Assessment of the range of the HIV-1 infectivity enhancing effect of individual human semen specimen and the range of inhibition by EGCG

Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted

Germ Cell Maintenance and Sustained Testosterone and Precursor Hormone Production in Human Prepubertal Testis Organ Culture with Tissues from Boys 7 Years+ under Conditions from Adult Testicular Tissue

Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples (n = 3; patient ages 7, 9, and 12 years). Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period

Activation of the macroautophagy pathway by Yersinia enterocolitica promotes intracellular multiplication and egress of yersiniae from epithelial cells.

The virulence strategy of pathogenic Yersinia spp. involves cell-invasive as well as phagocytosis-preventing tactics to enable efficient colonisation of the host organism. Enteropathogenic yersiniae display an invasive phenotype in early infection stages, which facilitates penetration of the intestinal mucosa. Here we show that invasion of epithelial cells by Yersinia enterocolitica is followed by intracellular survival and multiplication of a subset of ingested bacteria. The replicating bacteria were enclosed in vacuoles with autophagy-related characteristics, showing phagophore formation, xenophagy, and recruitment of cytoplasmic autophagosomes to the bacteria-containing compartments. The subsequent fusion of these vacuoles with lysosomes and concomitant vesicle acidification were actively blocked by Yersinia. This resulted in increased intracellular proliferation and detectable egress of yersiniae from infected cells. Notably, deficiency of the core autophagy machinery component FIP200 impaired the development of autophagic features at Yersinia-containing vacuoles as well as intracellular replication and release of bacteria to the extracellular environment. These results suggest that Y. enterocolitica may take advantage of the macroautophagy pathway in epithelial cells to create an autophagosomal niche that supports intracellular bacterial survival, replication, and, eventually, spread of the bacteria from infected cells

M45 targets NEMO to autophagosomes.

<p>(A) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture. (B) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows. (C) NIH-3T3 cells stably expressing Flag-NEMO were mock infected or infected with MCMV-M45-HA or MCMV-ΔM45 at an MOI of 7. Seven hpi, cells were fixed and analyzed as described for panel B. (D) NIH-3T3 cells were transfected with expression plasmids for Flag-NEMO and M45-HA or empty vector as indicated. Plasmids encoding GFP-tagged LC3, Rab5, or Rab7 were cotransfected. 24 hours posttransfection cells were fixed and used for anti-Flag immunofluorescence staining.</p

M45 targets RIP1 and NEMO, two crucial components of the inflammatory cascade.

<p>M45 targets RIP1 and NEMO, two crucial components of the inflammatory cascade.</p

The C-terminal RNR R1 homology domain of M45 is required for the inhibition of NF-κB activation and interaction with NEMO.

<p>(A) Schematic representation of M45 truncation mutants used in this and a previous <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002517#ppat.1002517-Mack1" target="_blank">[36]</a> study. The C-terminal RNR R1 homology domain is shown in white, the unique N terminus in grey. The RHIM is marked black. (B) NIH-3T3 cells were transduced with retroviruses expressing full length M45, truncated M45 proteins, or GFP. After stimulation with IL-1β (20 ng/ml, 15 min), IκBα levels were determined by immunoblotting. (C) 293A cells were transfected with plasmids encoding Flag-tagged NEMO and HA-tagged full-length M45, truncated M45, or an unrelated MCMV control protein (m142), respectively. Lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates and the whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies. (D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).</p