Vaginal microbiome-hormonal contraceptive interactions associate with the mucosal proteome and HIV acquisition

Alterations to the mucosal environment of the female genital tract, such as genital inflammation, have been associated with increased HIV acquisition in women. As the microbiome and hormonal contraceptives can affect vaginal mucosal immunity, we hypothesized these components may interact in the context of HIV susceptibility. Using previously published microbiome data from 685 women in the CAPRISA-004 trial, we compared relative risk of HIV acquisition in this cohort who were using injectable depot medroxyprogesterone acetate (DMPA), norethisterone enanthate (NET-EN), and combined oral contraceptives (COC). In women who were Lactobacillus-dominant, HIV acquisition was 3-fold higher in women using DMPA relative to women using NET-EN or COC (OR: 3.27; 95% CI: 1.24–11.24, P = 0.0305). This was not observed in non-Lactobacillus-dominant women (OR: 0.95, 95% CI: 0.44–2.15, P = 0.895) (interaction P = 0.0686). Higher serum MPA levels associated with increased molecular pathways of inflammation in the vaginal mucosal fluid of Lactobacillus-dominant women, but no differences were seen in non-Lactobacillus dominant women. This study provides data suggesting an interaction between the microbiome, hormonal contraceptives, and HIV susceptibility.     

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.     

The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors

Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes ( murA and murZ ) encoding UDP- N -acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro , consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA , but remained almost unchanged following inactivation of murZ . Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA . Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.     

The Effect of Lubricants on Powder Flowability for Pharmaceutical Application

Pharmaceutical tablets are manufactured through a series of batch steps finishing with compression into a form using a tablet press. Lubricants are added to the powder mixture prior to the tabletting step to ensure that the tablet is ejected properly from the press. The addition of lubricants also affects tablet properties and can affect the behavior of the powder mixture. The objective of this research was to investigate the effect of lubricants on powder flowability as flowability into the tablet press is critical. Four lubricants (magnesium stearate, magnesium silicate, stearic acid, and calcium stearate) were mixed, in varying amounts, with spray-dried lactose. In addition, magnesium stearate was also mixed with placebo granules from a high-shear granulator. Measurements based on avalanche behavior indicated flowability potential and dynamic density and were more sensitive to changes in the mixture and provided a more accurate and reproducible indication of flowability than traditional static measurements. Of the tested lubricants, magnesium stearate provided the best increase in flowability even in the low amounts commonly added in formulations.     

Multi-dose Romidepsin Reactivates Replication Competent SIV in Post-antiretroviral Rhesus Macaque Controllers

Viruses that persist despite seemingly effective antiretroviral treatment (ART) and can reinitiate infection if treatment is stopped preclude definitive treatment of HIV-1 infected individuals, requiring lifelong ART. Among strategies proposed for targeting these viral reservoirs, the premise of the “shock and kill” strategy is to induce expression of latent proviruses [for example with histone deacetylase inhibitors (HDACis)] resulting in elimination of the affected cells through viral cytolysis or immune clearance mechanisms. Yet, ex vivo studies reported that HDACis have variable efficacy for reactivating latent proviruses, and hinder immune functions. We developed a nonhuman primate model of post-treatment control of SIV through early and prolonged administration of ART and performed in vivo reactivation experiments in controller RMs, evaluating the ability of the HDACi romidepsin (RMD) to reactivate SIV and the impact of RMD treatment on SIV-specific T cell responses. Ten RMs were IV-infected with a SIVsmmFTq transmitted-founder infectious molecular clone. Four RMs received conventional ART for >9 months, starting from 65 days post-infection. SIVsmmFTq plasma viremia was robustly controlled to <10 SIV RNA copies/mL with ART, without viral blips. At ART cessation, initial rebound viremia to ~10⁶ copies/mL was followed by a decline to < 10 copies/mL, suggesting effective immune control. Three post-treatment controller RMs received three doses of RMD every 35–50 days, followed by in vivo experimental depletion of CD8⁺ cells using monoclonal antibody M-T807R1. RMD was well-tolerated and resulted in a rapid and massive surge in T cell activation, as well as significant virus rebounds (~10⁴ copies/ml) peaking at 5–12 days post-treatment. CD8⁺ cell depletion resulted in a more robust viral rebound (10⁷ copies/ml) that was controlled upon CD8⁺ T cell recovery. Our results show that RMD can reactivate SIV in vivo in the setting of post-ART viral control. Comparison of the patterns of virus rebound after RMD administration and CD8⁺ cell depletion suggested that RMD impact on T cells is only transient and does not irreversibly alter the ability of SIV-specific T cells to control the reactivated virus.     

The influence of fluid shear on the structure and material properties of sulphate-reducing bacterial biofilms

Biofilms of sulphate-reducing Desulfovibrio sp. EX265 were grown in square section glass capillary flow cells under a range of fluid flow velocities from 0.01 to 0.4 m/s (wall shear stress, τ_w, from 0.027 to 1.0 N/m²). In situ image analysis and confocal scanning laser microscopy revealed biofilm characteristics similar to those reported for aerobic biofilms. Biofilms in both flow cells were patchy and consisted of cell clusters separated by voids. Length-to-width ratio measurements (l _c:w _c) of biofilm clusters demonstrated the formation of more “streamlined” biofilm clusters (l _c:w _c=3.03) at high-flow velocity (Reynolds number, Re, 1200), whereas at low-flow velocity (Re 120), the l _c:w _c of the clusters was approximately 1 (l _c:w _c of 1 indicates no elongation in the flow direction). Cell clusters grown under high flow were more rigid and had a higher yield point (the point at which the biofilm began to flow like a fluid) than those established at low flow and some biofilm cell aggregates were able to relocate within a cluster, by travelling in the direction of flow, before attaching more firmly downstream. Received 01 February 2002/ Accepted in revised form 16 July 2002     

Echinococcus granulosus: secretory activity of the rostellum of the adult cestode in situ in the dog.

Secretary activities associated with the rostellum of adult Echinococcus granulosus were studied using histological, histochemical, and ultrastructural techniques, following rapid fixation of the cestodes in situ in the small intestine of the anaesthetised dog. Studies concentrated on the host-parasite interface from 30 to 35 days postinfection. At this time, contraction of the muscular rostellar pad appeared to be associated with extension of the apical rostellum into a crypt of Lieberkühn. Crypt invasion by the apical rostellum coincided with morphological changes and secretory activity in a group of modified tegumental cells, previously referred to as the rostellar gland. Secretory material, a cystine-rich protein, was observed in the nuclei and cytoplasm of the rostellar gland cells. Release of this material into the interface was seen only following crypt invasion by the apical rostellum. Although the mechanism of release is not clear, it may be analagous to holocrine secretory mechanisms, since apparent degeneration of the rostellar gland region was associated with secretion. Possible functional activities of the secretion associated with hook formation, nutrition, regulation, adhesion, and protection are discussed.     

Primary type II alveolar epithelial cells present microbial antigens to antigen-specific CD4+ T cells

Type II alveolar epithelial cells (AEC) can produce various antimicrobial and proinflammatory effector molecules. This, together with their abundance and strategic location, suggests a role in host defense against pulmonary pathogens. We report that murine type II AEC, like their human counterparts, express class II major histocompatibility complex (MHC). Using a murine model of pulmonary tuberculosis, we find that type II AEC become activated and have increased cell surface expression of class II MHC, CD54, and CD95 following infection. Type II AEC use the class II MHC pathway to process and present mycobacterial antigens to immune CD4+ T cells isolated from mice infected with Mycobacterium tuberculosis. Therefore, not only can type II AEC contribute to the pulmonary immunity by secreting chemokines that recruit inflammatory cells to the lung, but they can also serve as antigen-presenting cells. Although type II AEC are unlikely to prime na?ve T cells, their ability to present antigens to T cells demonstrates that they can participate in the effector phase of the immune response. This represents a novel role for type II AEC in the immunological response to pulmonary pathogens.     

NATIVE MICHIGAN PLANTS STIMULATE SOIL MICROBIAL SPECIES CHANGES AND PAH REMEDIATION AT A LEGACY STEEL MILL

A 1.3-acre phytoremediation site was constructed to mitigate polyaromatic hydrocarbon (PAH) contamination from a former steel mill in Michigan. Soil was amended with 10% (v/v) compost and 5% (v/v) poultry litter. The site was divided into twelve 11.89 m X 27.13 m plots, planted with approximately 35,000 native Michigan perennials, and soils sampled for three seasons. Soil microbial density generally increased in subplots of Eupatorium perfoliatum (boneset), Aster novae-angliae (New England aster), Andropogon gerardii (big bluestem), and Scirpus atrovirens (green bulrush) versus unplanted subplots. Using enumeration assays with root exudates, PAH degrading bacteria were greatest in soils beneath plants. Initially predominant, Arthrobacter were found capable of degrading a PAH cocktail in vitro, especially upon the addition of root exudate. Growth of some Arthrobacter isolates was stimulated by root exudate. The frequency of Arthrobacter declined in planted subplots with a concurrent increase in other species, including secondary PAH degraders Bacillus and Nocardioides. In subplots supporting only weeds, an increase in Pseudomonas density and little PAH removal were observed. This study supports the notion that a dynamic interplay between the soil, bacteria, and native plant root secretions likely contributes to in situ PAH phytoremediation.