Epidemiologic studies have suggested that the practice of receptive anal intercourse (RAI) is not confined to men who have sex with men (MSM). It seems that a significant group of women also practice RAI. Recent data from Kenya and the United States have documented a lifetime incidence of RAI of 35% to 40%.1,2 As a consequence, it is likely that vaginal microbicides may be used as sexual lubricants to facilitate RAI or in the hope of preventing HIV transmission. It is thus important to assess the rectal safety of vaginal microbicides as part of the development path for a candidate vaginal microbicide. In addition, similar studies are required as rectal specific microbicides are developed.
The first step in moving toward phase 1 rectal safety studies is to determine which endpoints should be used as indices to assess the rectal safety of candidate microbicides. Study endpoints might include symptom history, direct inspection of anorectal mucosal tissue (via anoscopy or endoscopy), histologic examination of rectal biopsies, or more complex assays of inflammation such as flow cytometric assessment of mucosal mononuclear cell (MMC) populations or tissue cytokine levels. An additional problem is that there may be regional heterogeneity in some or all the putative mucosal parameters. The anorectal tissue directly contacted during RAI (within 10 cm from the anal verge) might be quite different from those areas not directly traumatized but potentially in contact with seminal ejaculate (eg, proximal sigmoid colon).
Unfortunately, there are few data on the normal range of these parameters in individuals practicing RAI and essentially no data on the biologic stability of these endpoints over the short to medium term. Although some guidance can be acquired from the development of vaginal safety parameters,3 the compartments differ significantly, necessitating rectal-specific safety parameter development.
The purpose of this study was to conduct a detailed evaluation of the short-term stability of a range of mucosal parameters in rectal biopsies obtained from HIV-seronegative and -seropositive men who were or were not practicing RAI.
Study participants included 8 HIV-seronegative participants (4 who practiced RAI [group 1] and 4 who did not practice RAI [group 2]) and 8 HIV-1-seropositive participants (4 with a plasma RNA viral load of ≥10,000 copies/mL [group 3] and 4 with a plasma RNA viral load ≤50 copies/mL [group 4]). Inclusion criteria were (1) age ≥18 years; (2) for participants practicing RAI, an average of 1 to 2 episodes per week for the past 2 months; (3) for participants not practicing RAI, no episodes of RAI in the previous 2 months; (4) HIV status confirmed serologically; and (5) for HIV-seropositive participants, a plasma viral load ≥10,000 copies/mL (group 3) or ≤50 copies/mL (group 4) of HIV-1 RNA. Exclusion criteria included (1) a history of inflammatory bowel disease, rectal malignancy, or rectal surgery (including hemorrhoidectomy in the past 6 months); (2) a diagnosed bleeding disorder, including hemophilia and thrombocytopenia; (3) a prosthetic heart valve or diagnosis of a valvular disorder; (4) 3 or more outbreaks of perianal herpes simplex type 2 (HSV-2) in the previous 12 months or 1 or more HSV-2 outbreaks in the previous 6 months; (5) active diarrheal disease, aspirin, or nonsteroidal anti-inflammatory use; (6) positive rectal cultures for Chlamydia trachomatis or Neisseria gonorrhoeae at the time of screening; (7) parenteral antibiotics in the past 15 days; and (8) unprotected anal intercourse in the previous 3 months (groups 1 and 2). HIV-positive individuals and seronegative healthy controls were recruited through flyers and community referrals. All participants gave informed consent, and the Institutional Review Board at the David Geffen School of Medicine at UCLA (Los Angeles, CA) approved the research protocol.
Collection of Intestinal Tissue Samples
After a preparatory enema (Fleet enema; C.B. Fleet Company, Lynchburg, VA), rectosigmoid biopsies were collected endoscopically using a flexible sigmoidoscope as previously described.4,5 In brief, the rectosigmoid colon was sampled at 10 and 30 cm from the anal verge, with 10 biopsies acquired at each site (20 total biopsies). Endoscopy was performed on 3 occasions at 2-weekly intervals. Study participants kept sexual diaries, which allowed calculation of the approximate time between the last episode of RAI (groups 1, 3, and 4) and the endoscopy/biopsy collection performed at weeks 2 and 4. The mean time for the week 2 visit was 4 days (range: 0.5 to 7.5 days), and that for the week 4 visit was 3.6 days (range: 0.5 to 15.5 days).
Histopathologic scoring of inflammation was carried out on formalin-fixed paraffin-embedded colonic mucosal biopsy samples. A single pathologist (GC) with specialty training in gastrointestinal pathology performed the qualitative and quantitative histologic assessments. The pathologist was blinded to sample group.
Qualitative Assessment of Mucosal Inflammation
A qualitative score of chronic active inflammation was adapted from a validated colonic inflammation scoring system used in ulcerative colitis,6 as summarized in Table 1.
Quantitative Assessment of Mucosal Inflammation
Measurements were made on a representative sample of mucosa spanning 10 epithelial crypts. A manual count was made of all lamina propria lymphocytes, plasma cells, eosinophils, degranulating eosinophils, neutrophils, and intraepithelial lymphocytes. Lamina propria mononuclear cells lacking plasma cell differentiation or definitive monocyte/macrophage characteristics were counted as lymphocytes, and plasma cells were counted separately.
Mucosal Cytokine Messenger RNA
Cytokine messenger RNA (mRNA) for RANTES, interferon-γ (IFNγ), and interleukin-10 (IL-10) was measured in RNA extracted from endoscopic biopsies using a previously described technique.7 Briefly, primers optimized for real-time polymerase chain reaction (PCR) were designed using the complementary DNA (cDNA) sequences of the gene of interest from the Genbank database in conjunction with Primer Express software (Applied Biosystems, Foster City, CA). All assays were performed in triplicate, and a mean was calculated from the 3 measurements obtained. Results are reported as copies of cytokine mRNA standardized per 106 copies of β-actin.
Isolation of Mononuclear Cells
MMCs were isolated from intestinal biopsies using enzymatic digestion as previously published.8 Biopsies were incubated in 20 to 25 mL of RPMI/7.5% fetal calf serum containing 0.5 mg/mL of collagenase type II (Sigma-Aldrich, St. Louis, MO) for 30 minutes in a 37°C water bath, with intermittent shaking. The entire suspension was then passed through a sterile plastic strainer (Falcon 2350, Franklin Lakes, NJ) to remove free cells and concentrate the remaining tissue fragments. Free cells were immediately washed twice in medium to remove excess collagenase before being resuspended in 500 to 1000 μL of media and set aside on ice. Free cells were combined at the completion of the digestion, and the single-cell suspensions were then used for flow cytometric studies.
Monoclonal antibodies used included anti-CD4 and human leukocyte antigen D-related (HLA-DR)-fluorescein isothiocyanate (FITC); CD38-, CXCR4-, and CCR5-phycoerythrin (PE), CD4- and CD45-peridin chlorophyll protein (PER-cp), and CCR5 and CXCR4-allophycocyanin (APC). All monoclonal antibodies were supplied by BD Immunocytometry Systems (BDIS, Mountain View, CA). Analysis was carried out on a FACSCalibur flow cytometer (BDIS) with analysis using CellQuestPro software (BDIS) as previously described.4 Quadrant settings were determined by experience for well-defined populations, such as T-cell subsets. For those populations that were less well defined, such as CD38 and coreceptors, historical quadrant settings, which had been established using isotype controls, and consistent target voltages were used. Percentage values for the stained subsets were recorded.
Collection of Samples
Rectal secretions were collected using cellulose sponges (Ultracell Medical Technologies, North Stonington, CT) applied to the rectal wall via an anoscope. Sterile sponges were premoistened with 50 μL of phosphate-buffered saline (PBS; Gibco BRL, Gaithersburg, MD) and attached to a 2-mL plastic transfer pipette (Fisher Scientific, Pittsburgh, PA). The pipette and sponge were introduced into the rectum via the anoscope and held against the rectal mucosa under direct vision for 5 minutes. Samples were maintained on ice, transported back to the laboratory, and frozen at −80°C until batch processing was carried out. Samples with visible blood were discarded.
Processing of Samples
Sponges were thawed on ice, and the sponge tips were transferred to a 2-mL Spin-X column (Corning, Corning, NY) from which the acetate membrane had been removed. Absorbed rectal secretions were eluted twice with a total volume of 250 μL of cold elution buffer (PBS containing 0.25% bovine serum albumin [BSA]; Sigma Chemicals, St. Louis, MO), 1% Igepal (Sigma Chemicals), and 1× protease inhibitor cocktail (Sigma Chemicals) from the sponges by centrifugation (10,000 rpm for 30 minutes at 4°C). The recovered eluate was transferred to a preweighed 1.5-mL Eppendorf tube (Fisher Scientific, Pittsburgh, PA) and reweighed. The recovered volume of secretion was calculated by subtracting the recovered volume from that recovered from control sponges run in parallel. Duplicate samples were pooled, frozen, and retrieved in batches for further analysis.
Quantification of Immunoglobulins
Total IgG and total IgA were quantified in the eluted rectal secretions by enzyme-linked immunosorbent assay (ELISA).9,10 Samples were run in duplicate, along with a positive control sample, for which performance characteristics and acceptable ranges had been previously established. Values in (ng/mL) were extrapolated from the relevant standard curves, and the means were calculated for each sample. Quality control was ensured by the performance of the positive control.
Because the calculation of total immunoglobulin content in rectal secretion samples, based on the volume of sample retrieved, might be unreliable, we decided to quantify the total protein in each sample and compare its utility as an assay denominator. The Coomassie Dry Blue Protein Assay (Pierce, Rockford, IL) was performed in accordance with the manufacturer's instructions, and a standard curve, prepared from an albumin standard (Pierce) was used to extrapolate the quantity of total protein (μg/mL). For the purposes of analysis, the immunoglobulin results were expressed as (1) total immunoglobulin per sponge, (2) total immunoglobulin standardized by eluate volume, or (3) total immunoglobulin standardized per microgram of total protein in the sample.
Mixed linear models11 were used to analyze each of the parameters. Specifically, the data for each parameter were modeled as equal to the sum of group by biopsy site-fixed effects, subject-specific random effects, and subject-by-time-specific random effects (errors). Because of sample size limitations, the subject-specific random effects were assumed to have a common variance (τ2) across the 4 groups; in contrast, the error terms were modeled as having differing variances in each of the 4 groups (σ12, σ22, σ32, and σ42). The resulting intraclass correlations (ICCs), defined as τ2/(τ2 + σk2) for the kth groups, were used to quantify the stability of the parameters over time. ICC values of 0.75 or greater are described as “strong” stability, and ICC values of 0.5 are described as “moderate” stability in the following sections. Table 2 reports unadjusted P values for overall group and site differences. In addition, statistical significance of group by site differences are referred to in several places in the text of the Results section; the corresponding P values were adjusted for multiple comparisons using the simulation-based approach of Edwards and Berry.12 For the experimental design used here, mixed models generalize the alternative repeated-measures ANOVA by allowing for intermittent missing values (the data analyzed here had a small number) and group-specific error variances. This model uses 96 data points (4 subjects in each of 4 groups by 3 time points by 2 biopsy sites) to estimate 8 mean parameters and 5 variance parameters. Analyses were implemented using the mixed procedure in SAS, version 9.1 (SAS Institute, Cary, NC); the code to estimate the model is as follows:
Twenty-three participants were screened, and 16 were enrolled into the study. Study demographics are presented in Table 3. Participant retention was 100%. For the participants in groups 1, 3, and 4, the mean interval between the last episode of RAI and the week 2 endoscopy was 4 days (range: 0.5 to 7.5 days), and for the week 4 endoscopy, the mean interval was 3.6 days (range: 0.5 to 15.5 days).
Endoscopic examination and collection of colorectal biopsies were performed using a flexible endoscope, without sedation, and were well tolerated. Commonly reported adverse events included mild abdominal discomfort, flatulence, and rectal bleeding. These symptoms were mild and transient and did not prevent participants from completing all 3 scheduled endoscopic procedures.
The findings from most histologic samples were reported as normal. Only 4 (4.2%) of the 96 samples were scored as >grade 1. The abnormal samples came from group 1 (n = 2) and group 4 (n = 2). In all cases, abnormality was only seen at 1 site on a single occasion. Two participants had evidence of rectal spirochetosis (groups 1 and 3).
Plasma cells were the most frequent cell type seen in the intestinal sections (mean across the entire study population at 10 and 30 cm was 121 cells per 10 crypts assessed); lymphocytes were less common (mean of 103 cells), eosinophils were also uncommon (mean of 13 cells), and neutrophils were <1 cell per 10 crypts (see Table 2). Analysis of the stability (ICC) of quantitative histology across the entire study determined that the ICC value was low (range: 0.05 to 0.12) for all individual cell types and for the total lamina propria cell count. ICC values were also low across the individual groups (Table 4).
Mucosal Cytokine mRNA
Cytokine mRNA for RANTES, IFNγ, and IL-10 were detected in all the tissue samples. There were no significant differences between the 4 groups, although the highest mean values for RANTES and IFNγ mRNA were in group 3 (HIV-1-positive individuals with a plasma viral load ≥10,000 copies/mL) and the lowest mean value for IL-10 across groups was also in group 3. IFNγ mRNA expression was significantly higher in the 10-cm samples compared with the 30-cm samples in group 4; otherwise, there were no significant differences in individual cytokine mRNA expression within subjects (and within groups) between 10 and 30 cm. Cytokine mRNA levels were quite stable, with ICC values ≥0.7.
No blood was seen on any of the sponges. All 3 techniques used to quantify immunoglobulin levels generated equivalent results (data not shown), and no differences were seen across groups. Overall ICC values were 0.32 and 0.56 for IgG and IgA, respectively (total immunoglobulin measurement).
Significant differences were seen between the HIV-positive (groups 3 and 4) and HIV-seronegative (groups 1 and 2) participants. The following phenotypic markers were significantly lower in groups 3 and 4: CD4, CD16, CD56, CD4+/CCR5+, and CD4+/CXCR4+/CCR5+. The following phenotypic markers were significantly higher in groups 3 and 4: CD8, CD4+/CD38+, CD8+/CD38+, CD4+/HLA-DR+, and CD8+/HLA-DR+. Relative fluorescent intensity (RFI) of CD38 expressed on CD4+ and CD8+ lymphocytes was significantly increased in groups 3 and 4 (P < 0.01). The ICC for dual-stained CD4+/CD8+ lymphocytes was >0.8. The ICC for dual-stained CD4+/CD38+ lymphocytes was 0.58.
Modest but statistically significant differences in the prevalence of cell phenotype were seen between the samples collected at 10 and 30 cm (see Table 5). Cells expressing CD3, CD4, CD8, CD4+/CCR5+, and HLA-DR+/dendritic cell specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN)+ lineage-negative cells were more common in the samples collected at 30 cm, although there was variability by group (see Table 2). In contrast, cells expressing CD19 were more common in the 10-cm samples.
The purpose of this study was to evaluate the biologic variability and 4-week stability of a series of rectal mucosal parameters that might be monitored in phase 1 rectal mucosal studies. The parameters evaluated included histology, mucosal inflammatory cytokines (mRNA), mucosal cell phenotype, and secreted mucosal immunoglobulins. Three repeated endoscopic assessments, including the collection of 20 biopsies from the distal colon, conducted on 16 participants over a 4-week period were safe and well tolerated by the study participants. Study retention was 100%. Qualitative histology was reported as normal (grade 0) in most samples (>85%). Quantitative histology had a poor ICC score (≤0.12), IgG had an intermediate score of 0.56, but mucosal cytokine mRNA and selected cell phenotype scores were more stable (≥0.70).
As part of the process of developing vaginal, and subsequently rectal, microbicides, it is necessary to define a rectal safety evaluation framework analogous to that seen for vaginal microbicides.3,13 Unfortunately, no such algorithm currently exists for rectal safety. In addition, there is significant uncertainty about what endpoints should be measured in rectal safety studies, and their inherent biologic variability, stability, and difference between insertive and receptive partners at baseline.
The rectal mucosa is a highly susceptible target for HIV transmission. The vaginal mucosa offers significant protection from the mechanical stresses associated with sexual intercourse. In contrast, the rectal epithelium is a single layer of columnar cells that has little capacity to withstand any trauma. The subepithelial lamina propria contains abundant target cells expressing the HIV receptors and coreceptors, many of which have a highly activated phenotype.4 These histologic differences might lead to the rectal mucosa having increased susceptibility to microbicide-induced toxicity. Moreover, a product that had the ability to induce “immunologic toxicity” might only enrich this fertile soil further. Such toxicity could include induction of mucosal proinflammatory cytokine release, recruitment of new target cells, upregulation of HIV receptors on resident cells, and/or an increase in the activation status of target cells. All these changes might increase mucosal susceptibility to HIV infection,14 as is seen with mucosal inflammation associated with sexually transmitted infections.15 These forms of injury/changes are not likely to be detected through assessment of symptoms, signs, endoscopic appearance, or standard histology. Initial phase 1 studies need adjunct immunologic studies, in addition to more conventional safety parameters, to exclude potential immunologic toxicity of the introduced microbicidal product.
Murine and macaque studies of Nonoxynol-9 (N-9) have demonstrated profound epithelial damage after rectal administration of the vaginal formulation of N-9.16,17 Human studies have been less consistent. Tabet et al18 showed mild histologic abnormality in participants receiving rectal N-9. More dramatic changes were seen in 2 subsequent studies in which a rectal sloughing assay was used together with histology as early safety endpoints.19,20 Future phase 1 rectal safety studies need to include assessments at these early time points. It is hoped that N-9 may be a uniquely toxic product, because recent nonhuman primate studies involving rectal administration of vaginal microbicide candidates have failed to demonstrate similar epithelial changes, although the breadth of safety endpoints has been limited.21-23
In this study, we aimed to establish baseline ranges and stability of a variety of parameters without the confounder of an introduced study product. Histologic assessment, repeatedly conducted over 4 weeks using a modified qualitative scale, demonstrated that most specimens had no evidence of abnormal inflammation, including sections collected from HIV-positive participants and those practicing RAI. Two participants had evidence of rectal spirochetosis, a common benign finding in homosexual men.24 It is possible that the qualitative technique used in this study lacked the sensitivity to detect subtle changes in mucosal histology. It would be important to evaluate this approach in subjects exposed to a known injurious agent such as N-9. Surprisingly, the quantitative assessment of histology, despite being laborious and time-consuming, did not seem to be stable over the course of the study and did not provide additional discriminating data between the groups of subjects. Quantitative histology does not seem to be a useful technique to include in phase 1 rectal safety studies.
Cytokine mRNA has previously been characterized in intestinal mucosa using reverse transcriptase (RT) PCR, but usually in the setting of diseases such as inflammatory bowel disease.25,26 In this study, cytokine mRNA seemed to be a stable index within and between groups, and thus a useful parameter for future studies. There were no significant differences in cytokine mRNA expression across groups, although IFNγ and RANTES expression was highest in the group 3 participants (HIV-positive with detectable viral load), a finding observed in a previous study from our group.7
Immunoglobulin secretion was moderately stable for IgG (ICC scores of 0.56) but less so for IgA (0.32). The 3 different approaches to standardization all gave similar results (data not shown).
The most significant differences between the 4 groups were found in analyzing MMC phenotypes. As expected, the participants with HIV infection and a plasma viral load ≥10,000 copies/mL (group 3) had the most dramatic reduction in mucosal CD4+ T cells compared with the seronegative participants in groups 1 and 2 (P < 0.001). These individuals were fairly recently infected, with a mean duration of infection of 25 months, and had not started treatment with antiretroviral therapy (ART). In contrast, those HIV-positive participants with a plasma viral load ≤50 copies per mL (group 4) had CD4 counts that were higher than those of group 3 and not significantly different from those of groups 1 and 2. These participants had been infected for a mean of 159 months. These findings are in accordance with recent published data on the kinetics of mucosal CD4 lymphopenia associated with HIV infection and the partial recovery seen with ART.27-30
Similar reductions in CCR5 receptor expression on mucosal CD4+ T cells were seen in group 3, but these changes were not significant. Another feature of the HIV-infected participants in group 3 was that mucosal T cells (CD4+ and CD8+) had significantly higher levels of CD38, a T-cell activation marker previously associated with poor clinical prognosis.31,32 Despite these differences in group 3, the ICC values for all groups for CD4 and CD8 markers were >0.8 and seem to be stable over the time frame assessed in this study. Other subsets had lower ICC values, which are summarized in Table 4.
A reduction in natural killer (NK) cells, defined by CD16+ and CD56+, was seen in group 3. These changes have been noted before in patients with uncontrolled HIV-1 infection.33,34 Interestingly, in these studies, reduced NK function in peripheral blood mononuclear cells (PBMCs) is usually associated with reduced production of IFNγ.33,34 In our study, however, IFNγ levels were highest in group 3 patients. It is not clear why this discrepancy exists, although the PBMC data are derived from functional assays of isolated cells, whereas we used RT-PCR to measure cytokine mRNA in RNA derived from whole-tissue extracts.
In this study, we have demonstrated that there is regional heterogeneity in the expression of mucosal cell phenotypes. T-cell populations that are recognized as target cells for HIV were more common in the 30-cm samples, whereas cells bearing the B-cell marker CD19 were more common in the 10-cm samples. Cytokine expression displayed less variability, although IFNγ was increased in the 10-cm samples from group 4. Such regional heterogeneity might have implications for the design of microbicide trials and, potentially, for product development. These regional differences were modest, however, and need to be replicated in other studies.
The primary goal of this study was to evaluate the stability of certain mucosal parameters across a heterogeneous population of male trial participants. Qualitative histology, mucosal cytokine profiles, and selected T-cell subsets seem to be the most stable parameters that are of relevance in assessing the rectal safety profile of candidate microbicides. In addition, the study has confirmed the mucosal CD4 lymphopenia and partial reconstitution previously reported with treated HIV infection.30 The increased lymphocyte activation status defined by CD38 has been previously reported in peripheral blood lymphocytes, lymph nodes,35 and tonsil,36 but this is the first report of CD38 on intestinal MMCs, with apparent normalization in participants treated with ART.
There are a number of limitations to our study that need to be addressed in future studies. All the participants in this study received preparatory enemas with hyperosmolar products that have recently been reported to be associated with mucosal injury, although we did not see evidence of epithelial disruption on histology samples collected at 10 and 30 cm from the anal margin. It is also important to conduct studies with N-9 to determine which, if any, of these rectal parameters change with exposure to products that are known to be injurious to the rectal mucosa.17,19 We did not measure sloughing of rectal epithelium, which has been previously evaluated in simian and human studies,17,19 or measure fecal calprotectin, a neutrophil product that is increased in conditions characterized by mucosal inflammation.37,38 Another limitation of this study is that all the enrolled participants were male; thus, results cannot be generalized to women practicing RAI.
In summary, this study demonstrates that qualitative (not quantitative) histology, mucosal cytokine mRNA, and cell phenotypes are stable mucosal indices that should be useful as endpoints in phase 1 rectal safety studies. Other more variable parameters such as rectal immunoglobulins require further evaluation before being used in this context.
The authors thank the dedicated participants who enrolled in this study. They are also grateful to Xin Huang and Ying Zhou for assisting with the data analysis.
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