Cwp84, a Surface-associated Cysteine Protease, Plays a Role in the Maturation of the Surface Layer of Clostridium difficile

Clostridium difficile is a major and growing problem as a hospital-associated infection that can cause severe, recurrent diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood but undoubtedly involves protein components within the surface layer (S-layer), which play a role in adhesion. In C. difficile, the S-layer is composed of two principal components, the high and low molecular weight S-layer proteins, which are formed from the post-translational cleavage of a single precursor, SlpA. In the present study, we demonstrate that a recently characterized cysteine protease, Cwp84 plays a role in maturation of SlpA. Using a gene knock-out approach, we show that inactivation of the Cwp84 gene in C. difficile 630ΔErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDΔCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDΔCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDΔCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDΔCwp84 was found to be competent at causing disease with a similar pathology to the wild-type strain. The data show that whereas Cwp84 plays a role in the cleavage of SlpA, it is not an essential virulence factor and that bacteria expressing immature SlpA are able to cause disease.     

Antigen presented in the local lymph node by cells from dimethylbenzanthracene-treated murine epidermis activates suppressor cells

Application to skin depleted of LC by treatment with the chemical carcinogen DMBA of a dose of contact sensitizer optimal for inducing contact sensitivity activates transferrable suppressor cells. Excision of solvent- or DMBA-treated skin at various times following application of the contact sensitizer DNFB indicated that the fraction of antigen which leaves the skin within the first few hours induces tolerance. An initial signal inducing unresponsiveness, observed within 1/2 hr, was overturned 3-6 hr later. A more permanent tolerogenic signal in the DMBA- but not solvent-treated lymph node resulted from an epidermal cell from DMBA-treated skin presenting antigen to suppressor cells. Therefore it is likely that suppressor cells are activated in DMBA-treated mice by an epidermal cell which migrates to the local lymph node. Local lymph node cells from DMBA-treated mice also have a diminished ability to present antigen in vivo but they do not activate suppressor cells.     

Anti LAV/HTLV III antibodies in blood donors: preliminary results of pool screening of 5 samples

Serological performances obtained with five reagents for anti-LAV/HTLVIII antibody screening were compared in individual test and in pool. A panel of 55 selected samples and 2,079 pools prepared from 10,395 unselected samples of blood donors was studied. A first dilution is achieved by pooling, then a second dilution allows to obtain the exact working dilution recommended for the test. The sensitivity in pool method is always similar to the sensitivity in individual test, no false negative was observed. The specificity, evaluated on unselected samples, is slightly inferior to that observed in individual test: false-positive rate is in generally less than 3% compared to 2% average rate routinely observed with individual test. These results allow to propose this method, which save about 75% of the reagent cost, for blood donor screening in all laboratories and especially in developing countries.     

Survival and Conflict Behavior of American Black Bears after Rehabilitation

Wildlife agencies face difficult situations when orphaned or injured American black bear (Ursus americanus) cubs (<12 months old) or yearlings (≥12 and <24 months old) are captured. One option is bear rehabilitation, the care and feeding of cubs or yearlings in a semi-natural environment, followed by release. Unfortunately, the survival and movements of bears released from rehabilitation facilities are often poorly documented and the ultimate reasons for success or failure poorly understood. Our goal was to assess survival and post-release conflict of orphaned bear cubs and yearlings following release from a rehabilitation facility, Appalachian Bear Rescue (ABR), in Townsend, Tennessee, USA, from 2015–2016. We predicted that rehabilitated bears would survive at similar rates, die from similar causes, and engage in similar conflict behavior to wild conspecifics. We equipped 42 black bear cubs and yearlings from ABR with global positioning system-collars and released them in Great Smoky Mountains National Park or Cherokee National Forest, Tennessee and North Carolina, USA. Estimated annual survival using known-fate methods for all released bears was 0.93 ± 0.06 [SE]). Survival for 13 bears released as cubs was 0.64 ± 0.14, whereas none of the bears released as yearlings died within 1 year after release (n = 29). Survival of rehabilitated bears was similar to or higher than published rates for wild conspecifics. Three of 42 bears (7.1%) released from ABR engaged in conflict behavior up to 1 year following release, and those had spent time involved in conflict behavior with their mothers (e.g., approaching humans) prior to being orphaned. Despite not having the typical post-natal experience with their mothers, the bears in our study appeared to behave and survive similarly to their wild conspecifics. Rehabilitation is effective for managing orphaned or injured bears. Best survival occurred for bears released as yearlings; however, managers can maximize cub survival through fall releases when plentiful wild foods are available. © 2019 The Authors. The Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.     

Mental Health Following Acquisition of Disability in Adulthood—The Impact of Wealth

BackgroundAcquisition of a disability in adulthood has been associated with a reduction in mental health. We tested the hypothesis that low wealth prior to disability acquisition is associated with a greater deterioration in mental health than for people with high wealth.MethodsWe assess whether level of wealth prior to disability acquisition modifies this association using 12 waves of data (2001–2012) from the Household, Income and Labour Dynamics in Australia survey–a population-based cohort study of working-age Australians. Eligible participants reported at least two consecutive waves of disability preceded by at least two consecutive waves without disability (1977 participants, 13,518 observations). Fixed-effects linear regression was conducted with a product term between wealth prior to disability (in tertiles) and disability acquisition with the mental health component score of the SF–36 as the outcome.ResultsIn models adjusted for time-varying confounders, there was evidence of negative effect measure modification by prior wealth of the association between disability acquisition and mental health (interaction term for lowest wealth tertile: -2.2 points, 95% CI -3.1 points, -1.2, p<0.001); low wealth was associated with a greater decline in mental health following disability acquisition (-3.3 points, 95% CI -4.0, -2.5) than high wealth (-1.1 points, 95% CI -1.7, -0.5).ConclusionThe findings suggest that low wealth prior to disability acquisition in adulthood results in a greater deterioration in mental health than among those with high wealth.     

Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides.

We report the cloning and characterization of the gene product of the gene for the form II ribulose bisphosphate carboxylase from Rhodopseudomonas sphaeroides. We present evidence that the form II enzyme is encoded by a single gene in R. sphaeroides; however, this gene does hybridize to a second chromosomal locus.     

Effect of rAd5-Vector HIV-1 Preventive Vaccines on HIV-1 Acquisition: A Participant-Level Meta-Analysis of Randomized Trials

Background

Three phase 2b, double-blind, placebo-controlled, randomized efficacy trials have tested recombinant Adenovirus serotype-5 (rAd5)-vector preventive HIV-1 vaccines: MRKAd5 HIV-1 gag/pol/nef in Step and Phambili, and DNA/rAd5 HIV-1 env/gag/pol in HVTN505. Due to efficacy futility observed at the first interim analysis in Step and HVTN505, participants of all three studies were unblinded to their vaccination assignments during the study but continued follow–up. Rigorous meta-analysis can provide crucial information to advise the future utility of rAd5-vector vaccines.

Methods

We included participant-level data from all three efficacy trials, and three Phase 1–2 trials evaluating the HVTN505 vaccine regimen. We predefined two co-primary analysis cohorts for assessing the vaccine effect on HIV-1 acquisition. The modified-intention-to-treat (MITT) cohort included all randomly assigned participants HIV-1 uninfected at study entry, who received at least the first vaccine/placebo, and the Ad5 cohort included MITT participants who received at least one dose of rAd5-HIV vaccine or rAd5-placebo. Multivariable Cox regression models were used to estimate hazard ratios (HRs) of HIV-1 infection (vaccine vs. placebo) and evaluate HR variation across vaccine regimens, time since vaccination, and subgroups using interaction tests.

Findings

Results are similar for the MITT and Ad5 cohorts; we summarize MITT cohort results. Pooled across the efficacy trials, over all follow-up time 403 (n = 224 vaccine; n = 179 placebo) of 6266 MITT participants acquired HIV-1, with a non-significantly higher incidence in vaccine recipients (HR 1.21, 95% CI 0.99–1.48, P = 0.06). The HRs significantly differed by vaccine regimen (interaction P = 0.03; MRKAd5 HR 1.41, 95% CI 1.11–1.78, P = 0.005 vs. DNA/rAd5 HR 0.88, 95% CI 0.61–1.26, P = 0.48). Results were similar when including the Phase 1–2 trials. Exploratory analyses based on the efficacy trials supported that the MRKAd5 vaccine-increased risk was concentrated in Ad5-positive or uncircumcised men early in follow-up, and in Ad5-negative or circumcised men later. Overall, MRKAd5 vaccine-increased risk was evident across subgroups except in circumcised Ad5-negative men (HR 0.97, 95% CI 0.58−1.63, P = 0.91); there was little evidence that the DNA/rAd5 vaccine, that was tested in this subgroup, increased risk (HR 0.88, 95% CI 0.61–1.26, P = 0.48). When restricting the analysis of Step and Phambili to follow-up time before unblinding, 114 (n = 65 vaccine; n = 49 placebo) of 3770 MITT participants acquired HIV-1, with a non-significantly higher incidence in MRKAd5 vaccine recipients (HR 1.30, 95% CI 0.89–1.14, P = 0.18).

Interpretation and Significance

The data support increased risk of HIV-1 infection by MRKAd5 over all follow-up time, but do not support increased risk of HIV-1 infection by DNA/rAd5. This study provides a rationale for including monitoring plans enabling detection of increased susceptibility to infection in HIV-1 at-risk populations.