Infection of an HIV-1/SIVmac Chimeric Virus Having HIV-1 env to Macaque Monkeys via the Vaginal Cavity

We previously reported that an HIV-1/SIVmac chimeric virus (designated as NM-3rN) having HIV-1 env efficiently infected macaque monkeys by intravenous inoculation. In this study, this chimeric virus was atraumatically inoculated into the vaginal cavity of two rhesus and one cynomolgus monkeys. Although antibody response and detection of proviral genome by PCR were observed in both rhesus monkeys, virus recovery was only once from PBMC in one of them. In the cynomolgus monkey, no virus was recovered and proviral DNA detection was rare. Thus, vaginal inoculation with NM-3rN resulted in poor systemic infection, implying the presence of selective pressure while passing through mucosal membranes.     

MiniCD4 Microbicide Prevents HIV Infection of Human Mucosal Explants and Vaginal Transmission of SHIV162P3 in Cynomolgus Macaques

In complement to an effective vaccine, development of potent anti-HIV microbicides remains an important priority. We have previously shown that the miniCD4 M48U1, a functional mimetic of sCD4 presented on a 27 amino-acid stable scaffold, inhibits a broad range of HIV-1 isolates at sub-nanomolar concentrations in cellular models. Here, we report that M48U1 inhibits efficiently HIV-1_Ba-L in human mucosal explants of cervical and colorectal tissues. In vivo efficacy of M48U1 was evaluated in nonhuman primate (NHP) model of mucosal challenge with SHIV_162P3 after assessing pharmacokinetics and pharmacodynamics of a miniCD4 gel formulation in sexually matured female cynomolgus macaques. Among 12 females, half were treated with hydroxyethylcellulose-based gel (control), the other half received the same gel containing 3 mg/g of M48U1, one hour before vaginal route challenge with 10 AID₅₀ of SHIV_162P3. All control animals were infected with a peak plasma viral load of 10⁵–10⁶ viral RNA (vRNA) copies per mL. In animals treated with miniCD4, 5 out of 6 were fully protected from acquisition of infection, as assessed by qRT-PCR for vRNA detection in plasma, qPCR for viral DNA detection in PBMC and lymph node cells. The only infected animal in this group had a delayed peak of viremia of one week. These results demonstrate that M48U1 miniCD4 acts in vivo as a potent entry inhibitor, which may be considered in microbicide developments.     

Zinc Inhibits High Glucose-Induced Apoptosis in Peritoneal Mesothelial Cells

Zinc (Zn) plays an important role in influencing many types of apoptosis. However, its function in apoptosis in peritoneal mesothelial cells (PMCs) remains unknown. Here, we studied the effects of Zn on high glucose (HG)-induced apoptosis in rat PMCs (RPMCs) and examined the underlying molecular mechanisms. We found that Zn supplementation inhibited HG-induced RPMC apoptosis significantly, by attenuating reactive oxygen species (ROS) production, inhibiting HG-induced sFasR and sFasL over-expression, caspase-8 and caspase-3 activation, and inhibiting release of cytochrome c from mitochondria to the cytosol. Further analysis revealed that Zn supplementation facilitated cell survival through activation of the phosphatidylinositol 3-kinase/Akt signaling pathway and MAPK/ERK pathways. These results indicate that Zn can inhibit apoptosis in HG-induced RPMCs by several independent mechanisms, including an indirect antioxidative effect and probably by inhibition of caspase-8 and caspase-3 activation.     

Zinc Supplementation Inhibits Complement Activation in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. AMD is a multifactorial disorder but complement-mediated inflammation at the level of the retina plays a pivotal role. Oral zinc supplementation can reduce the progression of AMD but the precise mechanism of this protective effect is as yet unclear. We investigated whether zinc supplementation directly affects the degree of complement activation in AMD and whether there is a relation between serum complement catabolism during zinc administration and the complement factor H (CFH) gene or the Age-Related Maculopathy susceptibility 2 (ARMS2) genotype. In this open-label clinical study, 72 randomly selected AMD patients in various stages of AMD received a daily supplement of 50 mg zinc sulphate and 1 mg cupric sulphate for three months. Serum complement catabolism–defined as the C3d/C3 ratio–was measured at baseline, throughout the three months of supplementation and after discontinuation of zinc administration. Additionally, downstream inhibition of complement catabolism was evaluated by measurement of anaphylatoxin C5a. Furthermore, we investigated the effect of zinc on complement activation in vitro. AMD patients with high levels of complement catabolism at baseline exhibited a steeper decline in serum complement activation (p<0.001) during the three month zinc supplementation period compared to patients with low complement levels. There was no significant association of change in complement catabolism and CFH and ARMS2 genotype. In vitro zinc sulphate directly inhibits complement catabolism in hemolytic assays and membrane attack complex (MAC) deposition on RPE cells. This study provides evidence that daily administration of 50 mg zinc sulphate can inhibit complement catabolism in AMD patients with increased complement activation. This could explain part of the mechanism by which zinc slows AMD progression.

Trial Registration

The Netherlands National Trial Register NTR2605     

Effectiveness of Cellulose Sulfate Vaginal Gel for the Prevention of HIV Infection: Results of a Phase III Trial in Nigeria

Background

This trial evaluated the safety and effectiveness of 6% cellulose sulfate vaginal gel in preventing male-to-female vaginal transmission of HIV, gonorrhea and chlamydial infection.

Methods

This Phase III, double-blind, randomized, placebo-controlled trial was conducted between November 2004 and March 2007 in Lagos and Port Harcourt, Nigeria. We enrolled 1644 HIV-antibody negative women at high risk of HIV acquisition. Study participants were randomized 1∶1 to cellulose sulfate or placebo and asked to use gel plus a condom for each act of vaginal intercourse over one year of follow-up. The participants were evaluated monthly for HIV, gonorrhea and chlamydial infection, and for adverse events.

Results

The trial was stopped prematurely after the data safety monitoring board of a parallel trial concluded that cellulose sulfate might be increasing the risk of HIV. In contrast, we observed fewer infections in the active arm (10) than on placebo (13), a difference that was nonetheless not statistically significant (HR = 0.8, 95% CI 0.3–1.8; p = 0.56). Rates of gonorrhea and chlamydial infection were lower in the CS group but the difference was likewise not statistically significant (HR = 0.8, 95% CI 0.5–1.1; p = 0.19 for the combined STI outcome). Rates of adverse events were similar across study arms. No serious adverse events related to cellulose sulfate use were reported.

Conclusions

Cellulose sulfate gel appeared to be safe in the evaluated study population but we found insufficient evidence that it prevented male-to-female vaginal transmission of HIV, gonorrhea or chlamydial infection. The early closure of the trial compromised the ability to draw definitive conclusions about the effectiveness of cellulose sulfate against HIV.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00120770","term_id":"NCT00120770"}}NCT00120770     

Water-Filtered Infrared A Irradiation in Combination with Visible Light Inhibits Acute Chlamydial Infection

New therapeutic strategies are needed to overcome drawbacks in treatment of infections with intracellular bacteria. Chlamydiaceae are Gram-negative bacteria implicated in acute and chronic diseases such as abortion in animals and trachoma in humans. Water-filtered infrared A (wIRA) is short wavelength infrared radiation with a spectrum ranging from 780 to 1400 nm. In clinical settings, wIRA alone and in combination with visible light (VIS) has proven its efficacy in acute and chronic wound healing processes. This is the first study to demonstrate that wIRA irradiation combined with VIS (wIRA/VIS) diminishes recovery of infectious elementary bodies (EBs) of both intra- and extracellular Chlamydia (C.) in two different cell lines (Vero, HeLa) regardless of the chlamydial strain (C. pecorum, C. trachomatis serovar E) as shown by indirect immunofluorescence and titration by subpassage. Moreover, a single exposure to wIRA/VIS at 40 hours post infection (hpi) led to a significant reduction of C. pecorum inclusion frequency in Vero cells and C. trachomatis in HeLa cells, respectively. A triple dose of irradiation (24, 36, 40 hpi) during the course of C. trachomatis infection further reduced chlamydial inclusion frequency in HeLa cells without inducing the chlamydial persistence/stress response, as ascertained by electron microscopy. Irradiation of host cells (HeLa, Vero) neither affected cell viability nor induced any molecular markers of cytotoxicity as investigated by Alamar blue assay and Western blot analysis. Chlamydial infection, irradiation, and the combination of both showed a similar release pattern of a subset of pro-inflammatory cytokines (MIF/GIF, Serpin E1, RANTES, IL-6, IL-8) and chemokines (IL-16, IP-10, ENA-78, MIG, MIP-1α/β) from host cells. Initial investigation into the mechanism indicated possible thermal effects on Chlamydia due to irradiation. In summary, we demonstrate a non-chemical reduction of chlamydial infection using the combination of water-filtered infrared A and visible light.     

A p7 Ion Channel-derived Peptide Inhibits Hepatitis C Virus Infection in Vitro

Viral infection is an early stage of its life cycle and represents a promising target for antiviral drug development. Here we designed and characterized three peptide inhibitors of hepatitis C virus (HCV) infection based on the structural features of the membrane-associated p7 polypeptide of HCV. The three peptides exhibited low toxicity and high stability while potently inhibiting initial HCV infection and suppressed established HCV infection at non-cytotoxic concentrations in vitro. The most efficient peptide (designated H2-3), which is derived from the H2 helical region of HCV p7 ion channel, inhibited HCV infection by inactivating both intracellular and extracellular viral particles. The H2-3 peptide inactivated free HCV with an EC₅₀ (50% effective concentration) of 82.11 nm, which is >1000-fold lower than the CC₅₀ (50% cytotoxic concentration) of Huh7.5.1 cells. H2-3 peptide also bound to cell membrane and protected host cells from viral infection. The peptide H2-3 did not alter the normal electrophysiological profile of the p7 ion channel or block viral release from Huh7.5.1 cells. Our work highlights a new anti-viral peptide design strategy based on ion channel, giving the possibility that ion channels are potential resources to generate antiviral peptides.     

RNA特异腺苷脱氨酶1(p150亚型)抑制肝细胞脂质合成

目的:在油酸诱导的肝细胞脂肪变模型中,检测RNA特异腺苷脱氨酶1 p150亚型(ADAR1-p150)高表达细胞系中脂肪合成的变化。方法:利用本课题组前期摸索的油酸刺激人胚胎肝细胞L-02细胞系脂肪变的条件,q RT-PCR和Western-Blot检测油酸刺激组和对照组ADAR1-p150表达变化;将构建成功的ADAR1-p150过表达慢病毒载体GV166-ADAR1-p150及空载体病毒GV166-control感染L-02细胞,检测感染细胞中ADAR1-p150的m RNA和蛋白表达水平;通过油红O染色和BODIPY染色观察L-02 ADAR1-p150和L-02 control细胞中脂滴形成,并进一步利用高内涵系统检测其荧光强度,对脂滴合成作定量分析。结果:L-02细胞在油酸刺激后ADAR1-p150的m RNA和蛋白水平降低;成功构建ADAR1-p150过表达慢病毒载体GV166-ADAR1-p150及空载体病毒GV166-control,q RT-PCR及Western-Blot检测显示病毒转染GV166-ADAR1-p150后ADAR1-p150在细胞中的表达水平显著升高;油红O染色和BODIPY染色发现L-02 ADAR1-p150较L-02 control细胞胞质中脂滴数量减少。高内涵筛选系统检测提示L-02 ADAR1-p150组中脂滴的荧光强度明显较L-02 control组低。结论:成功构建ADAR1-p150过表达稳定转染L-02细胞系,并证实高表达ADAR1-p150能够抑制脂肪合成。     

MTN-001: Randomized Pharmacokinetic Cross-Over Study Comparing Tenofovir Vaginal Gel and Oral Tablets in Vaginal Tissue and Other Compartments

Background

Oral and vaginal preparations of tenofovir as pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) infection have demonstrated variable efficacy in men and women prompting assessment of variation in drug concentration as an explanation. Knowledge of tenofovir concentration and its active form, tenofovir diphosphate, at the putative vaginal and rectal site of action and its relationship to concentrations at multiple other anatomic locations may provide key information for both interpreting PrEP study outcomes and planning future PrEP drug development.

Objective

MTN-001 was designed to directly compare oral to vaginal steady-state tenofovir pharmacokinetics in blood, vaginal tissue, and vaginal and rectal fluid in a paired cross-over design.

Methods and Findings

We enrolled 144 HIV-uninfected women at 4 US and 3 African clinical research sites in an open label, 3-period crossover study of three different daily tenofovir regimens, each for 6 weeks (oral 300 mg tenofovir disoproxil fumarate, vaginal 1% tenofovir gel [40 mg], or both). Serum concentrations after vaginal dosing were 56-fold lower than after oral dosing (p<0.001). Vaginal tissue tenofovir diphosphate was quantifiable in ≥90% of women with vaginal dosing and only 19% of women with oral dosing. Vaginal tissue tenofovir diphosphate was ≥130-fold higher with vaginal compared to oral dosing (p<0.001). Rectal fluid tenofovir concentrations in vaginal dosing periods were higher than concentrations measured in the oral only dosing period (p<0.03).

Conclusions

Compared to oral dosing, vaginal dosing achieved much lower serum concentrations and much higher vaginal tissue concentrations. Even allowing for 100-fold concentration differences due to poor adherence or less frequent prescribed dosing, vaginal dosing of tenofovir should provide higher active site concentrations and theoretically greater PrEP efficacy than oral dosing; randomized topical dosing PrEP trials to the contrary indicates that factors beyond tenofovir’s antiviral effect substantially influence PrEP efficacy.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00592124","term_id":"NCT00592124"}}NCT00592124     

Amblyomma maculatum Feeding Augments Rickettsia parkeri Infection in a Rhesus Macaque Model: A Pilot Study

Rickettsia parkeri is an emerging eschar-causing human pathogen in the spotted fever group of Rickettsia and is transmitted by the Gulf coast tick, Amblyomma maculatum. Tick saliva has been shown to alter both the cellular and humoral components of the innate and adaptive immune systems. However, the effect of this immunomodulation on Rickettsia transmission and pathology in an immunocompetent vertebrate host has not been fully examined. We hypothesize that, by modifying the host immune response, tick feeding enhances infection and pathology of pathogenic spotted fever group Rickettsia sp. In order to assess this interaction in vivo, a pilot study was conducted using five rhesus macaques that were divided into three groups. One group was intradermally inoculated with low passage R. parkeri (Portsmouth strain) alone (n = 2) and another group was inoculated during infestation by adult, R. parkeri-free A. maculatum (n = 2). The final macaque was infested with ticks alone (tick feeding control group). Blood, lymph node and skin biopsies were collected at several time points post-inoculation/infestation to assess pathology and quantify rickettsial DNA. As opposed to the tick-only animal, all Rickettsia-inoculated macaques developed inflammatory leukograms, elevated C-reactive protein concentrations, and elevated TH1 (interferon-γ, interleukin-15) and acute phase inflammatory cytokines (interleukin-6) post-inoculation, with greater neutrophilia and interleukin-6 concentrations in the tick plus R. parkeri group. While eschars formed at all R. parkeri inoculation sites, larger and slower healing eschars were observed in the tick feeding plus R. parkeri group. Furthermore, dissemination of R. parkeri to draining lymph nodes early in infection and increased persistence at the inoculation site were observed in the tick plus R. parkeri group. This study indicates that rhesus macaques can be used to model R. parkeri rickettsiosis, and suggests that immunomodulatory factors introduced during tick feeding may enhance the pathogenicity of spotted fever group Rickettsia.     

A Peptide of Heparin Cofactor II Inhibits Endotoxin-Mediated Shock and Invasive Pseudomonas aeruginosa Infection

Sepsis and septic shock remain important medical problems with high mortality rates. Today''s treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII) has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn²⁺ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn²⁺ and Cd²⁺ chelator with capacity to bind 10 Zn²⁺ ions per C terminus. When AtHMA4 is expressed in a Zn²⁺-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn²⁺ and Cd²⁺ tolerance and lowered Zn²⁺ and Cd²⁺ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn²⁺ and Cd²⁺ chelator (sensor) and as a regulator of the efficiency of Zn²⁺ and Cd²⁺ export. The identification of a post-translational handle on Zn²⁺ and Cd²⁺ transport efficiency opens new perspectives for regulation of Zn²⁺ nutrition and tolerance in eukaryotes.     

Roflumilast Inhibits Respiratory Syncytial Virus Infection in Human Differentiated Bronchial Epithelial Cells

Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H₂O₂ and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H₂O₂ formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus.