Latent HIV-1 infection of resting CD4⁺ T cells in the humanized Rag2⁻/⁻ γc⁻/⁻ mouse

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4⁺ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4⁺ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2^−/− γ_c^−/− (hu-Rag2^−/− γ_c^−/−) mice, as in humans, resting CD4⁺ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4⁺ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4⁺ T cell reservoir of hu-Rag2^−/− γ_c^−/− mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments.     

Disaccharide Composition of Heparan Sulfates: Brain, Nervous Tissue Storage Organelles, Kidney, and Lung

Abstract: We have characterized the structural properties of heparan sulfates from brain and other tissues after de-polymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46–69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17–21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10–14%, together with smaller and approximately equall proportions (5–9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine-and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5–10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71 %) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large densecored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for ～ 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21 % unsulfated disaccharides, 10% mono-and disulfated disaccharides, and 69% trisulfated disaccharides. Our results therefore reveal significant differences among heparan sulfates from different sources, supporting other evidence that structural variations in heparan sulfate may be related to specific biological functions, such as the switching in the neural response from fibroblast growth factor-2 to fibro-blast growth factor-1 resulting from developmental changes in the glycosaminoglycan chains of a heparan sulfate proteoglycan.     

Dengue incidence in urban and rural Cambodia: results from population-based active fever surveillance, 2006-2008

Background

Dengue vaccines are now in late-stage development, and evaluation and robust estimates of dengue disease burden are needed to facilitate further development and introduction. In Cambodia, the national dengue case-definition only allows reporting of children less than 16 years of age, and little is known about dengue burden in rural areas and among older persons. To estimate the true burden of dengue in the largest province of Cambodia, Kampong Cham, we conducted community-based active dengue fever surveillance among the 0-to-19–year age group in rural villages and urban areas during 2006–2008.

Methods and Findings

Active surveillance for febrile illness was conducted in 32 villages and 10 urban areas by mothers trained to use digital thermometers combined with weekly home visits to identify persons with fever. An investigation team visited families with febrile persons to obtain informed consent for participation in the follow-up study, which included collection of personal data and blood specimens. Dengue-related febrile illness was defined using molecular and serological testing of paired acute and convalescent blood samples. Over the three years of surveillance, 6,121 fever episodes were identified with 736 laboratory-confirmed dengue virus (DENV) infections for incidences of 13.4–57.8/1,000 person-seasons. Average incidence was highest among children less than 7 years of age (41.1/1,000 person-seasons) and lowest among the 16-to-19–year age group (11.3/1,000 person-seasons). The distribution of dengue was highly focal, with incidence rates in villages and urban areas ranging from 1.5–211.5/1,000 person-seasons (median 36.5). During a DENV-3 outbreak in 2007, rural areas were affected more than urban areas (incidence 71 vs. 17/1,000 person-seasons, p<0.001).

Conclusion

The large-scale active surveillance study for dengue fever in Cambodia found a higher disease incidence than reported to the national surveillance system, particularly in preschool children and that disease incidence was high in both rural and urban areas. It also confirmed the previously observed focal nature of dengue virus transmission.     

Molecular biological approaches to the olfactory system: olfactory marker protein as a model

Detailed exploration of the chemistry of proteins and nucleicacids and of the mechanisms regulating gene expression havebecome commonplace with the advent of recent advances in molecularbiology. The olfactory system is an attractive model in whichto study a wide variety of cellular- and neurobiological questions.Thus, the application to this system of molecular biologicaltechniques should open wide areas for study. Our initial effortshave been directed at the olfactory marker protein (OMP). Wehave determined almost the complete amino acid sequence, isolated,characterized and sized the mRNA and demonstrated its translationin vitro. In addition, cloning of the cDNA for this proteinis underway. In this paper we present an overview of our initialefforts in the use of this technology as well as some indicationof the results which may be expected from its application inthe future.     

Terrestrial biosphere models need better representation of vegetation phenology: results from the North American Carbon Program Site Synthesis

Phenology, by controlling the seasonal activity of vegetation on the land surface, plays a fundamental role in regulating photosynthesis and other ecosystem processes, as well as competitive interactions and feedbacks to the climate system. We conducted an analysis to evaluate the representation of phenology, and the associated seasonality of ecosystem‐scale CO₂ exchange, in 14 models participating in the North American Carbon Program Site Synthesis. Model predictions were evaluated using long‐term measurements (emphasizing the period 2000–2006) from 10 forested sites within the AmeriFlux and Fluxnet‐Canada networks. In deciduous forests, almost all models consistently predicted that the growing season started earlier, and ended later, than was actually observed; biases of 2 weeks or more were typical. For these sites, most models were also unable to explain more than a small fraction of the observed interannual variability in phenological transition dates. Finally, for deciduous forests, misrepresentation of the seasonal cycle resulted in over‐prediction of gross ecosystem photosynthesis by +160 ± 145 g C m^?2 yr^?1 during the spring transition period and +75 ± 130 g C m^?2 yr^?1 during the autumn transition period (13% and 8% annual productivity, respectively) compensating for the tendency of most models to under‐predict the magnitude of peak summertime photosynthetic rates. Models did a better job of predicting the seasonality of CO₂ exchange for evergreen forests. These results highlight the need for improved understanding of the environmental controls on vegetation phenology and incorporation of this knowledge into better phenological models. Existing models are unlikely to predict future responses of phenology to climate change accurately and therefore will misrepresent the seasonality and interannual variability of key biosphere–atmosphere feedbacks and interactions in coupled global climate models.     

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans

After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix.     

Costs of Rapid HIV Screening in an Urban Emergency Department and a Nearby County Jail in the Southeastern United States

Emergency departments and jails provide medical services to persons at risk for HIV infection and are recommended venues for HIV screening. Our main objective in this study was to analyze the cost per new HIV diagnosis associated with the HIV screening program in these two venues. The emergency department’s parallel testing program was conducted at Grady Memorial Hospital in Atlanta, Georgia starting in 2008; the jail’s integrated testing program began at the Fulton County (GA) Jail in 2011. The two sites, four miles apart from one another, employed the same rapid HIV test. Ascertainment that cases were new differed by site; only the jail systematically checked identities against health department HIV registries. The program in the emergency department used dedicated HIV test counselors and made 242 diagnoses over a 40-month period at a cost of $2,981 per diagnosis. The jail program used staff nurses, and found 41 new HIV cases over 10.5 months at a cost of $6,688 per new diagnosis. Differences in methods for ascertainment of new diagnoses, previously undiagnosed HIV sero-positivity, and methodologies used for assessing program costs prevent concluding that one program was more economical than the other. Nonetheless, our findings show that testing in both venues yielded many new diagnoses, with the costs within the range reported in the literature.