Michael A. Kolber

Norepinephrine and GTP-γ-S increase myofilament Ca2+ sensitivity in α-toxin permeabilized arterial smooth muscle

Summary A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using α-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After α-toxin treatment the fibers developed tension as a function of Ca 2+ concentration (EC 50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5×10 −7 M Ca 2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca 2+ solution and a transient and maintained contraction in 5×10 −7 M Ca 2+ solution. GTP-γ-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca 2+ and maintained tension revealed that NE and GTP-γ-S cause increases in Ca 2+ sensitivity of myofilament shifting the EC 50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-γ-S causes an increase in myofilament Ca 2+ sensitivity and that G protein may be involved in receptor signal transduction system. α-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.

Preexposure Prophylaxis for HIV Infection Integrated With Municipal- and Community-Based Sexual Health Services

IMPORTANCE Several randomized clinical trials have demonstrated the efficacy of preexposure prophylaxis (PrEP) in preventing human immunodeficiency virus (HIV) acquisition. Little is known about adherence to the regimen, sexual practices, and overall effectiveness when PrEP is implemented in clinics that treat sexually transmitted infections (STIs) and community-based clinics serving men who have sex with men (MSM). OBJECTIVE To assess PrEP adherence, sexual behaviors, and the incidence of STIs and HIV infection in a cohort of MSM and transgender women initiating PrEP in the United States. DESIGN, SETTING, AND PARTICIPANTS Demonstration project conducted from October 1, 2012, through February 10, 2015 (last date of follow-up), among 557 MSM and transgender women in 2 STI clinics in San Francisco, California, and Miami, Florida, and a community health center in Washington, DC. Data were analyzed from December 18, 2014, through August 8, 2015. INTERVENTIONS A combination of daily, oral tenofovir disoproxil fumarate and emtricitabine was provided free of charge for 48 weeks. All participants received HIV testing, brief client-centered counseling, and clinical monitoring. MAIN OUTCOMES AND MEASURES Concentrations of tenofovir diphosphate in dried blood spot samples, self-reported numbers of anal sex partners and episodes of condomless receptive anal sex, and incidence of STI and HIV acquisition. RESULTS Overall, 557 participants initiated PrEP, and 437 of these (78.5%) were retained through 48 weeks. Based on the findings from the 294 participants who underwent measurement of tenofovir diphosphate levels, 80.0% to 85.6% had protective levels (consistent with ≥4 doses/wk) at follow-up visits. African American participants (56.8% of visits; P = .003) and those from the Miami site (65.1% of visits; P < .001) were less likely to have protective levels, whereas those with stable housing (86.8%; P = .02) and those reporting at least 2 condomless anal sex partners in the past 3 months (88.6%; P = .01) were more likely to have protective levels. The mean number of anal sex partners declined during follow-up from 10.9 to 9.3, whereas the proportion engaging in condomless receptive anal sex remained stable at 65.5% to 65.6%. Overall STI incidence was high (90 per 100 person-years) but did not increase over time. Two individuals became HIV infected during follow-up (HIV incidence, 0.43 [95% CI, 0.05-1.54] infections per 100 person-years); both had tenofovir diphosphate levels consistent with fewer than 2 doses/wk at seroconversion. CONCLUSIONS AND RELEVANCE The incidence of HIV acquisition was extremely low despite a high incidence of STIs in a large US PrEP demonstration project. Adherence was higher among those participants who reported more risk behaviors. Interventions that address racial and geographic disparities and housing instability may increase the impact of PrEP.

Cytochalasin B induces cellular DNA fragmentation.

Cellular DNA fragmentation can be induced in many biological instances without plasma membrane damage. The fungal metabolite, cytochalasin B, is capable of modifying numerous cellular functions related to DNA synthesis. In this work it is demonstrated that cytochalasin B is capable of inducing DNA fragmentation in a number of cells lines. This DNA fragmentation occurs before plasma membrane lysis and over a period of hours. Cytochalasin E and villin, agents that act on the microfilaments, also induce DNA fragmentation. Phorbol dibutyrate, a diacylglyceral analog, is able to inhibit cytochalasin B‐induced DNA fragmentation in a dose‐dependent fashion. These findings support the interpretation that cytochalasin B is inducing DNA fragmentation via its effect on the actin filaments.— Kolber, M. A., Broschat, K. O., Landa‐Gonzalez, B. Cytochalasin B induces cellular DNA fragmentation. FASEB J. 4: 3021‐3027; 1990.

High interest in preexposure prophylaxis among men who have sex with men at risk for HIV infection: baseline data from the US PrEP demonstration project.

Background:Preexposure prophylaxis (PrEP) is the first biomedical intervention with proven efficacy to reduce HIV acquisition in men who have sex with men (MSM) and transgender women. Little is known about levels of interest and characteristics of individuals who elect to take PrEP in real-world clinical settings. Methods:The US PrEP Demonstration Project is a prospective open-label cohort study assessing PrEP delivery in municipal sexually transmitted disease clinics in San Francisco and Miami and a community health center in Washington, DC. HIV-uninfected MSM and transgender women seeking sexual health services at participating clinics were assessed for eligibility and offered up to 48 weeks of emtricitabine/tenofovir for PrEP. Predictors of enrollment were assessed using a multivariable Poisson regression model, and characteristics of enrolled participants are described. Results:Of 1069 clients assessed for participation, 921 were potentially eligible and 557 (60.5%) enrolled. In multivariable analyses, participants from Miami (adjusted Relative Risk [aRR]: 1.53; 95% confidence interval [CI]: 1.33 to 1.75) or DC (aRR: 1.33; 95% CI: 1.2 to 1.47), those who were self-referred (aRR: 1.48; 95% CI: 1.32 to 1.66), those with previous PrEP awareness (aRR: 1.56; 95% CI: 1.05 to 2.33), and those reporting >1 episode of anal sex with an HIV-infected partner in the last 12 months (aRR: 1.20; 95% CI: 1.09 to 1.33) were more likely to enroll. Almost all (98%) enrolled participants were MSM, and at baseline, 63.5% reported condomless receptive anal sex in the previous 3 months. Conclusions:Interest in PrEP is high among a diverse population of MSM at risk for HIV infection when offered in sexually transmitted disease and community health clinics.

Hantavirus pulmonary syndrome in Florida: Association with the newly identified Black Creek Canal virus

Hantavirus pulmonary syndrome (HPS) is a recently recognized viral zoonosis. The first recognized cases were caused by a newly described hantavirus. Sin Nombre virus (previously known as Muerto Canyon virus), isolated from Peromyscus maniculatus (deer mouse). We describe a 33-year-old Floridian man who resided outside the ecologic range of P maniculatus but was found to have serologic evidence of a hantavirus infection during evaluation of azotemia associated with adult respiratory distress syndrome. Small mammal trapping conducted around this patients residence demonstrated the presence of antihantaviral antibodies in 13% of Sigmodon hispidus [cotton rat). Serologic testing using antigen derived from the Black Creek Canal hantavirus subsequently isolated from this rodent established that this patient was acutely infected with this new pathogenic American hantavirus. HPS is not confined to the geographical distribution of P maniculatus and should be suspected in individuals with febrile respiratory syndromes, perhaps associated with azotemia, throughout the continental United States.

Evidence for a role of phosphatidyl ethanolamine as a modulator of membrane-membrane contact.

SummaryPhosphatidyl ethanolamine (PE) is shown to be effective in producing membrane aggregation. The aggregation of PE and PE/PC (phosphatidyl choline) mixed vesicles was studied as a function of pH and cation composition of the medium. The kinetics and equilibria were studied in stopped-flow rapid mixing experiments, in which PE vesicles prepared at pH 9.2 were “jumped” to pH 7.H+ ions protonate PE− and promote vesicle aggregation in a cooperative fashion. Vesicles containing PC have a decreased tendency to aggregate compared to pure PE vesicles. The apparent rate constant for aggregation was about two orders of magnitude below that for diffusion controlled aggregation and was virtually the same for PE and PE/PC mixed vesicles.A theoretical description of equilibrium for vesicle aggregation is developed in terms of three parameters: the equilibrium constant for the protonation of PE (KA), the equilibrium constant for aggregation (Keq) and the number of PE molecules in an effective area that the two vesicles must interact in order to aggregate (Neff). These parameters are compared with values and trends expected for electrostatic calculations based on dipolar repulsion and short-range binding, to which hydrogen bonding may contribute. The results are interpreted in a self-consistent fashion to indicate: (i) that PE and PC mix randomly, (ii) that head-to-tail binding occurs between PE(PC) molecules on apposing vesicles, (iii) that electrostatic screening accounts for the decrease inKA as a function of the molar fraction of PC per vesicle, (iv) that the PE must be 90% protonated before aggregation can occur, and (v) that for all the lipid systems we considered, the point at which the extent of dimerization is half maximal is close to the physiological pH, indicating that PE may have a regulatory effect in the aggregation of biological systems.

IL-21 augments natural killer effector functions in chronically HIV-infected individuals

Objective:This study addresses the interleukin (IL)-21 effects on resting peripheral blood natural killer (NK) cells in chronically HIV-infected individuals. Design:The effects of IL-21 on perforin expression, proliferation, degranulation, interferon (IFN)-γ production, cytotoxicity and induction of STAT phosphorylation in NK cells were determined in vitro. Methods:Peripheral blood mononuclear cells from HIV-infected and healthy individuals were incubated in vitro for 6 h, 24 h or 5 days with IL-21 or IL-15. Percentages of perforin, IFN-γ, CD107a, NKG2D and STAT3–5 positive cells were determined within NK cell populations. K562 cells were used as target cells in NK cytotoxicity assay. Results:Frequency of CD56dim cells in chronically HIV-infected individuals was diminished. Perforin expression in CD56dim and CD56bright was comparable in healthy and HIV-infected individuals. IL-15 upregulated perforin expression primarily in CD56bright NK cells, whereas IL-21 upregulated perforin in both NK subsets. IL-21 and IL-15 upregulated CD107a and IFN-γ, as well as NK cytotoxicity. IL-15 predominantly activated STAT5, whereas IL-21 activated STAT5 and STAT3. IL-15, but not IL-21 increased NK cell proliferation in uninfected and HIV-infected individuals. Conclusion:IL-21 augments NK effector functions in chronically HIV-infected individuals and due to its perforin enhancing properties it has potential for immunotherapy or as a vaccine adjuvant.

Genotypic analysis of plasma HIV-1 RNA after influenza vaccination of patients with previously undetectable viral loads

Objective In this study we evaluated the possibility that plasma viral load elevations secondary to influenza vaccination in HIV-1-seropositive individuals with previously undetectable viral loads (< 200 copies/ml) could develop resistance-bearing mutations in the viral reverse transcriptase (RT) and protease regions. Methods Thirty-four patients with undetectable viral burdens on highly active antiretroviral therapy (HAART) were evaluated for elevations in plasma viral load 2 and 4 weeks post-influenza vaccination. Plasma from patients whose viral load increased after vaccination was subject to genotypic resistance analysis by the line probe assay (LiPA) to determine whether primary resistance-bearing mutations developed during this period and at follow-up. Stored plasma was used to evaluate whether RT or protease mutations existed pre-vaccination. Results Seven out of 34 patients were found to experience elevations in their viral load after influenza vaccination. Two of the patients revealed evidence of primary RT or protease mutations not demonstrated in earlier pre-vaccination samples. One patient failed therapy after vaccination, and one patient revealed post-vaccination viral load elevations that eventually led to the progressive development of primary zidovudine mutations. Conclusion Evidence is presented that supports the contention that a small subset of patients who experience viral load elevations after influenza vaccination can develop mutational changes in the RT region of the viral genome either acutely or after a failure of the viral load to return to undetectable levels.

Cutting edge: novel vaccination modality provides significant protection against mucosal infection by highly pathogenic simian immunodeficiency virus.

Vaccine-induced protection against infection by HIV or highly pathogenic and virulent SIV strains has been limited. In a proof-of-concept study, we show that a novel vaccine approach significantly protects rhesus macaques from mucosal infection by the highly pathogenic strain SIVmac251. We vaccinated three cohorts of 12 macaques each with live, irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig. Cohort 1 was vaccinated with cells secreting gp96SIVIg carrying SIV peptides. In addition, Cohort 2 received recombinant envelope protein SIV-gp120. Cohort 3 was injected with cells secreting gp96-Ig (no SIV Ags) vaccines. Cohort 2 was protected from infection. After seven rectal challenges with highly pathogenic SIVmac251, the hazard ratio was 0.27, corresponding to a highly significant, 73% reduced risk for viral acquisition. The apparent success of the novel vaccine modality recommends further study.

Gp96SIVIg immunization induces potent polyepitope specific, multifunctional memory responses in rectal and vaginal mucosa

The ER-resident chaperone gp96, when released by cell lysis, induces an immunogenic chemokine signature and causes innate immune activation of DC and NK cells. Here we show that intraperitoneal immunization with a genetically engineered, secreted form of gp96, gp96-Ig chaperoning SIV antigens, induces high levels of antigen specific CD8 CTL in the rectal and vaginal mucosa of Rhesus macaques. The frequency of SIV Gag- and SIV Tat-tetramer positive CD8 CTL in the intestinal mucosa reached 30-50% after the third immunization. Tetramer positive CD8 CTL expressed appropriate functional (granzyme B) and migration markers (CD103). The polyepitope specificity of the mucosal CD8 and CD4 response is evident from a strong, multifunctional cytokine response upon stimulation with peptides covering the gag, tat and env proteins. Induction of powerful mucosal effector CD8 CTL responses by cell-based gp96(SIV)-Ig immunization may provide a pathway to the development of safe and effective SIV/HIV vaccines.