Peter A. Anton

Enhanced levels of functional HIV-1 co-receptors on human mucosal T cells demonstrated using intestinal biopsy tissue

ObjectiveTo examine compartmental differences in co-receptor expression on CD4 lymphocytes between blood and gut using endoscopic biopsies. DesignMucosal and peripheral CD4 T cells from healthy controls were compared for co-receptor expression and vulnerability to infection by HIV-1. MethodsExpression of CCR5 and CXCR4 was quantified by flow cytometry on isolated mucosal CD4 lymphocytes obtained from endoscopic biopsies and blood from healthy controls. Vulnerability to in vitro infection by both R5 and X4 strains was assessed by measuring p24. ResultsBiopsies yielded sufficient lymphocytes for flow cytometric characterization and infectivity studies. The percentage of mucosal CD4 T lymphocytes that expressed CCR5 and the per cell expression of CCR5 were both significantly increased compared with that in peripheral blood CD4 T lymphocytes. CXCR4 was expressed on the majority of CD4 lymphocytes in both compartments. In vitro infection of mucosal mononuclear cells supported greater viral replication of both R5 and X4 strains than peripheral blood mononuclear cells. ConclusionsEnhanced expression of CXCR4 and CCR5 on CD4 lymphocytes in normal intestinal mucosa predicts increased vulnerability to infection by both R5 and X4 HIV-1. Endoscopic biopsies provide a useful mucosal tissue sampling technique to identify compartmental immunologic differences that may be exploited by HIV-1 in establishing initial mucosal infection.

A Preponderance of CCR5+CXCR4+ Mononuclear Cells Enhances Gastrointestinal Mucosal Susceptibility to Human Immunodeficiency Virus Type 1 Infection

ABSTRACT The gastrointestinal mucosa harbors the majority of the bodys CD4+ cells and appears to be uniquely susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We undertook this study to examine the role of differences in chemokine receptor expression on infection of mucosal mononuclear cells (MMCs) and peripheral blood mononuclear cells (PBMCs) by R5- and X4-tropic HIV-1. We performed in vitro infections of MMCs and PBMCs with R5- and X4-tropic HIV-1, engineered to express murine CD24 on the infected cells surface, allowing for quantification of HIV-infected cells and their phenotypic characterization. A greater percentage of MMCs than PBMCs are infected by both R5- and X4-tropic HIV-1. Significant differences exist in terms of chemokine receptor expression in the blood and gastrointestinal mucosa; mucosal cells are predominantly CCR5+ CXCR4+, while these cells make up less than 20% of the peripheral blood cells. It is this cell population that is most susceptible to infection with both R5- and X4-tropic HIV-1 in both compartments. Regardless of whether viral isolates were derived from the blood or mucosa of HIV-1-infected patients, HIV-1 p24 production was greater in MMCs than in PBMCs. Further, the chemokine receptor tropism of these patient-derived viral isolates did not differ between compartments. We conclude that, based on these findings, the gastrointestinal mucosa represents a favored target for HIV-1, in part due to its large population of CXCR4+ CCR5+target cells and not to differences in the virus that it contains.

Patchiness of mucosal inflammation in treated ulcerative colitis: A prospective study

Conventional wisdom dictates that ulcerative colitis affects contiguous areas of the colon and is most severe in the rectum, and that the finding of rectal sparing or patchy involvement should raise suspicions of Crohns disease. We and others have noted occasional rectal sparing and patchy involvement in patients with ulcerative colitis. Therefore, we prospectively studied the prevalence of patchiness, including rectal sparing, in treated cases of ulcerative colitis. Consecutive patients with longstanding ulcerative colitis were studied. The left colon was divided into three zones for scoring degree of activity, and biopsy specimens from each zone were graded for histologic activity by a blinded observer. Patchiness by endoscopy or histology was defined as (1) frank rectal sparing (normal appearance endoscopically; absence of inflammation of the lamina propria and crypts histologically); (2) areas of greater inflammation proximally than distally; or (3) discrete areas of patchiness endoscopically within any one zone. Of 39 patients evaluated, 17 (44%) had endoscopic evidence of patchiness, including 5 (13%) with rectal sparing. Thirteen (33%) had histologic evidence of patchiness, including 6 (15%) with rectal sparing. Both endoscopic and histologic patchiness were seen in 9 patients (23%). The patchy and nonpatchy groups did not differ in regard to the use of rectal therapy. In patients with treated ulcerative colitis, the finding of rectal sparing or patchiness should not necessarily indicate a change in the diagnosis to Crohns disease.

A randomized, placebo‐controlled trial of calcium supplementation for decreased bone density in corticosteroid‐using patients with inflammatory bowel disease: a pilot study

Background: Patients with inflammatory bowel disease (IBD) have a high prevalence of osteoporosis. A number of studies have found that corticosteroid use is associated with the development of osteoporosis in these patients. Calcium supplementation may be of benefit in corticosteroid‐induced osteoporosis and calcium may be a nutrient that patients with IBD lack. Aim: To test the benefit of calcium supplementation on bone density in a pilot study over a 1‐year period, in a group of corticosteroid‐using patients with IBD, in a randomized, double‐blind, placebo‐controlled treatment study.

Prevention of SIV rectal transmission and priming of T cell responses in macaques after local pre-exposure application of tenofovir gel.

Background The rectum is particularly vulnerable to HIV transmission having only a single protective layer of columnar epithelium overlying tissue rich in activated lymphoid cells; thus, unprotected anal intercourse in both women and men carries a higher risk of infection than other sexual routes. In the absence of effective prophylactic vaccines, increasing attention is being given to the use of microbicides and preventative antiretroviral (ARV) drugs. To prevent mucosal transmission of HIV, a microbicide/ARV should ideally act locally at and near the virus portal of entry. As part of an integrated rectal microbicide development programme, we have evaluated rectal application of the nucleotide reverse transcriptase (RT) inhibitor tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy) propyl] adenine monohydrate), a drug licensed for therapeutic use, for protective efficacy against rectal challenge with simian immunodeficiency virus (SIV) in a well-established and standardised macaque model. Methods and Findings A total of 20 purpose-bred Indian rhesus macaques were used to evaluate the protective efficacy of topical tenofovir. Nine animals received 1% tenofovir gel per rectum up to 2 h prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given tenofovir gel 2 h after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a noncloned, virulent stock of SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from peripheral blood mononuclear cells (PBMC), quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive quantitative competitive (qc) RT-PCR, and presence of SIV-specific serum antibodies by ELISA. We report here a significant protective effect (p = 0.003; Fisher exact probability test) wherein eight of nine macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving tenofovir gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. We found a strong positive association between the concentration of tenofovir in the plasma 15 min after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged tenofovir-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of tenofovir. Intriguingly, in the absence of seroconversion, Gag-specific gamma interferon (IFN-γ)-secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/106 PBMC to 261 spot forming cells (SFC)/106 PBMC. Conclusions These results indicate that colorectal pretreatment with ARV drugs, such as tenofovir, has potential as a clinically relevant strategy for the prevention of HIV transmission. We conclude that plasma tenofovir concentration measured 15 min after rectal administration may serve as a surrogate indicator of protective efficacy. This may prove to be useful in the design of clinical studies. Furthermore, in vitro intestinal explants served as a model for drug distribution in vivo and susceptibility to virus infection. The finding of T cell priming following exposure to virus in the absence of overt infection is provocative. Further studies would reveal if a combined modality microbicide and vaccination strategy is feasible by determining the full extent of local immune responses induced and their protective potential.

An international survey of the use and attitudes regarding alternative medicine by patients with inflammatory bowel disease.

Objective:There is a perception of increasing and widespread use of alternative medicine for inflammatory bowel disease (IBD). We assessed the usage of alternative therapies among patients with IBD, whether there were similar or contrasting variables that were predictive of such use, and contrasted the use in four different centers in North America and Europe.Methods:Patients in four IBD centers completed a self-administered questionnaire regarding alternative medicine. The centers were in Cork, Los Angeles, Stockholm, and Winnipeg. Patient demographics, the use of 18 types of alternative medicine, and attitudes towards alternative and conventional medicine were compared. A multiple logistic regression analysis was used.Results:Fifty-one percent of 289 patients used some form of alternative medicine. The percentages of use by site were Cork = 31%, Los Angeles = 68%, Stockholm = 32%, and Winnipeg = 57%. The six most commonly used therapies in descending order were: exercise (28%), prayer (18%), counseling (13%), massage (11%), chiropractic (11%), and relaxation (10%). Only 7% used acupuncture or homeopathy and 5% used herbal medicine. The highest odds ratios (confidence intervals [CIs]) for using any form of alternative medicine were associated with: being single 3.1 (1.7–5.7), Los Angeles patient 4.4 (2.3–8.3), Winnipeg patient 2.7 (1.3–5.9), and an increase of alternative medicine use of 2.7% for every M.D. visit (CI, 2–11%/visit). The patient age, gender, disease diagnosis, or duration of disease were not predictive of any type of alternative medicine use. Regarding attitudes, respondents from Cork were most favorable toward alternative medicine use and least favorable toward conventional medicine. Based on attitudes, subjects were more likely to use alternative medicine if they were not satisfied with conventional therapy, viewed hospitals as dangerous places, thought that alternative medicine practitioners should have a role in hospitals, and felt their medical situation was hopeless.Conclusions:Fifty-one percent of respondents used some form of alternative medicine. The use was greater among the North American patients than the European ones. Respondents were more likely to use alternative medicine if they were single, in a higher income bracket, and an urban dweller.

Susceptibility of Lewis and Fischer rats to stress-induced worsening of TNB-colitis: protective role of brain CRF

We assessed the role of central corticotropin-releasing factor (CRF) in stress-induced worsening of colitis in inbred rat strains with hypo (Lewis/N) and hyper (Fischer344/N) CRF responses to stress. Intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNB) induced colitis of similar severity in both strains as assessed on day 7 by macroscopic scoring, histological evaluation, tissue myeloperoxidase (MPO) activity, and decrease in food intake and body weight. Colitis was inhibited by daily intracerebroventricular injections of CRF in both strains. Chronic stress (3 h/day, water avoidance or wrap restraint on alternate days for 6 days) aggravated colitis more in Lewis than Fischer rats (71 and 22% further increase in MPO activity, respectively). The CRF antagonist astressin injected intracerebroventricularly enhanced the colitis response to stress and caused mortality in both strains. Fischer rats had higher plasma corticosterone levels 20 min after stress alone on day 1 and after TNB plus stress on days 1 and 3 compared with Lewis. These data show that central CRF restrains the proinflammatory action of stress in experimental colitis.We assessed the role of central corticotropin-releasing factor (CRF) in stress-induced worsening of colitis in inbred rat strains with hypo (Lewis/N) and hyper (Fischer344/N) CRF responses to stress. Intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNB) induced colitis of similar severity in both strains as assessed on day 7 by macroscopic scoring, histological evaluation, tissue myeloperoxidase (MPO) activity, and decrease in food intake and body weight. Colitis was inhibited by daily intracerebroventricular injections of CRF in both strains. Chronic stress (3 h/day, water avoidance or wrap restraint on alternate days for 6 days) aggravated colitis more in Lewis than Fischer rats (71 and 22% further increase in MPO activity, respectively). The CRF antagonist astressin injected intracerebroventricularly enhanced the colitis response to stress and caused mortality in both strains. Fischer rats had higher plasma corticosterone levels 20 min after stress alone on day 1 and after TNB plus stress on days 1 and 3 compared with Lewis. These data show that central CRF restrains the proinflammatory action of stress in experimental colitis.

Ex vivo culture of human colorectal tissue for the evaluation of candidate microbicides.

Objectives:Establishment of an in vitro model to evaluate rectal safety and the efficacy of microbicide candidates. Design:An investigation and characterization of human colorectal explant culture for screening candidate microbicides to prevent rectal transmission of HIV-1 infection. Methods:Human colorectal explants were cultured at the liquid–air interface on gelfoam rafts. Phenotypic characterization of HIV-1 target cells was performed by fluorescence-activated cell sorter analysis. HIV-1 infection was determined by the measurement of p24 antigen release, viral RNA, and proviral DNA accumulation. Results:Colorectal explant CD4 T cells expressed higher CCR5 and CXCR4 levels compared with blood. Minor differences between the rectal and sigmoid colon were observed with a trend for slightly higher CCR5 and HLA-DR expression in cells from the sigmoid colon. Favourable culture conditions were established for colorectal tissue. Although tissue structure degenerated with time, CD4: CD8 cell ratios remained constant, and tissue supported productive HIV-1 infection. The ability of candidate microbicides to inhibit R5 HIV-1 infection was evaluated. Polyanion candidates, PRO2000 and dextrin sulphate, provided 99% protection at 1 μg/ml and 1 mg/ml, respectively, equivalent to 1/5000 and 1/40 of the vaginal formulations. The nucleotide reverse transcriptase inhibitor (NRTI) 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) provided protection at concentrations 1000-fold lower (10 μg/ml) than the proposed vaginal formulation (1%). Furthermore, non-NRTI UC-781 and TMC-120 provided greater than 99% inhibition at 3.3 or 0.33 μg/ml, respectively. No products demonstrated toxicity to rectal mucosa at inhibitory concentrations. Conclusion:Colorectal explant culture was shown to be a useful tool for the preclinical evaluation of potential microbicides. The data suggest that rectally applied microbicides might provide protection from HIV-1 transmission.

Binding and Transfer of Human Immunodeficiency Virus by DC-SIGN+ Cells in Human Rectal Mucosa

ABSTRACT The role of DC-SIGN on human rectal mucosal dendritic cells is unknown. Using highly purified human rectal mucosal DC-SIGN+ cells and an ultrasensitive real-time reverse transcription-PCR assay to quantify virus binding, we found that HLA-DR+/DC-SIGN+ cells can bind and transfer more virus than the HLA-DR+/DC-SIGN− cells. Greater than 90% of the virus bound to total mucosal mononuclear cells (MMCs) was accounted for by the DC-SIGN+ cells, which comprise only 1 to 5% of total MMCs. Significantly, anti-DC-SIGN antibodies blocked 90% of the virus binding when more-physiologic amounts of virus inoculum were used. DC-SIGN expression in the rectal mucosa was significantly correlated with the interleukin-10 (IL-10)/IL-12 ratio (r = 0.58, P < 0.002; n = 26) among human immunodeficiency virus (HIV)-positive patients. Ex vivo and in vitro data implicate the role of IL-10 in upregulating DC-SIGN expression and downregulating expression of the costimulatory molecules CD80/CD86. Dendritic cells derived from monocytes (MDDCs) in the presence of IL-10 render the MDDCs less responsive to maturation stimuli, such as lipopolysaccharide and tumor necrosis factor alpha, and migration to the CCR7 ligand macrophage inflammatory protein 3β. Thus, an increased IL-10 environment could render DC-SIGN+ cells less immunostimulatory and migratory, thereby dampening an effective immune response. DC-SIGN and the IL-10/IL-12 axis may play significant roles in the mucosal transmission and pathogenesis of HIV type 1.

Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues

For most viruses, there is a need for antimicrobials that target unique viral molecular properties. Acyclovir (ACV) is one such drug. It is activated into a human herpesvirus (HHV) DNA polymerase inhibitor exclusively by HHV kinases and, thus, does not suppress other viruses. Here, we show that ACV suppresses HIV-1 in HHV-coinfected human tissues, but not in HHV-free tissue or cell cultures. However, addition of HHV-6-infected cells renders these cultures sensitive to anti-HIV ACV activity. We hypothesized that such HIV suppression requires ACV phosphorylation by HHV kinases. Indeed, an ACV monophosphorylated prodrug bypasses the HHV requirement for HIV suppression. Furthermore, phosphorylated ACV directly inhibits HIV-1 reverse transcriptase (RT), terminating DNA chain elongation, and can trap RT at the termination site. These data suggest that ACV anti-HIV-1 activity may contribute to the response of HIV/HHV-coinfected patients to ACV treatment and could guide strategies for the development of new HIV-1 RT inhibitors.