Microhabitats associated with solar energy development alter demography of two desert annuals.

Political and economic initiatives intended to increase energy production while reducing carbon emissions are driving demand for solar energy. Consequently, desert regions are now targeted for development of large-scale photovoltaic solar energy facilities. Where vegetation communities are left intact or restored within facilities, ground-mounted infrastructure may have negative impacts on desert-adapted plants because it creates novel rainfall runoff and shade conditions. We used experimental solar arrays in the Mojave Desert to test how these altered conditions affect population dynamics for a closely related pair of native annual plants: rare Eriophyllum mohavense and common E. wallacei. We estimated aboveground demographic rates (seedling emergence, survivorship, and fecundity) over 7 yr and used seed bank survival rates from a concurrent study to build matrix models of population growth in three experimental microhabitats. In drier years, shade tended to reduce survival of the common species, but increase survival of the rare species. In a wet year, runoff from panels tended to increase seed output for both species. Population growth projections from microhabitat-specific matrix models showed stronger effects of microhabitat under wetter conditions, and relatively little effect under dry conditions (lack of rainfall was an overwhelming constraint). Performance patterns across microhabitats in the wettest year differed between rare and common species. Projected growth of E. mohavense was substantially reduced in shade, mediated by negative effects on aboveground demographic rates. Hence, the rare species were more susceptible to negative effects of panel infrastructure in wet years that are critical to seed bank replenishment. Our results suggest that altered shade and water runoff regimes associated with energy infrastructure will have differential effects on demographic transitions across annual species and drive population-level processes that determine local abundance, resilience, and persistence.

Top-down and sideways: Herbivory and cross-ecosystem connectivity shape restoration success at the salt marsh-upland ecotone.

Wetland restoration provides remarkable opportunities to understand vegetation dynamics and to inform success of future projects through rigorous restoration experiments. Salt marsh restoration typically focuses on physical factors such as sediment dynamics and elevation. Despite many demonstrations of strong top-down effects on salt marshes, the potential for consumers to affect salt marsh restoration projects has rarely been quantified. Recently, major restoration projects at the Elkhorn Slough National Estuarine Research Reserve in central California, USA provided an opportunity to examine how herbivory influences restoration success. We quantified the strength of consumer effects by comparing caged to uncaged plantings, and compared effects among plant species and sites. We used camera traps to detect which herbivores were most common and how their abundance varied spatially. Beyond characterizing consumer effects, we also tested management strategies for reducing negative effects of herbivory at the restoration sites, including caging, mowing, and acoustic playbacks of predator sounds. We found extremely strong consumer effects at sites with extensive stands of exotic forbs upland of the high marsh; uncaged restoration plants suffered heavy herbivory and high mortality, while most caged plants survived. Brush rabbits (Sylvilagus bachmani) were by far the most frequent consumers of these high marsh plants. Our work thus provides the first evidence of mammal consumers affecting salt marsh restoration success. Mowing of tall exotic forb cover adjacent to the marsh at one restoration site greatly reduced consumption, and nearly all monitored plantings survived at a second restoration site where construction had temporarily eliminated upland cover. Playbacks of predator sounds did not significantly affect restoration plantings, but restoration efforts in marsh communities vulnerable to terrestrial herbivory may benefit from concurrent restoration of predator communities in the upland habitats surrounding the marsh. A landscape approach is thus critical for recognizing linkages between terrestrial and marine vegetation.

Simulated Photovoltaic Solar Panels Alter the Seed Bank Survival of Two Desert Annual Plant Species.

Seed bank survival underpins plant population persistence but studies on seed bank trait-environment interactions are few. Changes in environmental conditions relevant to seed banks occur in desert ecosystems owing to solar energy development. We developed a conceptual model of seed bank survival to complement methodologies using in-situ seed bank packets. Using this framework, we quantified the seed bank survival of two closely related annual desert plant species, one rare (Eriophyllum mohavense) and one common (Eriophyllum wallacei), and the seed bank-environment interactions of these two species in the Mojave Desert within a system that emulates microhabitat variation associated with solar energy development. We tracked 4860 seeds buried across 540 seed packets and found, averaged across both species, that seed bank survival was 21% and 6% for the first and second growing seasons, respectively. After two growing seasons, the rare annual had a significantly greater seed bank survival (10%) than the common annual (2%). Seed bank survival across both species was significantly greater in shade (10%) microhabitats compared to runoff (5%) and control microhabitats (3%). Our study proffers insight into this early life-stage across rare and common congeners and their environmental interactions using a novel conceptual framework for seed bank survival.

Comparative Assessment of Small and Large Intestine Biopsies for Ex Vivo HIV-1 Pathogenesis Studies.

Ex vivo mucosal explants have become a mainstay of HIV-1 studies using human tissue. In this study, we examine the baseline phenotypic and virologic differences between biopsies derived from the small intestine (SI) and large intestine (LI) for use in ex vivo explant studies. To do this, we collected endoscopic mucosal biopsies from both SI and LI from the same healthy, HIV-seronegative participants. Mucosal mononuclear cell phenotypes and quantity were compared using flow cytometry. Comparative HIV-1 infectibility of the explants was assessed using an ex vivo explant HIV-1 infection assay. We found that all biopsies had similar numbers of T cells per biopsy. While the percentage of CD4+ T cells from SI biopsies expressed significantly more activation markers (CD38, HLA-DR) and HIV coreceptors (CXCR4, CCR5), the absolute numbers of activated CD4+ T cells were similar between both sites. LI explants, however, supported more efficient HIV-1 infection, as evidenced by earlier rise in p24 accumulation and greater percent of infected explants at limiting infectious doses. These results suggest that explants from LI biopsies support more efficient HIV-1 infection than SI biopsies, despite similar numbers of available, activated HIV-1 target cells. These findings highlight important differences in LI and SI explants, which must be considered in designing and interpreting ex vivo HIV-1 infection studies, and suggest that factors within the tissue other than target cell number and activation state may play a role in regulating HIV-1 infection.

Human Semen or Seminal Plasma Does Not Enhance HIV-1BaL Ex Vivo Infection of Human Colonic Explants.

To determine whether human whole semen (WS) and seminal plasma (SP) either previously frozen or freshly acquired altered ex vivo infectibility of human colonic explants or was associated with histology or toxicity changes, which may influence mucosal HIV-1 transmission in vivo. Pooled human semen samples were freshly obtained from study volunteers (never frozen) and from commercial sources (frozen/thawed). Endoscopically acquired rectal biopsies were evaluated for toxicity following titered ex vivo WS/SP exposure by histological grading and by MTT assay. The ex vivo HIV-1 biopsy challenge model was used to evaluate effects of exposure to either previously frozen or freshly acquired WS/SP on HIVBaL infectibility at a range of viral inocula (104-100 TCID50). To evaluate the effects at lower viral inocula of HIV-1 (10-2-102), experiments in the presence or absence of WS/SP were also performed utilizing TZM-bl cells. MTT assays and histological scoring demonstrated no tissue degradation of biopsies when exposed for 2 h to concentrations of 10% or 100% of either fresh or previously frozen WS/SP. Ex vivo biopsy HIV-1 challenge experiments showed no differences in the presence of freshly acquired or previously frozen/thawed WS/SP compared with control; no differences were seen with lower infectious titers on TZM-bl cells. Within the limits of assay sensitivity and variability, these data show no toxicity or significant enhancement of HIV-1 infectibility of human rectal mucosa using the colorectal explant model with either pooled fresh or frozen/thawed nonautologous human semen.

Analytical Advances in the Ex Vivo Challenge Efficacy Assay.

The ex vivo challenge assay is being increasingly used as an efficacy endpoint during early human clinical trials of HIV prevention treatments. There is no standard methodology for the ex vivo challenge assay, although the use of different data collection methods and analytical parameters may impact results and reduce the comparability of findings between trials. In this analysis, we describe the impact of data imputation methods, kit type, testing schedule and tissue type on variability, statistical power, and ex vivo HIV growth kinetics. Data were p24 antigen (pg/ml) measurements collected from clinical trials of candidate microbicides where rectal (n = 502), cervical (n = 88), and vaginal (n = 110) tissues were challenged with HIV-1BaL ex vivo. Imputation of missing data using a nonlinear mixed effect model was found to provide an improved fit compared to imputation using half the limit of detection. The rectal virus growth period was found to be earlier and of a relatively shorter duration than the growth period for cervical and vaginal tissue types. On average, only four rectal tissue challenge assays in each treatment and control group would be needed to find a one log difference in p24 to be significant (alpha = 0.05), but a larger sample size was predicted to be needed for either cervical (n = 21) or vaginal (n = 10) tissue comparisons. Overall, the results indicated that improvements could be made in the design and analysis of the ex vivo challenge assay to provide a more standardized and powerful assay to compare efficacy of microbicide products.

A multi-compartment single and multiple dose pharmacokinetic comparison of rectally applied tenofovir 1% gel and oral tenofovir disoproxil fumarate.

This Phase 1, randomized, two-site (United States), double-blind, placebo-controlled study enrolled 18 sexually abstinent men and women. All received a single 300-mg dose of oral tenofovir disoproxil fumarate (TDF) and were then randomized 2:1 to receive single and then seven daily rectal exposures of vaginally-formulated tenofovir (TFV) 1% gel or a hydroxyethyl cellulose (HEC) placebo gel. Blood, colonic biopsies and rectal and vaginal mucosal fluids were collected after the single oral TDF, the single topical TFV gel dose, and after 7 days of topical TFV gel dosing for extracellular analysis of TFV and intracellular analysis of the active metabolite tenofovir diphosphate (TFVdp) in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMC), including CD4+ and CD4- cell subsets. With a single rectal dose, TFV plasma concentrations were 24­33 fold lower and half-life was 5 h shorter compared to a single oral dose (p = 0.02). TFVdp concentrations were also undetectable in PBMCs with rectal dosing. Rectal tissue exposure to both TFV and TFVdp was 2 to 4-log10 higher after a single rectal dose compared to a single oral dose, and after 7 daily doses, TFVdp accumulated 4.5 fold in tissue. TFVdp in rectal tissue homogenate was predictive (residual standard error, RSE = 0.47) of tissue MMC intracellular TFVdp concentration, with the CD4+ cells having a 2-fold higher TFVdp concentration than CD4- cells. TFV concentrations from rectal sponges was a modest surrogate indicator for both rectal tissue TFV and TFVdp (RSE = 0.67, 0.66, respectively) and plasma TFV (RSE = 0.38). TFV penetrates into the vaginal cavity after oral and rectal dosing, with rectal dosing leading to higher vaginal TFV concentrations (p<0.01). Trial registration: ClinicalTrials.gov NCT00984971.

Correlation between compartmental tenofovir concentrations and an ex vivo rectal biopsy model of tissue infectibility in the RMP-02/MTN-006 phase 1 study.

OBJECTIVES: This study was designed to assess the dose-response relationship between tissue, blood, vaginal and rectal compartment concentrations of tenofovir (TFV) and tenofovir diphosphate (TFVdp) and ex vivo rectal HIV suppression following oral tenofovir disoproxil fumarate (TDF) and rectal administration of TFV 1% vaginally-formulated gel. DESIGN: Phase 1, randomized, two-site (US), double-blind, placebo-controlled study of sexually-abstinent males and females. METHODS: Eighteen participants received a single 300 mg exposure of oral TDF and were then randomized 2∶1 to receive a single then seven-daily rectal exposures of TFV 1% gel (40 mg TFV per 4 ml gel application) or hydroxyethyl-cellulose (HEC) placebo gel. Blood and rectal biopsies were collected for pharmacokinetic TDF and TFVdp analyses and ex vivo HIV-1 challenge. RESULTS: There was a significant fit for the TFVdp dose-response model for rectal tissue (p = 0.0004), CD4+MMC (p<0.0001), CD4-MMC (p<0.0001), and TotalMMC (p<0.0001) compartments with r2 ranging 0.36-0.64. Higher concentrations of TFVdp corresponded with lower p24, consistent with drug-mediated virus suppression. The single oral treatment failed to provide adequate compartment drug exposure to reach the EC50 of rectal tissue TFVdp predicted to be necessary to suppress HIV in rectal tissue. The EC50 for CD4+MMC was within the single topical treatment range, providing evidence that a 1% topical, vaginally-formulated TFV gel provided in-vivo doses predicted to provide for 50% efficacy in the ex vivo assay. The 7-daily topical TFV gel treatment provided TFVdp concentrations that reached EC90 biopsy efficacy for CD4-MMC, CD4+MMC and TotalMMC compartments. CONCLUSION: The TFVdp MMC compartment (CD4+, CD4- and Total) provided the best surrogate for biopsy infectibility and the 7-daily topical TFV gel treatment provided the strongest PK profile for HIV suppression. ClinicalTrials.gov NCT00984971.

Seminal plasma HIV-1 RNA concentration is strongly associated with altered levels of seminal plasma interferon-γ, interleukin-17, and interleukin-5.

Seminal plasma HIV-1 RNA level is an important determinant of the risk of HIV-1 sexual transmission. We investigated potential associations between seminal plasma cytokine levels and viral concentration in the seminal plasma of HIV-1-infected men. This was a prospective, observational study of paired blood and semen samples from 18 HIV-1 chronically infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood plasma were measured and analyzed using simple linear regressions to screen for associations between cytokines and seminal plasma HIV-1 levels. Forward stepwise regression was performed to construct the final multivariate model. The median HIV-1 RNA concentrations were 4.42 log10 copies/ml (IQR 2.98, 4.70) and 2.96 log10 copies/ml (IQR 2, 4.18) in blood and seminal plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 RNA level (p<0.0001) was most strongly associated with seminal plasma HIV-1 RNA level. After controlling for blood HIV-1 RNA level, seminal plasma HIV-1 RNA level was positively associated with interferon (IFN)-γ (p=0.03) and interleukin (IL)-17 (p=0.03) and negatively associated with IL-5 (p=0.0007) in seminal plasma. In addition to blood HIV-1 RNA level, cytokine profiles in the male genital tract are associated with HIV-1 RNA levels in semen. The Th1 and Th17 cytokines IFN-γ and IL-17 are associated with increased seminal plasma HIV-1 RNA, while the Th2 cytokine IL-5 is associated with decreased seminal plasma HIV-1 RNA. These results support the importance of genital tract immunomodulation in HIV-1 transmission.

Antigen-presenting cell candidates for HIV-1 transmission in human distal colonic mucosa defined by CD207 dendritic cells and CD209 macrophages.

A common route for HIV-1 infection is sexual transmission across colorectal mucosa, which is thought to be 10-2,000 times more vulnerable to infection than that of the female genital tract. Mucosal surfaces are the first line of defense against many pathogens but the antigen-presenting cells (APCs), key regulators of innate immunity and determinants of adaptive immunity, are not well defined in these target tissues. Using immunohistochemistry, dendritic cells expressing Langerin (CD207(+)), a lectin known to bind and internalize HIV-1, were detected in the periphery of colonic glands and sparsely scattered in the submucosa similarly in colorectal mucosa. This cell type, well known in skin, has generally not been reported in colonic/rectal mucosa. Unexpectedly, the largest APC population observed was a macrophage-like population expressing the well-characterized tissue macrophage markers CD68 and CD163. Confocal microscopy of these cells revealed colocalization of CD209 (DC-SIGN), a presumed dendritic cell marker believed to facilitate HIV-1 transmission, but not other dendritic cell markers. These results show evidence of the unconfirmed presence of Langerhans cells in colorectal mucosa and a predominance of macrophage-like APCs that express CD209 (DC-SIGN). These findings define potential target cells in the pathogenesis of HIV-1 transmission, which may have key implications for the study of early transmission events in normal colorectal mucosa, as well as other infectious diseases and primary immune diseases involving the gut.

Nonreproducibility of "snap-frozen" rectal biopsies for later use in ex vivo explant infectibility studies.

Sexual transmission accounts for the majority of new HIV infections worldwide with sexually exposed cervicovaginal and colorectal mucosae being primary sites of infection. Two recent Phase 1 rectal microbicide trials included, as an ancillary endpoint, suppression of ex vivo HIV infection of in vivo microbicide-exposed rectal mucosal tissue biopsies. Both trials demonstrated significant suppression of biopsy infectibility in drug-exposed versus placebo-exposed tissue. This potential early biomarker of efficacy has raised the feasibility of utilizing "snap-frozen" tissue samples, acquired at multiple trial sites to be shipped for central processing, providing a mechanism to correlate tissue drug concentrations with a functional index of HIV prevention. While previous reports have indicated acceptable comparability of fresh versus freeze-thawed cervicovaginal tissue samples, no similar evaluations with colorectal tissue biopsies have been done. In this study, rectal biopsies from healthy, HIV-seronegative participants were assessed for structural integrity (histology), viability (MTT assays), and tissue infectibility to compare results from fresh versus combinations of freeze/thaw protocols. Results indicated that while all protocols showed equivalent viability with fresh samples (MTT), histology documented poor preservation of tissue integrity following freezing. Infectibility results from freeze-thawed colorectal tissue were markedly lower (usually<25% of fresh samples) and varied greatly and unpredictably. Centralized colorectal tissue infectibility assays using biopsies from remote trial sites cannot currently be supported under these protocols.

First phase 1 double-blind, placebo-controlled, randomized rectal microbicide trial using UC781 gel with a novel index of ex vivo efficacy.

OBJECTIVES: Successful control of the HIV/AIDS pandemic requires reduction of HIV-1 transmission at sexually-exposed mucosae. No prevention studies of the higher-risk rectal compartment exist. We report the first-in-field Phase 1 trial of a rectally-applied, vaginally-formulated microbicide gel with the RT-inhibitor UC781 measuring clinical and mucosal safety, acceptability and plasma drug levels. A first-in-Phase 1 assessment of preliminary pharmacodynamics was included by measuring changes in ex vivo HIV-1 suppression in rectal biopsy tissue after exposure to product in vivo. METHODS: HIV-1 seronegative, sexually-abstinent men and women (N = 36) were randomized in a double-blind, placebo-controlled trial comparing UC781 gel at two concentrations (0.1%, 0.25%) with placebo gel (1∶1∶1). Baseline, single-dose exposure and a separate, 7-day at-home dosing were assessed. Safety and acceptability were primary endpoints. Changes in colorectal mucosal markers and UC781 plasma drug levels were secondary endpoints; ex vivo biopsy infectibility was an ancillary endpoint. RESULTS: All 36 subjects enrolled completed the 7-14 week trial (100% retention) including 3 flexible sigmoidoscopies, each with 28 biopsies (14 at 10 cm; 14 at 30 cm). There were 81 Grade 1 adverse events (AEs) and 8 Grade 2; no Grade 3, 4 or procedure-related AEs were reported. Acceptability was high, including likelihood of future use. No changes in mucosal immunoinflammatory markers were identified. Plasma levels of UC781 were not detected. Ex vivo infection of biopsies using two titers of HIV-1(BaL) showed marked suppression of p24 in tissues exposed in vivo to 0.25% UC781; strong trends of suppression were seen with the lower 0.1% UC781 concentration. CONCLUSIONS: Single and 7-day topical rectal exposure to both concentrations of UC781 were safe with no significant AEs, high acceptability, no detected plasma drug levels and no significant mucosal changes. Ex vivo biopsy infections demonstrated marked suppression of HIV infectibility, identifying a potential early biomarker of efficacy. (Registered at ClinicalTrials.gov; #NCT00408538).

Characterization of baseline intestinal mucosal indices of injury and inflammation in men for use in rectal microbicide trials (HIV Prevention Trials Network-056).

OBJECTIVES: The purpose of this study was to evaluate the biologic stability of mucosal parameters that might be used as endpoints in phase 1 rectal safety studies. METHODS: Sixteen male participants were enrolled into 4 groups defined by HIV status, viral load, and sexual activity. Each participant underwent 3 flexible sigmoidoscopies at 2-week intervals with collection of blood, intestinal biopsies, and rectal secretions. Intestinal histology, phenotypic characterization of mucosal mononuclear cells, cytokine messenger RNA (mRNA) profiles (RANTES, interferon-gamma [IFNgamma], and interleukin-10), and immunoglobulin secretion were assessed. Intraclass correlation (ICC) was calculated to assess endpoint stability. RESULTS: Qualitative histology demonstrated minimal inflammation in >95% of biopsies and remained stable throughout the study period. ICC for the tissue cytokine mRNA measurements and several T-cell phenotypic markers was >0.7, indicating stability over time. Mucosal CD4 lymphopenia was seen in the HIV-positive participants and was more pronounced in those with higher viral loads. Modest differences were observed for cytokine expression (IFNgamma) and T-cell phenotype (CD3, CD4, CD8, CD19, CD4/CCR5, and CD4/CD38) between the tissue samples collected at 10 and 30 cm. CONCLUSIONS: These data help to provide a rationale for the selection of endpoints for future phase 1 rectal safety studies.