Temporal and spatial characterization of HIV/SIV infection at anorectal mucosa using rhesus macaque rectal challenge model.

The study described herein is a continuation of our work in which we developed a methodology to identify small foci of transduced cells following rectal challenge of rhesus macaques with a non-replicative luciferase reporter virus. In the current study, the wild-type virus was added to the inoculation mix and twelve rhesus macaques were necropsied 2-4 days after the rectal challenge to study the changes in infected cell phenotype as the infection progressed. Relying on luciferase reporter we noted that both anus and rectum tissues are susceptible to the virus as early as 48h after the challenge. Small regions of the tissue containing luciferase-positive foci were further analyzed microscopically and were found to also contain cells infected by wild-type virus. Phenotypic analysis of the Env and Gag positive cells in these tissues revealed the virus can infect diverse cell populations, including but not limited to Th17 T cells, non Th17 T cells, immature dendritic cells, and myeloid-like cells. The proportions of the infected cell types, however, did not vary much during the first four days of infection when anus and rectum tissues were examined together. Nonetheless, when the same data was analyzed on a tissue-specific basis, we found significant changes in infected cell phenotypes over the course of infection. For anal tissue, a statistically significant increase in infection was observed for Th17 T cells and myeloid-like cells, while in the rectum, the non-Th17 T cells showed the biggest temporal increase, also of statistical significance.

Th17 T Cells and Immature Dendritic Cells Are the Preferential Initial Targets after Rectal Challenge with a Simian Immunodeficiency Virus-Based Replication-Defective Dual-Reporter Vector.

Understanding the earliest events of human immunodeficiency virus (HIV) sexual transmission is critical to developing and optimizing HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intrarectally challenged with a single-round simian immunodeficiency virus (SIV)-based dual reporter that expresses luciferase and near-infrared fluorescent protein 670 (iRFP670) upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 h after the rectal challenge. Detailed phenotypic analysis revealed that, on average, 62% of transduced cells are CCR6-positive (CCR6+) T cells-the vast majority of which express RORγT, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission. IMPORTANCE Men and women who participate in unprotected receptive anal intercourse are at high risk of acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

Anatomic Distribution of Intravenously Injected IgG Takes Approximately 1 Week to Achieve Stratum Corneum Saturation in Vaginal Tissues.

i.v. injected Abs have demonstrated protection against simian HIV infection in rhesus macaques, paving the way for the Antibody Mediated Prevention trial in which at-risk individuals for HIV received an i.v. infusion of the HIV broadly neutralizing Ab VRC01. However, the time needed for these Abs to fully distribute and elicit protection at mucosal sites is still unknown. In this study, we interrogate how long it takes for Abs to achieve peak anatomical levels at the vaginal surface following i.v. injection. Fluorescently labeled VRC01 and/or Gamunex-C were i.v. injected into 24 female rhesus macaques (Macaca mulatta) with vaginal tissues and plasma acquired up to 2 wk postinjection. We found that Ab delivery to the vaginal mucosa occurs in two phases. The first phase involves delivery to the submucosa, occurring within 24 h and persisting beyond 1 wk. The second phase is the delivery through the stratified squamous epithelium, needing ∼1 wk to saturate the stratum corneum. This study has important implications for the efficacy of immunoprophylaxis targeting pathogens at the mucosa.

Glycogen Levels in Undiluted Genital Fluid and Their Relationship to Vaginal pH, Estrogen, and Progesterone.

BACKGROUND: Colonization of the female lower genital tract with Lactobacillus provides protection against STIs and adverse pregnancy outcomes. Growth of genital Lactobacillus is postulated to depend on epithelial cell-produced glycogen. However, the amount of cell-free glycogen in genital fluid available for utilization by Lactobacillus is not known. METHODS: Eighty-five genital fluid samples from 7 pre-menopausal women taken over 4-6 weeks were obtained using the Instead SoftCup® (EvoFem, Inc., San Diego, CA, USA) by consented donors. Cell-free glycogen and glucose in genital fluids and estrogen and progesterone in blood were quantified. FINDINGS: Glycogen ranged from 0.1-32 µg/µl. There were significant differences between women in glycogen over the observation period. There was a strong negative correlation between glycogen and vaginal pH (r = -0.542, p<0.0001). In multivariable analysis, free glycogen levels were significantly negatively associated with both vaginal pH and progesterone (p < 0.001 and p = 0.004, respectively). Estrogen, glucose, age, sexual intercourse 24 hours prior to visit, and days after the initial visit were not significantly associated with free glycogen levels. CONCLUSION: Cell-free glycogen concentrations can be very high, up to 3% of genital fluid, and are strongly associated with acidic vaginal pH. However, the fluctuations in glycogen levels in individuals and differences between individuals do not appear to be associated with estrogen.

Calcium-dependent membrane association of a flagellar calcium sensor does not require calcium binding.

Flagellar calcium-binding protein (FCaBP) is a dually acylated Ca(2+) sensor in the Trypanosoma cruzi flagellar membrane that undergoes a massive conformational change upon Ca(2+) binding. It is similar to neuronal Ca(2+) sensors, like recoverin, which regulate their binding partners through a calcium acyl switch mechanism. FCaBP is washed out of permeabilized cells with buffers containing EDTA, indicating Ca(2+)-dependent flagellar membrane association. We hypothesized that, like recoverin, FCaBP projects its acyl groups in the presence of Ca(2+), permitting flagellar membrane and binding partner association and that it sequesters the acyl groups in low Ca(2+), disassociating from the membrane and releasing its binding partner to perform a presumed enzymatic function. The X-ray crystal structure of FCaBP suggests that the acyl groups are always exposed, so we set out to test our hypothesis directly. We generated T. cruzi transfectants expressing FCaBP or Ca(2+)-binding mutant FCaBP(E151Q/E188Q) and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBP(E151Q/E188Q) were found to associate with lipid rafts, indicating the Ca(2+)-independence of this association. To our initial surprise, FCaBP(E151Q/E188Q), like wildtype FCaBP, exhibited Ca(2+)-dependent flagellar membrane association, even though this protein does not bind Ca(2+) itself [16]. One possible explanation for this is that FCaBP(E151Q/E188Q), like some other Ca(2+) sensors, may form dimers and that dimerization of FCaBP(E151Q/E188Q) with endogenous wildtype FCaBP might explain its Ca(2+)-dependent localization. Indeed both proteins are able to form dimers in the presence and absence of Ca(2+). These results suggest that FCaBP possesses two distinct Ca(2+)-dependent interactions-one involving a Ca(2+)-induced change in conformation and another perhaps involving binding partner association.

Vaginal challenge with an SIV-based dual reporter system reveals that infection can occur throughout the upper and lower female reproductive tract.

The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection.

Molecular determinants of ciliary membrane localization of Trypanosoma cruzi flagellar calcium-binding protein.

The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.

Unusual high rate of asymptomatic maternal parvovirus B19 infection associated with severe fetal outcome.

OBJECTIVE: To study the role of asymptomatic maternal parvo B19 infection in severe fetal outcome in Province of Vojvodina. METHODS: One hundred seventy-six pregnant women (13-25 weeks of gestation) were divided in two groups - patients with symptoms of imminent spontaneous abortion and poor pregnancy outcome and patients with normal course of pregnancy. Double serum samples were analyzed to quantify IgM and IgG to parvovirus B19. RESULTS: Among pregnant women with symptoms of spontaneous abortion, we found significantly higher percentage of acute parvovirus B19 infection. CONCLUSIONS: Asymptomatic parvo B19 infection is associated with poor fetal outcome much more than we presumed previously.

Acylation-dependent export of Trypanosoma cruzi phosphoinositide-specific phospholipase C to the outer surface of amastigotes.

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.

Composition and sensory function of the trypanosome flagellar membrane.

A cilium is an extension of the cell that contains an axonemal complex of microtubules and associated proteins bounded by a membrane which is contiguous with the cell body membrane. Cilia may be nonmotile or motile, the latter having additional specific roles in cell or fluid movement. The term flagellum refers to the motile cilium of free-living single cells (e.g. bacteria, archaea, spermatozoa, and protozoa). In eukaryotes, both nonmotile and motile cilia possess sensory functions. The ciliary interior (cilioplasm) is separated from the cytoplasm by a selective barrier that prevents passive diffusion of molecules between the two domains. The sensory functions of cilia reside largely in the membrane and signals generated in the cilium are transduced into a variety of cellular responses. In this review we discuss the structure and biogenesis of the cilium, with special attention to the trypanosome flagellar membrane, its lipid and protein composition and its proposed roles in sensing and signaling.

Molecular mechanisms of protein and lipid targeting to ciliary membranes.

Cilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and differentiation. The discovery that a number of human diseases, collectively known as ciliopathies, result from defective cilium function has expanded interest in these structures. Among the many properties of cilia, motility and intraflagellar transport have been most extensively studied. The latter is the process by which multiprotein complexes associate with microtubule motors to transport structural subunits along the axoneme to and from the ciliary tip. By contrast, the mechanisms by which membrane proteins and lipids are specifically targeted to the cilium are still largely unknown. In this Commentary, we review the current knowledge of protein and lipid targeting to ciliary membranes and outline important issues for future study. We also integrate this information into a proposed model of how the cell specifically targets proteins and lipids to the specialized membrane of this unique organelle.

Anthropological aspect of death in dialyzed patients.

The aim of this article is to compare the incidence of thanatophobia in dialysed patients having Balkan endemic nephropathy (BEN) with a control group (N18) members where some of them have chronic renal failure (CRF), but not (BEN). We examined thanatophobia on a sample of 753 dialysed patients with chronic renal failure (CRF) in Bosnia and Herzegovina (B&H) during the period from 1st January 2000 to 31st December 2006. The first group is a cohort consisted of 348 patients with Balkan endemic nephropathy (BEN), and the control group consisted of 405 randomly selected patients with different diagnoses of CRF (N18). The measurement instruments used were: General data list, Eysenck's Personality Questionnaire (EPQ), Beck's Anxiety Inventory (BAI), Hamilton's Depression Rating Scale (HDRS), and Mini-Mental State Examination (MMSE). Univariante and multivariante statistical analyses were carried out. From the multivariante analysis, the highest correlations with thanatophobia were found in these variables: avoidance of dialysis in BEN group: R=0.985, OR=0.358, CI=0.483-0.728 (95%), and in control group: R=0.550, OR=0.935, CI=0.615-0.830 (95%), age, years on dialysis, education, pervasive fear with statistical significance P=0.001. BEN group differentiates from control group: BAI-total (R=1.110, OR=0.578 (95%), CI=0.770-0.890, P=0.001), HDRS-total (R=0.995, OR=1.290 (95%), CI=1.180-1.920 P=0.001. BEN group have lower scores than the control group in MMSE-total: (R=0.430, OR=0.023 (95%), CI=0.034-2.850, P=0.001) which represents the organic part of anxiety. Thanatophobia is present in both groups, but it is more frequent in the BEN (11.70%) than in control group (7.50%). We found that thanatophobia occurs before dialysis, and that it is structured as a pervasive fear of death and is associated with endemia, years spent on dialysis, and avoidance of dialysis.

[Thanatophobia in the patients on dialysis].

BACKGROUND/AIM: Thanatophobia is an exaggerated, specific, structured fear of death. It appears in childhood and continues to grow over the years, and in the old age it is accompanied with nosophobia and other mental disorders. The aim of this study was to analyze thanatophobia in dialysed patients which was in the direct connection with a basic disorder, and the influence of this disorder on functioning and the quality of life of the patients on dialysis. METHODS: In the study we examined 753 patients from the chronic program of haemodialysis in a period from 1999 to 2004. The patiens were classified in two groups: 348 randomized patients with Balkan Endemic Nephropathy (BEN), and the control group (N18) of patients with terminal renal insufficiency, and other diagnoses (n = 405). Since the study was a comparative, cross-sectional one, the patients were tested by the appropriate questionnaires for anxiety, depression and general mental functioning. Statistical analysis was done by the standard descriptive and analytic statistic methods. RESULTS: Based on socio-demographics data we revealed a highly significant difference regarding the place of living between the groups BEN and N18 (chi2 = 23.970; p < 0.01), the frequency of occurrence of renal comorbidity (chi2 = 23.970; p < 0.01), the frequency of familly renal comorbidity in siblings (chi2 = 23.970; p < 0.01), and the frequency of migrationes (chi2 = 4.874; p < 0.01). According to psychiatry scales, the patiens from the BEN group were significantly more anxious and depressive than those from the control group. CONCLUSION: The signs of thanatophobia were revealed in both examined groups, but significantly more in the patients with BEN than in those with other nephrologic diseases. Thanatophobia starts before dialysis, and dialysis structures it into fear of death which is in a direct connection with the basic disorder. This intensive fear may be connected with dementia and depression, but also with other mental disorders.

An atomic mutation cycle for exploring RNA's 2'-hydroxyl group.

The 2'-hydroxyl group fulfills numerous structural and functional roles in RNA, including those of hydrogen bond donor and acceptor. While loss of function upon 2'-deoxynucleotide substitution establishes the importance of specific 2'-hydroxyl groups within RNA, this approach provides no information about how these hydroxyl groups impart their functional contribution. We use an atomic mutation cycle to evaluate the functional importance of the 2'-hydroxyl group's hydrogen atom. Using the Tetrahymena ribozyme reaction, we challenge the cycle to expose the catalytic contribution of the cleavage site 2'-hydroxyl group and its associated hydrogen bond network. The results establish the viability of this cycle as an approach to reveal 2'-hydroxyl groups that donate functionally significant hydrogen bonds.