Do Diagnosis Delays Impact Receipt of Test Results? Evidence from the HIV Early Infant Diagnosis Program in Uganda

Background

There is scant evidence on the association between diagnosis delays and the receipt of test results in HIV Early Infant Diagnosis (EID) programs. We determine the association between diagnosis delays and other health care system and patient factors on result receipt.

Methods

We reviewed 703 infant HIV test records for tests performed between January 2008 and February 2009 at a regional referral hospital and level four health center in Uganda. The main outcome was caregiver receipt of the test result. The primary study variable was turnaround time (time between sample collection and result availability at the health facility). Additional variables included clinic entry point, infant age at sample collection, reported HIV status and receipt of antiretroviral prophylaxis for prevention of mother-to-child transmission. We conducted a pooled analysis in addition to separate analyses for each facility. We estimated the relative risk of result receipt using modified Poisson regression with robust standard errors.

Results

Overall, the median result turnaround time, was 38 days. 59% of caregivers received infant test results. Caregivers were less likely to receive results at turnaround times greater than 49 days compared to 28 days or fewer (ARR = 0.83; 95% CI = 0.70–0.98). Caregivers were more likely to receive results at the PMTCT clinic (ARR = 1.81; 95% CI = 1.40–2.33) and less likely at the pediatric ward (ARR = 0.54; 95% CI = 0.37–0.81) compared to the immunization clinic. At the level four health center, result receipt was half as likely among infants older than 9 months compared to 3 months and younger (ARR= 0.47; 95% CI = 0.25–0.93).

Conclusion

In this study setting, we find evidence that longer turnaround times, clinic entry point and age at sample collection may be associated with receipt of infant HIV test results.     

Downregulation of MicroRNA-9 in iPSC-Derived Neurons of FTD/ALS Patients with TDP-43 Mutations

Transactive response DNA-binding protein 43 (TDP-43) is a major pathological protein in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are many disease-associated mutations in TDP-43, and several cellular and animal models with ectopic overexpression of mutant TDP-43 have been established. Here we sought to study altered molecular events in FTD and ALS by using induced pluripotent stem cell (iPSC) derived patient neurons. We generated multiple iPSC lines from an FTD/ALS patient with the TARDBP A90V mutation and from an unaffected family member who lacked the mutation. After extensive characterization, two to three iPSC lines from each subject were selected, differentiated into postmitotic neurons, and screened for relevant cell-autonomous phenotypes. Patient-derived neurons were more sensitive than control neurons to 100 nM straurosporine but not to other inducers of cellular stress. Three disease-relevant cellular phenotypes were revealed under staurosporine-induced stress. First, TDP-43 was localized in the cytoplasm of a higher percentage of patient neurons than control neurons. Second, the total TDP-43 level was lower in patient neurons with the A90V mutation. Third, the levels of microRNA-9 (miR-9) and its precursor pri-miR-9-2 decreased in patient neurons but not in control neurons. The latter is likely because of reduced TDP-43, as shRNA-mediated TDP-43 knockdown in rodent primary neurons also decreased the pri-miR-9-2 level. The reduction in miR-9 expression was confirmed in human neurons derived from iPSC lines containing the more pathogenic TARDBP M337V mutation, suggesting miR-9 downregulation might be a common pathogenic event in FTD/ALS. These results show that iPSC models of FTD/ALS are useful for revealing stress-dependent cellular defects of human patient neurons containing rare TDP-43 mutations in their native genetic contexts.     

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) ¹. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex ², is the most common site of human stroke ³, and ischemia within its territory can result in extensive dysfunction or death ^1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.     

Enhanced Lipid Oxidation and Maintenance of Muscle Insulin Sensitivity Despite Glucose Intolerance in a Diet-Induced Obesity Mouse Model

Background

Diet-induced obesity is a rising health concern which can lead to the development of glucose intolerance and muscle insulin resistance and, ultimately, type II diabetes mellitus. This research investigates the associations between glucose intolerance or muscle insulin resistance and tissue specific changes during the progression of diet-induced obesity.

Methodology

C57BL/6J mice were fed a normal or high-fat diet (HFD; 60% kcal fat) for 3 or 8 weeks. Disease progression was monitored by measurements of body/tissue mass changes, glucose and insulin tolerance tests, and ex vivo glucose uptake in intact muscles. Lipid metabolism was analyzed using metabolic chambers and ex vivo palmitate assays in intact muscles. Skeletal muscle, liver and adipose tissues were analyzed for changes in inflammatory gene expression. Plasma was analyzed for insulin levels and inflammatory proteins. Histological techniques were used on muscle and liver cryosections to assess metabolic and morphological changes.

Principal Findings/Conclusions

A rapid shift in whole body metabolism towards lipids was observed with HFD. Following 3 weeks of HFD, elevated total lipid oxidation and an oxidative fiber type shift had occurred in the skeletal muscle, which we propose was responsible for delaying intramyocellular lipid accumulation and maintaining muscle’s insulin sensitivity. Glucose intolerance was present after three weeks of HFD and was associated with an enlarged adipose tissue depot, adipose tissue inflammation and excess hepatic lipids, but not hepatic inflammation. Furthermore, HFD did not significantly increase systemic or muscle inflammation after 3 or 8 weeks of HFD suggesting that early diet-induced obesity does not cause inflammation throughout the whole body. Overall these findings indicate skeletal muscle did not contribute to the development of HFD-induced impairments in whole-body glucose tolerance following 3 weeks of HFD.     

Trypsin Potentiates Human Fibrocyte Differentiation

Trypsin-containing topical treatments can be used to speed wound healing, although the mechanism of action is unknown. To help form granulation tissue and heal wounds, monocytes leave the circulation, enter the wound tissue, and differentiate into fibroblast-like cells called fibrocytes. We find that 20 to 200 ng/ml trypsin (concentrations similar to those used in wound dressings) potentiates the differentiation of human monocytes to fibrocytes in cell culture. Adding trypsin inhibitors increases the amount of trypsin needed to potentiate fibrocyte differentiation, suggesting that the potentiating effect is dependent on trypsin proteolytic activity. Proteases with other site specificities such as pepsin, endoprotease GluC, and chymotrypsin do not potentiate fibrocyte differentiation. This potentiation requires the presence of albumin in the culture medium, and tryptic fragments of human or bovine albumin also potentiate fibrocyte differentiation. These results suggest that topical trypsin speeds wound healing by generating tryptic fragments of albumin, which in turn potentiate fibrocyte differentiation.     

Spatial Pattern Dynamics of 3D Stem Cell Loss of Pluripotency via Rules-Based Computational Modeling

Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells from all germ lineages, making them a potentially robust cell source for regenerative medicine therapies, but difficulties in predicting and controlling ESC differentiation currently limit the development of therapies and applications from such cells. A common approach to induce the differentiation of ESCs in vitro is via the formation of multicellular aggregates known as embryoid bodies (EBs), yet cell fate specification within EBs is generally considered an ill-defined and poorly controlled process. Thus, the objective of this study was to use rules-based cellular modeling to provide insight into which processes influence initial cell fate transitions in 3-dimensional microenvironments. Mouse embryonic stem cells (D3 cell line) were differentiated to examine the temporal and spatial patterns associated with loss of pluripotency as measured through Oct4 expression. Global properties of the multicellular aggregates were accurately recapitulated by a physics-based aggregation simulation when compared to experimentally measured physical parameters of EBs. Oct4 expression patterns were analyzed by confocal microscopy over time and compared to simulated trajectories of EB patterns. The simulations demonstrated that loss of Oct4 can be modeled as a binary process, and that associated patterns can be explained by a set of simple rules that combine baseline stochasticity with intercellular communication. Competing influences between Oct4+ and Oct4− neighbors result in the observed patterns of pluripotency loss within EBs, establishing the utility of rules-based modeling for hypothesis generation of underlying ESC differentiation processes. Importantly, the results indicate that the rules dominate the emergence of patterns independent of EB structure, size, or cell division. In combination with strategies to engineer cellular microenvironments, this type of modeling approach is a powerful tool to predict stem cell behavior under a number of culture conditions that emulate characteristics of 3D stem cell niches.     

A Hybrid Likelihood Model for Sequence-Based Disease Association Studies

In the past few years, case-control studies of common diseases have shifted their focus from single genes to whole exomes. New sequencing technologies now routinely detect hundreds of thousands of sequence variants in a single study, many of which are rare or even novel. The limitation of classical single-marker association analysis for rare variants has been a challenge in such studies. A new generation of statistical methods for case-control association studies has been developed to meet this challenge. A common approach to association analysis of rare variants is the burden-style collapsing methods to combine rare variant data within individuals across or within genes. Here, we propose a new hybrid likelihood model that combines a burden test with a test of the position distribution of variants. In extensive simulations and on empirical data from the Dallas Heart Study, the new model demonstrates consistently good power, in particular when applied to a gene set (e.g., multiple candidate genes with shared biological function or pathway), when rare variants cluster in key functional regions of a gene, and when protective variants are present. When applied to data from an ongoing sequencing study of bipolar disorder (191 cases, 107 controls), the model identifies seven gene sets with nominal p-values0.05, of which one MAPK signaling pathway (KEGG) reaches trend-level significance after correcting for multiple testing.     

Life History Changes in Coral Fluorescence and the Effects of Light Intensity on Larval Physiology and Settlement in Seriatopora hystrix

Fluorescence is common in both coral adult and larval stages, and is produced by fluorescent proteins that absorb higher energy light and emit lower energy light. This study investigated the changes of coral fluorescence in different life history stages and the effects of parental light environment on larval fluorescence, larval endosymbiotic dinoflagellate abundance, larval size and settlement in the brooding coral Seriatopora hystrix. Data showed that coral fluorescence changed during development from green in larvae to cyan in adult colonies. In larvae, two green fluorescent proteins (GFPs) co-occur where the peak emission of one GFP overlaps with the peak excitation of the second GFP allowing the potential for energy transfer. Coral larvae showed great variation in GFP fluorescence, dinoflagellate abundance, and size. There was no obvious relationship between green fluorescence intensity and dinoflagellate abundance, green fluorescence intensity and larval size, or dinoflagellate abundance and larval size. Larvae of parents from high and low light treatments showed similar green fluorescence intensity, yet small but significant differences in size, dinoflagellate abundance, and settlement. The large variation in larval physiology combined with subtle effects of parental environment on larval characteristics seem to indicate that even though adult corals produce larvae with a wide range of physiological capacities, these larvae can still show small preferences for settling in similar habitats as their parents. These data highlight the importance of environmental conditions at the onset of life history and parent colony effects on coral larvae.