Endothelial nitric oxide pathways in the pathophysiology of dengue: a prospective observational study.

Background: Dengue can cause increased vascular permeability that may lead to hypovolemic shock. Endothelial dysfunction may underlie this; however the association of endothelial nitric oxide pathways with disease severity is unknown. Methods: We performed a prospective observational study in two Vietnamese hospitals, assessing patients presenting early (<72 hours fever) and patients hospitalized with warning signs or severe dengue. The reactive hyperaemic index (RHI), which measures endothelium-dependent vasodilation and is a surrogate marker of endothelial function and NO bioavailability was evaluated using peripheral artery tonometry (EndoPAT) and plasma levels of L-arginine, Arginase-1 and ADMA were measured at serial time-points. The main outcome of interest was plasma leakage severity. Results: 314 patients were enrolled, median age of the participants was 21 (IQR 13-30) years. No difference was found in the endothelial parameters between dengue and other febrile illness (OFI). Considering dengue patients, the RHI was significantly lower for patients with severe plasma leakage compared to those with no leakage (1.46 vs. 2.00, P<0.001), over acute time-points, apparent already in the early febrile phase (1.29 vs. 1.75, P=0.012). RHI correlated negatively with arginase-1, and positively with L-arginine (P=0.001). Endothelial dysfunction/NO bioavailability is associated with worse plasma leakage, occurs early in dengue illness and correlates with hypoargininaemia and high arginase-1 levels

Regional variation in hospitalization for stroke among Asians/Pacific Islanders in the United States: a nationwide retrospective cohort study

BACKGROUND: In Asia, stroke incidence varies dramatically from country to country. Little is known about stroke incidence in Asians/Pacific Islanders in the US, where regional heterogeneity in Asian/Pacific Islander sub-populations is great. We sought to characterize both the national and regional incidences of first and recurrent hospitalized acute ischemic stroke, subarachnoid hemorrhage, and intracerebral hemorrhage in Asians/Pacific Islanders compared to non-Hispanic whites. METHODS: We used the National Inpatient Sample of the 1997 Healthcare Cost and Utilization Project. It is a 20% stratified sample of hospitalizations to nonfederal hospitals in the US. National and regional projections were made using sampling weights specific for patients and hospitals. We identified stroke subtypes using previously validated ICD-9 codes. Age-adjusted incidence rates were calculated using the direct method with the US population in 2000 as the standard. RESULTS: There were 169,386 stroke hospitalizations in the database. Nationally, compared to whites, Asians/Pacific Islanders were more likely to have subarachnoid hemorrhage (incidence rate ratio {RR} female: 1.53, 95% CI 1.41–1.65; male RR: 1.13, 95% CI 1.00–1.27) and intracerebral hemorrhage (female RR 1.29, 95% CI 1.22–1.36; male RR: 1.58, 95% CI 1.50–1.67). However, when examined by geographic regions, Asians/Pacific Islanders had higher incidence rates of subarachnoid hemorrhage and intracerebral hemorrhage predominantly in the West, and lower rates of stroke elsewhere. CONCLUSION: Stroke incidence varies 3-fold among Asians/Pacific Islanders residing in different US regions. Geographic variation is less dramatic in whites. Whether genetic or cultural differences are responsible for dramatic heterogeneity among Asian/Pacific Islander populations is unclear and deserves further study

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies

Energy and macronutrient intakes in preschool children in urban areas of Ho Chi Minh City, Vietnam

Background: An increasing prevalence of overweight and obesity has been documented in preschool children in Ho Chi Minh City (HCMC), Vietnam. However, little is known about what preschool children in HCMC eat or how well their nutrient intake meets nutrient recommendations. This study aims to describe the energy and macronutrient intake and compare these nutrient intakes with the recommendations for Vietnamese children aged four to five years. Methods: The data comes from the baseline measurement of a one year follow-up study on obesity in 670 children attending kindergartens in HCMC. Dietary information for each child at the school and home settings was collected using Food Frequency Questionnaires (FFQs), by interviewing teachers and parents or main caregivers. The average energy and nutrient intake in a day was calculated. The proportion of children with energy intake from macronutrients meeting or exceeding the recommendations was estimated based on the 2006 recommended daily allowance (RDA) for Vietnamese children in this age group. Results: The dietary intake of the participants contained more energy from protein and fat, particularly animal protein and fat, and less energy from carbohydrates, than the RDA. Most children (98.1%) had mean energy intake from protein greater than the recommended level of 15%, and no child obtained energy from animal fat that was in accordance with the recommendation of less than 30% of the total fat intake. Nearly one half of children (46.5%) consumed less than the advised range of mean energy intake from carbohydrate (60%–70%). Conclusion: In this preschool child population in HCMC, in which obesity is emerging as major public health problem, there is an imbalance in dietary intake. Healthy eating programs need to be developed as a part of an obesity prevention program for young children in HCMC

Increased Mobility of Metal Oxide Nanoparticles Due to Photo and Thermal Induced Disagglomeration

Significant advances have been made on our understanding of the fate and transport of engineered nanomaterials. One unexplored aspect of nanoparticle aggregation is how environmental stimuli such as light exposure and temperature variations affect the mobility of engineered nanoparticles. In this study, TiO2, ZnO, and CeO2 were chosen as model materials for investigating the mobility of nanoparticles under three external stimuli: heat, light and sonication. Sunlight and high power sonication were able to partially disagglomerate metal oxide clusters, but primary particles bonded by solid state necks were left intact. A cycle of temperature increase from 25°C to 65°C and then decrease back was found to disagglomerate the compact clusters in the heating phase and reagglomerate them as more open fractal structures during the cooling phase. A fractal model summing the pair-wise DLVO interactions between primary particles within two fractal agglomerates predicts weak attractions on the order of a few kT. Our study shows that common environmental stimuli such as light exposure or temperature variation can disagglomerate nanoparticle clusters and enhance their mobility in open waters. This phenomenon warrants attention since it is likely that metal oxide nanoparticles will experience these natural stimuli during their transport in the environment

Leishmania Mitochondrial Peroxiredoxin Plays a Crucial Peroxidase-Unrelated Role during Infection: Insight into Its Novel Chaperone Activity

Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx−) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx− was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx− were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47phox−/− and B6.RAG2−/− IFN-γ−/− mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx−. A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx− were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity

A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells

A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4- Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies

Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment