Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials

The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method

Estimating contributions of pelagic and benthic pathways to consumer production in coupled marine food webs

1. Pelagic and benthic systems usually interact, but their dynamics and production rates differ. Such differences influence the distribution, reproductive cycles, growth rates, stability and productivity of the consumers they support. Consumer preferences for, and dependence on, pelagic or benthic production are governed by the availability of these sources of production and consumer life history, distribution, habitat, behavioural ecology, ontogenetic stage and morphology. 2. Diet studies may demonstrate the extent to which consumers feed on prey in pelagic or benthic environments. But they do not discriminate benthic production directly supported by phytoplankton from benthic production recycled through detrital pathways. The former will track the dynamics of phytoplankton production more closely than the latter. We develop and apply a new analytical method that uses carbon (C) and sulfur (S) natural abundance stable isotope data to assess the relative contribution of pelagic and benthic pathways to fish consumer production. 4. For 13 species of fish that dominate community biomass in the northern North Sea (estimated >90% of total biomass), relative modal use of pelagic pathways ranged from <25% to >85%. Use of both C and S isotopes as opposed to just C reduced uncertainty in relative modal use estimates. Temporal comparisons of relative modal use of pelagic and benthic pathways revealed similar ranking of species dependency over four years, but annual variation in relative modal use within species was typically 10-40%. 5. For the total fish consumer biomass in the study region, the C and S method linked approximately 70% and 30% of biomass to pelagic and benthic pathways respectively. As well as providing a new method to define consumers’ links to pelagic and benthic pathways our results demonstrate that a substantial proportion of fish biomass, and by inference production, in the northern North Sea is supported by production that has passed through transformations on the seabed

Benthic-pelagic coupling and trophic relationships in northern Baltic Sea food webs

Understanding marine ecosystem structure and functioning is crucial in supporting sustainable management of natural resources and monitoring the health of marine ecosystems. The current study utilized stable isotope (SI) mixing models and trophic position models to examine energy flow, trophic relationships, and benthic-pelagic coupling between food web components. Roughly 1900 samples from different trophic levels in the food web, collected during 2001-2010 from four northern and central sub-basins of the Baltic Sea, were analyzed for SI ratios of carbon and nitrogen. Trophic structure of the food webs among the sub-basins was consistent, but there were differences between the proportions of energy in different trophic levels that had originated from the benthic habitat. Mysids and amphipods served as important links between the benthic and pelagic ecosystems. Much (35-65%) of their energy originated from the benthic zone but was transferred to higher trophic levels in the pelagic food web by consumption by herring (Clupea harengus). One percent to twenty-four percent of the energy consumption of apex seal predators (Halichoerus grypus and Pusa hispida) and predatory fish (Salmo salar) was derived from benthic zone. Diets of mysids and amphipods differed, although some overlap in their dietary niches was observed. The food web in the Gulf of Finland was more influenced by the benthic subsystem than food webs in the other sub-basins. The baseline levels of delta C-13 and delta N-15 differed between sub-basins of the Baltic Sea, indicating differences in the input of organic matter and nutrients to each sub-basin.peerReviewe

A phase 1 randomized, open label, rectal safety, acceptability, pharmacokinetic, and pharmacodynamic study of three formulations of tenofovir 1% Gel (the CHARM-01 study)

Objectives: The CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study. Methods: Participants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial. Results: All three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/106 cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection. Conclusions: All three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection. Trial Registration: ClinicalTrials.gov NCT01575405

Can spontaneous spinal epidural haematoma be managed safely without operation? a report of four cases

The presentation, investigation, and management of four patients with spontaneous spinal epidural haematoma is presented. In each case the diagnosis was made by MRI. At the time of diagnosis spontaneous recovery had started in each patient and therefore they were all treated conservatively. In each case follow up MRI confirmed rapid reduction in the size of the haematoma and no underlying cause was demonstrated. The presentation, diagnosis, and rationale for treatment are discussed. Conservative treatment is safe in some cases of spinal epidural haematoma if early neurological recovery has started.