine disorder (study group), and 70 patients were endocrine negative (control group). Endocrine disorders diagnosed were thyroid disorders (32.5%), diminished ovarian reserve (23.8%), insulin resistance (22.5%), PCOS (15.2%), hyperprolactinaemia (13.4%), obesity (12.1%), hypogonadotropic hypogonadism (0.8%) and congenital adrenal hyperplasia (0.2%). Before the IVF treatment, systematic endocrine laboratory examinations were performed in all patients. Higher age, BMI and FSH were found in the study group, while AMH level was lower. There were no differences in LH, E2, prolactin, TSH, FT3, FT4, TT, DHEAS, androstendione, 17-OHP and SHBG level between the study and control groups. The study group had higher baseline glucose, baseline insulin, 120-min glucose and 120-min insulin level after oral glucose tolerance test. With no difference in the IVF cycles performed, pregnancy rate was lower in the study group (61.43% vs. 34.16%; p = 0.003), and this difference (p = 0.0151) remained in age-corrected rates, as well. The analyses were also performed in individual endocrinology groups. The prevalence of endocrine disorders is high in females participating in IVF programs, and they are often accompanying each other. Even after proper correction, the presence of the endocrine disorder negatively influences the pregnancy rate in IVF treatment

Special aspects of pregnancy care in PCOS patients

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