Results of a phase 1, randomized, placebo-controlled first-in-human trial of griffithsin formulated in a carrageenan vaginal gel.

HIV pre-exposure prophylaxis (PrEP) is dominated by clinical therapeutic antiretroviral (ARV) drugs. Griffithsin (GRFT) is a non-ARV lectin with potent anti-HIV activity. GRFT's preclinical safety, lack of systemic absorption after vaginal administration in animal studies, and lack of cross-resistance with existing ARV drugs prompted its development for topical HIV PrEP. We investigated safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of PC-6500 (0.1% GRFT in a carrageenan (CG) gel) in healthy women after vaginal administration. This randomized, placebo-controlled, parallel group, double-blind first-in-human phase 1 study enrolled healthy, HIV-negative, non-pregnant women aged 24-45 years. In the open label period, all participants (n = 7) received single dose of PC-6500. In the randomized period, participants (n = 13) were instructed to self-administer 14 doses of PC-6500 or its matching CG placebo (PC-535) once daily for 14 days. The primary outcomes were safety and PK after single dose, and then after 14 days of dosing. Exploratory outcomes were GRFT concentrations in cervicovaginal fluids, PD, inflammatory mediators and gene expression in ectocervical biopsies. This trial is registered with ClinicalTrials.gov, number NCT02875119. No significant adverse events were recorded in clinical or laboratory results or histopathological evaluations in cervicovaginal mucosa, and no anti-drug (GRFT) antibodies were detected in serum. No cervicovaginal proinflammatory responses and no changes in the ectocervical transcriptome were evident. Decreased levels of proinflammatory chemokines (CXCL8, CCL5 and CCL20) were observed. GRFT was not detected in plasma. GRFT and GRFT/CG in cervicovaginal lavage samples inhibited HIV and HPV, respectively, in vitro in a dose-dependent fashion. These data suggest GRFT formulated in a CG gel is a safe and promising on-demand multipurpose prevention technology product that warrants further investigation.

Blocking α4ß7 integrin delays viral rebound in SHIVSF162P3-infected macaques treated with anti-HIV broadly neutralizing antibodies.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4ß7 with an anti-α4ß7 monoclonal antibody (Rh-α4ß7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4ß7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4ß7 or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α4ß7 and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α4ß7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

Delayed vaginal SHIV infection in VRC01 and anti-α4ß7 treated rhesus macaques.

VRC01 protects macaques from vaginal SHIV infection after a single high-dose challenge. Infusion of a simianized anti-α4ß7 mAb (Rh-α4ß7) just prior to, and during repeated vaginal exposures to SIVmac251 partially protected macaques from vaginal SIV infection and rescued CD4+ T cells. To investigate the impact of combining VRC01 and Rh-α4ß7 on SHIV infection, 3 groups of macaques were treated with a suboptimal dosing of VRC01 alone or in combination with Rh-α4ß7 or with control antibodies prior to the initiation of weekly vaginal exposures to a high dose (1000 TCID50) of SHIVAD8-EO. The combination Rh-α4ß7-VRC01 significantly delayed SHIVAD8-EO vaginal infection. Following infection, VRC01-Rh-α4ß7-treated macaques maintained higher CD4+ T cell counts and exhibited lower rectal SIV-DNA loads compared to controls. Interestingly, VRC01-Rh-α4ß7-treated macaques had fewer IL-17-producing cells in the blood and the gut during the acute phase of infection. Moreover, higher T cell responses to the V2-loop of the SHIVAD8-EO envelope in the VRC01-Rh-α4ß7 group inversely correlated with set point viremia. The combination of suboptimal amounts of VRC01 and Rh-α4ß7 delayed infection, altered antiviral immune responses and minimized CD4+ T cell loss. Further exploration of the effect of combining bNAbs with Rh-α4ß7 on SIV/HIV infection and antiviral immune responses is warranted and may lead to novel preventive and therapeutic strategies.

Mucosal Susceptibility to Human Immunodeficiency Virus Infection in the Proliferative and Secretory Phases of the Menstrual Cycle.

Factors underlying HIV acquisition in women remain incompletely understood. This study evaluated ex vivo mucosal HIV-1BaL infection (ectocervix, endocervix), T cell frequencies and phenotype (ectocervix, endocervix, peripheral blood), and HIV-1BaL-induced tissue immune responses (ectocervix) in the proliferative and secretory phases of the menstrual cycle using samples obtained from women undergoing hysterectomies. Tissue infectivity (number of productively infected explants) and infection level following 500 and/or fifty 50% tissue culture infectious dose (TCID50) HIV-1BaL challenge were similar in the proliferative and secretory phases. Although not associated with infection outcomes, higher frequencies of HIV target CD4+α4ß7+ T cells, and stronger HIV-1BaL-induced proinflammatory responses were detected in ectocervix in the secretory versus proliferative phase. Independently of the cycle phase, serum E2 concentrations were inversely associated with ectocervical and endocervical tissue infection levels following high-dose 500 TCID50 HIV-1BaL challenge, with frequencies of CD4+α4ß7+ T cells in endocervix, and with HIV-induced interleukin (IL)2R and IL4 in ectocervix. Although serum P4 concentrations and P4/E2 ratios were neither associated with tissue infection level nor infectivity, high P4 concentrations and/or P4/E2 ratios correlated with high frequencies of CD4+α4ß7+ T cells in ectocervix, low frequencies of CD4+CD103+ blood T cells, low CD4+LFA-1+ T cells in endocervix, and high proinflammatory (IL1ß, IL17, tumor necrosis factor α) ectocervical tissue responses to HIV-1BaL. The data suggest an inhibitory effect of E2 on mucosal HIV infection, provide insights into potential mechanisms of E2-mediated anti-HIV activity, and highlight P4-associated immune changes in the mucosa.

Integrin α4ß7 Blockade Preferentially Impacts CCR6+ Lymphocyte Subsets in Blood and Mucosal Tissues of Naive Rhesus Macaques.

Infusion of a simianized anti-α4ß7 mAb (Rh-α4ß7) just before and following SIV infection protected rhesus macaques from developing AIDS and partially from vaginal SIV acquisition. Recently, short-term treatment with Rh-α4ß7 in combination with cART was found to lead to prolonged viral suppression after withdrawal of all therapeutic interventions. The humanized form of Rh-α4ß7, vedolizumab, is a highly effective treatment for inflammatory bowel disease. To clarify the mechanism of action of Rh-α4ß7, naive macaques were infused with Rh-α4ß7 and sampled in blood and tissues before and after treatment to monitor several immune cell subsets. In blood, Rh-α4ß7 increased the CD4+ and CD8+ T cell counts, but not B cell counts, and preferentially increased CCR6+ subsets while decreasing CD103+ and CD69+ lymphocytes. In mucosal tissues, surprisingly, Rh-α4ß7 did not impact integrin α4+ cells, but decreased the frequencies of CCR6+ and CD69+ CD4+ T cells and, in the gut, Rh-α4ß7 transiently decreased the frequency of memory and IgA+ B cells. In summary, even in the absence of inflammation, Rh-α4ß7 impacted selected immune cell subsets in different tissues. These data provide new insights into the mechanisms by which Rh-α4ß7 may mediate its effect in SIV-infected macaques with implications for understanding the effect of treatment with vedolizumab in patients with inflammatory bowel disease.

Experimental Oral Herpes Simplex Virus-1 (HSV-1) Co-infection in Simian Immunodeficiency Virus (SIV)-Infected Rhesus Macaques.

Herpes simplex virus 1 and 2 (HSV-1/2) similarly initiate infection in mucosal epithelia and establish lifelong neuronal latency. Anogenital HSV-2 infection augments the risk for sexual human immunodeficiency virus (HIV) transmission and is associated with higher HIV viral loads. However, whether oral HSV-1 infection contributes to oral HIV susceptibility, viremia, or oral complications of HIV infection is unknown. Appropriate non-human primate (NHP) models would facilitate this investigation, yet there are no published studies of HSV-1/SIV co-infection in NHPs. Thus, we performed a pilot study for an oral HSV-1 infection model in SIV-infected rhesus macaques to describe the feasibility of the modeling and resultant immunological changes. Three SIV-infected, clinically healthy macaques became HSV-1-infected by inoculation with 4 × 108 pfu HSV-1 McKrae on buccal, tongue, gingiva, and tonsils after gentle abrasion. HSV-1 DNA was shed in oral swabs for up to 21 days, and shedding recurred in association with intra-oral lesions after periods of no shedding during 56 days of follow up. HSV-1 DNA was detected in explant cultures of trigeminal ganglia collected at euthanasia on day 56. In the macaque with lowest baseline SIV viremia, SIV plasma RNA increased following HSV-1 infection. One macaque exhibited an acute pro-inflammatory response, and all three animals experienced T cell activation and mobilization in blood. However, T cell and antibody responses to HSV-1 were low and atypical. Through rigorous assessesments, this study finds that the virulent HSV-1 strain McKrae resulted in a low level HSV-1 infection that elicited modest immune responses and transiently modulated SIV infection.

MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal Mucosa and SHIV-RT in Rectal Mucosa.

The Population Council's microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad-spectrum microbicide against HIV, herpes simplex virus (HSV), and human papillomavirus. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection, and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and human papillomavirus in murine models. Recently, we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 coinfection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC, and compared quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay readouts for monitoring SHIV-RT infection. MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosas and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by 1-step simian immunodeficiency virus gag quantitative RT-PCR and p27 enzyme-linked immunosorbent assay demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of quantitative RT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

A Novel Microbicide/Contraceptive Intravaginal Ring Protects Macaque Genital Mucosa against SHIV-RT Infection Ex Vivo.

Women need multipurpose prevention products (MPTs) that protect against sexually transmitted infections (STIs) and provide contraception. The Population Council has developed a prototype intravaginal ring (IVR) releasing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (M), zinc acetate (ZA), carrageenan (CG) and levonorgestrel (LNG) (MZCL IVR) to protect against HIV, HSV-2, HPV and unintended pregnancy. Our objective was to evaluate the anti-SHIV-RT activity of MZCL IVR in genital mucosa. First, macaque vaginal tissues were challenged with SHIV-RT in the presence of (i) MIV-150 ± LNG or (ii) vaginal fluids (VF); available from studies completed earlier) collected at various time points post insertion of MZCL and MZC IVRs. Then, (iii) MZCL IVRs (vs. LNG IVRs) were inserted in non-Depo Provera-treated macaques for 24h and VF, genital biopsies, and blood were collected and tissues were challenged with SHIV-RT. Infection was monitored with one step SIV gag qRT-PCR or p27 ELISA. MIV-150 (LCMS/MS, RIA), LNG (RIA) and CG (ELISA) were measured in different compartments. Log-normal generalized mixed linear models were used for analysis. LNG did not affect the anti-SHIV-RT activity of MIV-150 in vitro. MIV-150 in VF from MZC/MZCL IVR-treated macaques inhibited SHIV-RT in vaginal mucosa in a dose-dependent manner (p<0.05). MIV-150 in vaginal tissue from MZCL IVR-treated animals inhibited ex vivo infection relative to baseline (96%; p<0.0001) and post LNG IVR group (90%, p<0.001). No MIV-150 dose-dependent protection was observed, likely because of high MIV-150 concentrations in all vaginal tissue samples. In cervical tissue, MIV-150 inhibited infection vs. baseline (99%; p<0.05). No cervical tissue was available for MIV-150 measurement. Exposure to LNG IVR did not change tissue infection level. These observations support further development of MZCL IVR as a multipurpose prevention technology to improve women's sexual and reproductive health.

In Vitro Exposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa.

Our recent phase 1 trial demonstrated that PC-1005 gel containing 50 µM MIV-150, 14 mM zinc acetate dihydrate, and carrageenan (CG) applied daily vaginally for 14 days is safe and well tolerated. Importantly, cervicovaginal lavage fluid samples (CVLs) collected 4 or 24 h after the last gel application inhibited HIV-1 and human papillomavirus (HPV) in cell-based assays in a dose-dependent manner (MIV-150 for HIV-1 and CG for HPV). Herein we aimed to determine the anti-HIV and anti-herpes simplex virus 2 (anti-HSV-2) activity of PC-1005 in human cervical explants after in vitro exposure to the gel and to CVLs from participants in the phase 1 trial. Single HIV-1BaL infection and HIV-1BaL-HSV-2 coinfection explant models were utilized. Coinfection with HSV-2 enhanced tissue HIV-1BaL infection. In vitro exposure to PC-1005 protected cervical mucosa against HIV-1BaL (up to a 1:300 dilution) in single-challenge and cochallenge models. CG gel (PC-525) provided some barrier effect against HIV-1BaL at the 1:100 dilution in a single-challenge model but not in the cochallenge model. Both PC-1005 and PC-525 at the 1:100 dilution inhibited HSV-2 infection, pointing to a CG-mediated protection. MIV-150 and CG in CVLs inhibited HIV (single-challenge or cochallenge models) and HSV-2 infections in explants in a dose-dependent manner (P < 0.05). Stronger inhibition of HIV-1 infection by CVLs collected 4 h after the last gel administration was observed compared to infection detected in the presence of baseline CVLs. The anti-HIV and anti-HSV-2 activity of PC-1005 gel in vitro and CVLs in human ectocervical explants supports the further development of PC-1005 gel as a broad-spectrum on-demand microbicide.

MIV-150/zinc acetate gel inhibits cell-associated simian-human immunodeficiency virus reverse transcriptase infection in a macaque vaginal explant model.

The transmission of both cell-free and cell-associated immunodeficiency viruses has been demonstrated directly in multiple animal species and possibly occurs in humans, as suggested by genotyping of the infecting human immunodeficiency virus (HIV) in acutely infected women and in semen from their partners. Therefore, a microbicide may need to block both mechanisms of HIV transmission to achieve maximum efficacy. To date, most of the preclinical evaluation of candidate microbicides has been performed using cell-free HIV. New models of mucosal transmission of cell-associated HIV are needed to evaluate candidate microbicide performance. The MIV-150/zinc acetate/carrageenan (MZC) gel protects Depo-Provera-treated macaques against cell-free simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection when applied vaginally up to 8 h before challenge. We recently demonstrated the potent activity of MZC gel against cell-free SHIV-RT in macaque vaginal explants. In the current study, we established a cell-associated SHIV-RT infection model of macaque vaginal tissues and tested the activity of MZC gel in this model. MZC gel protected tissues against cell-associated SHIV-RT infection when present at the time of viral exposure or when applied up to 4 days prior to viral challenge. These data support clinical testing of the MZC gel. Overall, our ex vivo model of cell-associated SHIV-RT infection in macaque vaginal mucosa complements the cell-free infection models, providing tools for prioritization of products that block both modes of HIV transmission.

HSV-2-driven increase in the expression of α4ß7 correlates with increased susceptibility to vaginal SHIV(SF162P3) infection.

The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α4ß7 is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α4ß7 and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIVSF162P3 infection, had higher frequency of α4ß7high CD4+ T cells in the vaginal tissue and higher expression of α4ß7 and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIVSF162P3. HSV-2 infection increased the frequencies of α4ß7high CD4+ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α4ß7high CD4+ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4+ T cells and DCs that express high levels of α4ß7 is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α4ß7 positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission.

A MIV-150/zinc acetate gel inhibits SHIV-RT infection in macaque vaginal explants.

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC's activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51-62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65-74%) did not significantly differ from CG (32-45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.

Simian immunodeficiency virus interactions with macaque dendritic cells.

This chapter summarizes advances in the following areas: (1) dendritic cell (DC)-mediated simian immunodeficiency virus (SIV) transmission, (2) role of DCs in innate and adaptive immunity against SIV, and (3) approaches to harness DC function to induce anti-SIV responses. The nonhuman primate (NHP) model of human immunodeficiency virus (HIV) infection in rhesus macaques and other Asian NHP species is highly relevant to advance the understanding of virus-host interactions critical for transmission and disease pathogenesis. HIV infection is associated with changes in frequency, phenotype, and function of the two principal subsets of DCs, myeloid DCs and plasmacytoid DCs. DC biology during pathogenic SIV infection is strikingly similar to that observed in HIV-infected patients. The NHP models provide an opportunity to dissect the requirements for DC-driven SIV infection and to understand how SIV distorts the DC system to its advantage. Furthermore, the SIV model of mucosal transmission enables the study of the earliest events of infection at the portal of entry that cannot be studied in humans, and, importantly, the involvement of DCs. Nonpathogenic infection in African NHP hosts allows investigations into the role of DCs in disease control. Understanding how DCs are altered during SIV infection is critical to the design of therapeutic and preventative strategies against HIV.

The nonnucleoside reverse transcription inhibitor MIV-160 delivered from an intravaginal ring, but not from a carrageenan gel, protects against simian/human immunodeficiency virus-RT Infection.

We previously showed that a carrageenan (CG) gel containing 50 µM MIV-150 (MIV-150/CG) reduced vaginal simian/human immunodeficiency virus (SHIV)-RT infection of macaques (56%, p>0.05) when administered daily for 2 weeks with the last dose given 8 h before challenge. Additionally, when 100 mg of MIV-150 was loaded into an intravaginal ring (IVR) inserted 24 h before challenge and removed 2 weeks after challenge, >80% protection was observed (p<0.03). MIV-160 is a related NNRTI with a similar IC(50), greater aqueous solubility, and a shorter synthesis. To objectively compare MIV-160 with MIV-150, herein we evaluated the antiviral effects of unformulated MIV-160 in vitro as well as the in vivo protection afforded by MIV-160 delivered in CG (MIV-160/CG gel) and in an IVR under regimens used with MIV-150 in earlier studies. Like MIV-150, MIV-160 exhibited potent antiviral activity against SHIV-RT in macaque vaginal explants. However, formulated MIV-160 exhibited divergent effects in vivo. The MIV-160/CG gel offered no protection compared to CG alone, whereas the MIV-160 IVRs protected significantly. Importantly, the results of in vitro release studies of the MIV-160/CG gel and the MIV-160 IVR suggested that in vivo efficacy paralleled the amount of MIV-160 released in vitro. Hundreds of micrograms of MIV-160 were released daily from IVRs while undetectable amounts of MIV-160 were released from the CG gel. Our findings highlight the importance of testing different modalities of microbicide delivery to identify the optimal formulation for efficacy in vivo.

Whole genome microarray of the major pelvic ganglion after cavernous nerve injury: new insights into molecular profile changes after nerve injury.

UNLABELLED: What's known on the subject? and What does the study add? With the present study, we aimed to provide a global picture of the molecular processes that are activated by CN injury. The present study used genomic expression profiling to identify candidate genes that might be useful targets in the CN recovery process and, thus, the ultimate preservation of penile erection. Regeneration of the CN and axonal outgrowth clearly involve changes in multiple biochemical pathways that have never been investigated by microarray analysis. We analyzed global gene expression in the major pelvic ganglion at early stages (48 h and 14 days) after CN injury and focused on the detection of changes in genes related to nervous tissue repair and proliferation. The findings of the present study provide important insight into the molecular systems affected by CN injury and identify candidate genes that may be utilized for novel molecular-based therapies for the preservation and protection of the CN during RP. OBJECTIVES: To to examine the complexity of the many molecular systems involved in supporting cavernous nerve (CN) repair and regeneration in a rat model of bilateral crush injury utilizing a microarray analysis approach. Erectile dysfunction (ED) is a common clinical complication after prostate cancer treatment by radical prostatectomy, and recovery of erectile function can take as long as 2 years. There are gaps in our understanding of the autonomic pelvic innervation of the penis that still need to be addressed for the development of an adequate treatment strategy for post-prostatectomy ED. The molecular mechanisms of the intrinsic ability of CN to regenerate after an injury have not been elucidated. MATERIALS AND METHODS: We analyzed global gene expression in the major pelvic ganglion 48 h and 14 days after CN injury. Overall, a comparative analysis showed that 325 genes changed at the 48-h time point and 114 genes changed at 14 days. There were 60 changed genes in common with both time points. Using the Ingenuity Pathway Analysis® system (Ingenuity Systems, Inc., Redwood City, CA, USA), we were able to analyze the significantly changed genes that were unique and common to each time point by biological function. We focused on the detection of changes related to nervous tissue repair and proliferation, molecular networks of neurotrophic factors, stem cell regulation and synaptic transmission. RESULTS: There was strong evidence of the early mobilization of genes involved in repair and neuroprotection mechanisms (SERPINF1, IGF1, PLAU/PLAUR, ARG1). Genes related to nervous system development (ATF3 GJA1, PLAU, SERPINE1), nerve regeneration (SERPINE2, IGF1, ATF3, ARG1) and synaptic transmission (GJC1, GAL) were changed. Several genes related to proliferation as well as apoptosis (A2M, ATF3, C3, EGR4, FN1, GJA1, GAL) were also changed, possibly as part of a protective mechanism or the initiation of remodelling. CONCLUSIONS: The results obtained show that multiple biological processes are associated with injury and repair of the CN and provide a systematic genome-wide screen for neurotrophic and/or inhibitory pathways of nerve regeneration. These data identify the candidate genes that may be utilized in novel molecular-based therapies for the preservation and protection of the CN during radical prostatectomy.

Reversal of diabetic vasculopathy in a rat model of type 1 diabetes by opiorphin-related peptides.

Diabetes results in a myriad of vascular complications, often referred to as diabetic vasculopathy, which encompasses both microvascular [erectile dysfunction (ED), retinopathy, neuropathy, and nephropathy] and macrovascular complications (hypertension, coronary heart disease, and myocardial infarction). In diabetic animals and patients with ED, there is decreased opiorphin or opiorphin-related gene expression in corporal tissue. Both opiorphin and the rat homologous peptide sialorphin are found circulating in the plasma. In the present study, we investigated if diabetes induced changes in plasma sialorphin levels and if changes in these levels could modulate the biochemistry and physiology of vascular smooth muscle. We show that circulating sialorphin levels are reduced in a rat model of type I diabetes. Intracorporal injection of plasmids expressing sialorphin into diabetic rats restores sialorphin levels to those seen in the blood of nondiabetic animals and results in both improved erectile function and blood pressure. Sialorphin modulated the ability of C-type natriuretic peptide to relax both corporal and aortic smooth muscle strips and of bradykinin to regulate intracellular calcium levels in both corporal and aortic smooth muscle cells. We have previously shown that expression of genes encoding opiorphins is increased when erectile function is improved. Our findings thus suggest that by affecting circulating levels of opiorphin-related peptides, proper erectile function is not only an indicator but also a modulator of overall vascular health of a man.

Silencing MaxiK activity in corporal smooth muscle cells initiates compensatory mechanisms to maintain calcium homeostasis.

INTRODUCTION: The MaxiK potassium channel is regulated by voltage and intracellular calcium, and plays a critical role in regulating intracellular calcium concentration ([Ca(2+) ](i)), which is the ultimate determinant of smooth muscle tone. Tight control of corpus cavernosum smooth muscle (CCSM) tone is critically important and misregulation can result in erectile dysfunction. AIM: Because of the tight functional linkage of MaxiK and calcium channel activity, the aim of this study was to determine the effects of silencing and pharmacological inhibition of MaxiK on calcium homeostasis and intercellular calcium signaling in CCSM cells. METHODS: We compared changes in the basal intracellular [Ca(2+) ](i) and parameters defining intercellular calcium wave (ICW) spread in 48 hours MaxiK silenced CCSM cells vs. acute blockade of the channel with iberiotoxin. To analyze changes occurring in gene expression we performed micro-array analysis following MaxiK silencing for 48 hours. MAIN OUTCOME MEASURES: Changes in Fura-2 fluorescence intensities were measured to evaluate basal [Ca(2+) ](i) levels and ICW parameters. Microarray analysis of global gene expression was performed. RESULTS: Forty-eight hours after MaxiK silencing the basal [Ca(2+) ](i) , the ICW amplitude and spread among CCSM cells were not markedly different in silenced compared to mock transfected controls, whereas short-term blockade significantly increased basal [Ca(2+) ](i) level and amplified Ca(2+) signaling among CCSM cells. Micro-array analysis showed that several genes within Ca(2+) homeostasis and smooth muscle tone regulation pathways had significantly altered expression. CONCLUSIONS: Our results indicate that while short-term blockade of the MaxiK channel is associated with an increase in basal [Ca(2+) ](i), Ca(2+) homeostasis is restored during the 48 hours period following silencing. We hypothesize that the different pathways regulating [Ca(2+) ](i) and CCSM tone are linked through molecular crosstalk and that their coordinated regulation is part of a compensatory mechanism aimed to maintain Ca(2+) homeostasis and CCSM tone.

Testosterone regulates erectile function and Vcsa1 expression in the corpora of rats.

Vcsa1 plays an important role in the erectile physiology of the rat. We conducted experiments to determine if erectile function, testosterone levels and Vcsa1 expression were correlated. In orchiectomized rats, total testosterone in blood fell from an average of 4 ng/ml to <0.04 ng/ml. Erectile function was significantly lower compared to controls and Vcsa1 expression was significantly (>6-fold) decreased. Injection of orchiectomized animals with testosterone (2 mg in 100ml sesame oil every 4 days for 2 weeks) restored average levels of testosterone to 2 ng/ml, increased erectile function and significantly increased Vcsa1 expression. In isolated corporal cells there was testosterone dependent Vcsa1 expression. However, intracorporal injection of orchiectomized animals with a plasmid expressing Vcsa1 or its gene product Sialorphin (previously demonstrated to improve erectile function in old animals) gave no significant improvement in erectile function. Also, the ability of Sialorphin to reduce tension in corporal smooth muscle strips isolated from orchiectomized animals was impaired compared to controls.

Vcsa1 acts as a marker of erectile function recovery after gene therapeutic and pharmacological interventions.

PURPOSE: We identified molecular markers of erectile function, particularly those responding to erectile dysfunction treatment. MATERIALS AND METHODS: Sprague-Dawley retired breeder rats were intracorporeally injected with pVAX-hSlo, pSMAA-hSlo or the control plasmid pVAX. One week later the intracorporeal pressure-to-blood pressure ratio and gene expression were determined by microarray analysis and quantitative reverse transcriptase-polymerase chain reaction. Rat corporeal cells were transfected in vitro with pVAX-hSlo, pSMAA-hSlo or pVAX and the change in gene expression was determined. We also determined whether Vcsa1 expression was changed after pharmacotherapy using tadalafil. RESULTS: Animals treated with vectors expressing hSlo had significantly improved erectile function compared to that in controls, accompanied by changed expression of a subset of genes. Vcsa1 was one of the genes that was most changed in expression (the third of approximately 31,000 with greater than 10-fold up-regulation). Changes in gene expression were different than those observed in corporeal cells transfected in vitro, distinguishing gene expression changes that were a direct effect of hSlo over expression. When tadalafil was administered in retired breeder rats, the Vcsa1 transcript increased 4-fold in corporeal tissue compared to that in untreated controls. CONCLUSIONS: Our study identifies a set of genes that are changed in response to improved erectile function, rather than as a direct effect of treatment. We noted Vcsa1 may act as marker of the restoration of erectile function after gene transfer and pharmacotherapy.

Smooth-muscle-specific gene transfer with the human maxi-k channel improves erectile function and enhances sexual behavior in atherosclerotic cynomolgus monkeys.

BACKGROUND: Despite the advent of effective oral therapies for erectile dysfunction (ED), many patients are not successfully treated, and side effects have been documented. OBJECTIVE: To further evaluate the potential utility of naked DNA-based gene transfer as an attractive treatment option for ED. DESIGN, SETTING AND PARTICIPANTS: The effects of gene transfer on erectile function and sexual behavior were evaluated in eight male cynomolgus monkeys with ED secondary to moderately severe, diet-induced atherosclerosis. INTERVENTION: Following establishment of baseline characteristics, animals were subjected to intracavernous injection of a smooth-muscle-specific gene transfer vector (pSMAA-hSlo) encoding the pore-forming subunit of the human large-conductance, calcium-sensitive potassium channel (Maxi-K). MEASUREMENTS: For the sexual behavior studies, 2 wk of baseline data were obtained, and then animals were placed in the presence of estrogen-implanted females (n=2) three times per week for 30 min, and sexual behavior was recorded. The intracavernous pressure response to papaverine injection was also monitored. RESULTS AND LIMITATIONS: Dramatic changes in erectile function and sexual behavior were observed after intracorporal gene transfer. The frequency of partial (6±2 to 10±2) and full (2±1.5 to 5±1.4) erections were significantly increased, with a parallel 2-3-fold increase in the duration of the observed erections. The frequency and latency of ejaculation were increased and decreased, respectively. Frequency and duration of grooming by the female were increased, and the latency decreased. Increased latency and decreased frequency of body contact was also observed, and this is characteristic of the typical drop in consort intimacy that occurs after mating in most macaque species. In addition, an increased responsiveness to intracavernous papaverine injection was observed. CONCLUSIONS: The data indicate that intracorporal Maxi-K-channel gene transfer enhances erectile capacity and sexual behavior; the data imply that increased erectile function per se may lead to increased sexual function.