Rectal microbicides are needed to reduce the risk of HIV acquisition associated with unprotected receptive anal intercourse. The MTN-007 study was designed to assess the safety (general and mucosal), ...adherence, and acceptability of a new reduced glycerin formulation of tenofovir 1% gel.
Participants were randomized 1:1:1:1 to receive the reduced glycerin formulation of tenofovir 1% gel, a hydroxyethyl cellulose placebo gel, a 2% nonoxynol-9 gel, or no treatment. Each gel was administered as a single dose followed by 7 daily doses. Mucosal safety evaluation included histology, fecal calprotectin, epithelial sloughing, cytokine expression (mRNA and protein), microarrays, flow cytometry of mucosal T cell phenotype, and rectal microflora. Acceptability and adherence were determined by computer-administered questionnaires and interactive telephone response, respectively.
Sixty-five participants (45 men and 20 women) were recruited into the study. There were no significant differences between the numbers of ≥ Grade 2 adverse events across the arms of the study. Likelihood of future product use (acceptability) was 87% (reduced glycerin formulation of tenofovir 1% gel), 93% (hydroxyethyl cellulose placebo gel), and 63% (nonoxynol-9 gel). Fecal calprotectin, rectal microflora, and epithelial sloughing did not differ by treatment arms during the study. Suggestive evidence of differences was seen in histology, mucosal gene expression, protein expression, and T cell phenotype. These changes were mostly confined to comparisons between the nonoxynol-9 gel and other study arms.
The reduced glycerin formulation of tenofovir 1% gel was safe and well tolerated rectally and should be advanced to Phase 2 development.
ClinicalTrials.gov NCT01232803.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This study was designed to assess the safety, acceptability, pharmacokinetic (PK), and pharmacodynamic (PD) responses to rectal administration of tenofovir (TFV) 1% vaginally formulated gel and oral ...tenofovir disoproxil fumarate (TDF). This study was designed as a phase 1, randomized, two-site (United States), double-blind, placebo-controlled study of sexually abstinent men and women. Eighteen participants received a single 300-mg exposure of oral TDF and were then randomized 2:1 to receive a single and then seven daily exposures of rectal TFV or hydroxyethyl cellulose (HEC) placebo gel. Safety endpoints included clinical adverse events (AEs) and mucosal safety parameters. Blood and colonic biopsies were collected for PK analyses and ex vivo HIV-1 challenge. No serious AEs were reported. However, AEs were significantly increased with 7-day TFV gel use, most prominently with gastrointestinal AEs (p=0.002). Only 25% of participants liked the TFV gel. Likelihood of use "if somewhat protective" was ∼75% in both groups. Indices of mucosal damage showed minimal changes. Tissue TFV diphosphate (TFV-DP) C(max) 30 min after single rectal exposure was 6-10 times greater than single oral exposure; tissue TFV-DP was 5.7 times greater following 7-day versus single rectal exposure. In vivo exposure correlated with significant ex vivo tissue infectibility suppression single-rectal: p=0.12, analysis of covariance (ANCOVA) p=0.006; 7-day rectal: p=0.02, ANCOVA p=0.005. Tissue PK-PD was significantly correlated (p=0.002). We conclude that rectal dosing with TFV 1% gel resulted in greater TFV-DP tissue detection than oral dosing with reduced ex vivo biopsy infectibility, enabling PK-PD correlations. On the basis of increased gastrointestinal AEs, rectally applied, vaginally formulated TFV was not entirely safe or acceptable, suggesting the need for alternative rectal-specific formulations.
Defective cell-mediated immunity increases the risk of human papillomavirus-associated anal dysplasia and cancer. There is limited information on anal canal disease in patients with IBD.
The purpose ...of this study was to assess anal/vaginal human papillomavirus and anal dysplasia prevalence in patients with IBD.
Patients had an anal examination before routine colonoscopy.
The study was conducted at a tertiary IBD referral center.
We studied a convenience sample of sexually active male and female patients with IBD who were not on biological therapy.
Anal examination, anal and vaginal human papillomavirus testing, anal cytology, and high-resolution anoscopy/biopsy were carried out.
Anal and vaginal human papillomavirus types, anal cytology, and biopsy grade were measured.
Twenty-five male and 21 female evaluable participants, 31 with Crohn's disease, 14 with ulcerative colitis, and 1 with indeterminate colitis, were predominantly white (91.3%), treatment experienced (76.1%), an average age of 38.1 years (range, 22.0-66.0 y), and had an average length of IBD diagnosis of 9.3 years (range, 1.0-33.0 y). Eighteen (39.1%) had an abnormal perianal examination and 3 (6.5%) had an abnormal digital examination. Forty-one (89.1%) had anal human papillomavirus, 16 with a single type and 25 with multiple types (range, 2-5 types). Human papillomavirus type 16 was most common (65.2%), followed by human papillomavirus types 11 and 45 (37.0% each). Nineteen of 21 (90.5%) women had vaginal human papillomavirus. Overall, 21 (45.7%) had abnormal anal cytology. Thirty three (71.7%) had ≥1 anal biopsy (9 had multiple), with dysplasia diagnosed in 28 (60.9%) and high-grade and low-grade squamous intraepithelial lesions diagnosed in 4 (8.7%) and 24 (43.5%).
No control group was included, and no detailed sexual history was taken.
A high prevalence of anal and vaginal human papillomavirus and anal dysplasia was demonstrated in the study population outcomes. See Video Abstract at http://links.lww.com/DCR/A379.
•Multiple biopsies are required to accurately characterize gene expression in rectal tissue.•Inter/intra-subject gene expression had higher levels of variance than intra-biopsy ...variation.•Normalization of cytokines against reference genes failed to consistently reduce variance.
To determine the intra- and inter-subject variability of mucosal cytokine gene expression in rectal biopsies from healthy volunteers and to screen cytokine and chemokine mRNA as potential biomarkers of mucosal inflammation.
Rectal biopsies were collected from 8 participants (3 biopsies per participant) and 1 additional participant (10 biopsies). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to quantify IL-1β, IL-6, IL-12p40, IL-8, IFN-γ, MIP-1α, MIP-1β, RANTES, and TNF-α gene expression in the rectal tissue. The intra-assay, inter-biopsy and inter-subject variance was measured in the eight participants. Bootstrap re-sampling of the biopsy measurements was performed to determine the accuracy of gene expression data obtained for 10 biopsies obtained from one participant. Cytokines were both non-normalized and normalized using four reference genes (GAPDH, β-actin, β2 microglobulin, and CD45).
Cytokine measurement accuracy was increased with the number of biopsy samples, per person; four biopsies were typically needed to produce a mean result within a 95% confidence interval of the subject’s cytokine level approximately 80% of the time. Intra-assay precision (% geometric standard deviation) ranged between 8.2 and 96.9 with high variance between patients and even between different biopsies from the same patient. Variability was not greatly reduced with the use of reference genes to normalize data.
The number of biopsy samples required to provide an accurate result varied by target although 4 biopsy samples per subject and timepoint, provided for >77% accuracy across all targets tested. Biopsies within the same subjects and between subjects had similar levels of variance while variance within a biopsy (intra-assay) was generally lower. Normalization of inflammatory cytokines against reference genes failed to consistently reduce variance. The accuracy and reliability of mRNA expression of inflammatory cytokines will set a ceiling on the ability of these measures to predict mucosal inflammation. Techniques to reduce variability should be developed within a larger cohort of individuals before normative reference values can be validated.
BACKGROUNDThe quadrivalent human papillomavirus (qHPV) and 9 valent (nHPV) vaccine are licensed for males to prevent anal HPV–associated dysplasia and cancer caused by HPV types 6, 11, 16, and 18 ...(qHPV) and additional types 33, 35, 45, 52, and 58 (nHPV), respectively. Both conditions are common in HIV-infected and HIV-uninfected men who have sex with men (MSM). It is not well documented which anal HPV vaccine types are most prevalent in Southeast Asia.
METHODSA convenience sample of 400 anal swabs were obtained from 200 HIV-infected and 200 HIV-uninfected sexually active Bangkok MSM Cohort Study participants. After swab collection in PreservCyt (Cytyc Corp, Marlborough, MA), the media was stored at −80°C until processing. DNA was extracted, amplified by polymerase chain reaction, denatured, and then hybridized to probes for 37 HPV types and β-globin.
RESULTSThe mean participant age was 25.6 years (range, 18–55 years); the mean CD4 T-cell count was 410 cells/mm in the HIV-infected participants. Among all swab samples, 386 (192 HIV-positive and 194 HIV-negative) had adequate β-globin for HPV genotype testing. Anal HPV type was detected in 44.3% of participants whose samples underwent genotype testing. Both qHPV and nHPV types were more frequently detected in HIV-infected compared with HIV-uninfected (42.2% vs. 23.2% P < 0.01, 50.0% vs. 24.2% P < 0.01), respectively). There were no significant relationships between social behaviors (alcohol use, drug use) or sexual behaviors (number of partners, condom usage, sexual positioning) and anal HPV prevalence.
CONCLUSIONSThe prevalence of anal vaccine HPV types in Thai MSM was similar to that reported in MSM from Western populations and has a similar distribution by HIV status. Targeting young MSM with vaccination could offer protection against HPV vaccine types.
The ex vivo mucosal explant model is frequently used to test the efficacy of microbicides that have the potential for preventing HIV-1 transmission. The conventional assessment of product efficacy ...has been the extent of HIV-1 p24 suppression in supernatant fluids sampled up to day 14 after HIV-1 challenge ex vivo. The purpose of this study was to determine if measurement of HIV-1 nucleic acids by real-time PCR and HIV-1 integration by Alu-gag PCR provides advantages with regard to monitoring HIV-1 infection in explants. Rectal biopsies from HIV-1-negative individuals were challenged with 1 × 10(5) virions/ml of HIV-1BaL or HIV-1CH077 ex vivo. HIV-1 RNA and HIV-1 p24 in supernatant fluids and HIV-1 nucleic acids and integrated provirus in individual biopsies were measured at days 1-14 after infection. HIV-1 RNA and proviral DNA were measured by quantitative real-time PCR (qRT-PCR) while integrated virus was detected by Alu-gag PCR. Real-time PCR assays detecting HIV-1 DNA and RNA performed similarly provided that the infecting virus sequences were a good match with the sequences of the assay primers and probes. Increased HIV-1 nucleic acid levels and DNA integration were measurable on days 11 and 14 after infection. The magnitude of explant infection was similar after challenge with HIV-1BaL and HIV-1CH077, although the trajectory of infection was delayed in the HIV-1CH077-infected biopsies. In the majority of experiments, qRT-PCR did not appreciably shorten the time necessary to detect evidence of HIV-1 infection.
Human
solid tumors develop multiple genetic abnormalities that accumulate
progressively in individual cells during the course of tumor evolution.
We sought to determine whether there are specific ...sequences of
occurrence of these progressive evolutionary changes in human breast
cancers by performing correlated cell-by-cell measurements of cell DNA
content, p53 protein, Her-2/neu protein, and ras protein by
multiparameter flow cytometry in 56 primary tumor samples obtained at
surgery. In addition, p53 allelic loss and
Her-2/ neu gene amplification were determined by
fluorescence in situ hybridization in cells from the same
samples. We reasoned that if there is a specific order in which genetic
changes occur, the same early changes would be found consistently in
the cells with the fewest abnormalities. We reasoned further that
late-developing abnormalities would not occur alone in individual cells
but would almost always be found together with the early changes
inherited by the same cells. By these criteria, abnormalities involving
p53 generally occurred early in the course of development of invasive
breast cancers, whereas ras protein overexpression was found to be a
late-occurring phenomenon. Within individual tumors, cellular p53
overexpression was often observed alone in individual cells, whereas
ras protein overexpression was rarely observed in the absence of p53
overexpression and/or Her-2/neu overexpression in the same cells.
Furthermore, the intracellular level of each abnormally expressed
protein was found to increase progressively as new abnormalities were
acquired. Infiltrating ductal carcinomas exhibited characteristic
phenotypic patterns in which p53 allelic loss and/or p53
protein overexpression, Her-2/neu amplification and/or overexpression,
aneuploidy, and ras overexpression accumulated within individual cells.
However, this pattern was not a prominent feature of lobular breast
cancers. All six lobular breast cancers studied were diploid.
p53 allelic loss and/or early p53 overexpression, and late
ras cooverexpression in the same cells were less common in lobular
breast cancers than in infiltrating ductal carcinomas. Although
Her-2/neu overexpression was a common finding in lobular breast
cancers, Her-2/neu amplification was not observed in these
tumors.
A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular ...DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol‐fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p < 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p < 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti‐leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity oaf DNA staining with propidium iodide were dependent on paraformaldehyde concentration and Fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37°C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation‐induced effect is useful in identifying biologically distinct near‐diploid subpopulations in tumors.
Treatment of nonpolar ether extracts of human female blood with mild alkali produced more immunoassayable estradiol than the unhydrolyzed extract. Analysis of the serum extracts showed that the ...substance which released immunore-active estradiol after hydrolysis has chromatographic properties identical to those of fatty acid esters of estradiol esterified at carbon 17. The physiological role of these previously unknown endogenous esters might be inferred from their structural similarity to synthetic drugs used therapeutically for their prolonged estrogenic action.
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