Review article: RNA interference--potential therapeutic applications for the gastroenterologist.

BACKGROUND: A new technique of gene regulation, termed RNA interference, has emerged recently. RNA interference utilizes short double-stranded RNA to inhibit selectively gene expression of complementary RNA nucleotide sequences after transcription, but prior to translation. Gastrointestinal and hepatic disorders may be particularly amenable to therapeutic RNA interference intervention because of the relative ease of delivery of drugs to the gastrointestinal tract and liver. AIM: To examine the published literature for potential clinical uses of RNA interference in gastroenterology and speculate on future therapies for luminal disease. METHODS: Reports were identified using PubMed and the search term 'RNA interference', focusing on therapeutic uses related to gastrointestinal and liver disease. RESULTS: Cellular and animal models demonstrate the potential application of short-interfering RNA-based therapies for viral hepatitis and inflammatory bowel disease. With validation of specific targets and better in vivo delivery of short-interfering RNA, RNA interference may represent a new frontier for molecular-targeted therapy in gastroenterology and hepatology. CONCLUSIONS: Short-interfering RNA provides a novel and specific means to inhibit gene expression. Translation to the clinical arena will require further definition of side-effects, off-target effects and delivery systems. Ultimately, mucosally applied or endoscopically delivered short-interfering RNA could be one of the earliest clinical uses of short-interfering RNA therapy.

Increased turnover of T lymphocytes in HIV-1 infection and its reduction by antiretroviral therapy.

The mechanism of CD4(+) T cell depletion in human immunodeficiency virus (HIV)-1 infection remains controversial. Using deuterated glucose to label the DNA of proliferating cells in vivo, we studied T cell dynamics in four normal subjects and seven HIV-1-infected patients naive to antiretroviral drugs. The results were analyzed using a newly developed mathematical model to determine fractional rates of lymphocyte proliferation and death. In CD4(+) T cells, mean proliferation and death rates were elevated by 6.3- and 2.9-fold, respectively, in infected patients compared with normal controls. In CD8(+) T cells, the mean proliferation rate was 7.7-fold higher in HIV-1 infection, but the mean death rate was not significantly increased. Five of the infected patients underwent subsequent deuterated glucose labeling studies after initiating antiretroviral therapy. The lymphocyte proliferation and death rates in both CD4(+) and CD8(+) cell populations were substantially reduced by 5-11 weeks and nearly normal by one year. Taken together, these new findings strongly indicate that CD4(+) lymphocyte depletion seen in AIDS is primarily a consequence of increased cellular destruction, not decreased cellular production.

Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy.

Despite prolonged treatment with highly active antiretroviral therapy (HAART), infectious HIV-1 continues to replicate and to reside latently in resting memory CD4(+) T lymphocytes, creating a major obstacle to HIV-1 eradication. It is therefore not surprising to observe a prompt viral rebound after discontinuation of HAART. The nature of the rebounding virus, however, remains undefined. We now report on the genetic characterization of rebounding viruses in eight patients in whom plasma viremia was undetectable throughout about 3 years of HAART. Taking advantage of the extensive length polymorphism in HIV-1 env, we found that in five patients who did not show HIV-1 replication during treatment, the rebound virus was identical to those isolated from the latent reservoir. In three other patients, two of whom had been free of plasma viremia but had showed some residual viral replication, the rebound virus was genetically different from the latent reservoir virus, corresponding instead to minor viral variants detected during the course of treatment in lymphoid tissues. We conclude that in cases with apparent complete HIV-1 suppression by HAART, viral rebound after cessation of therapy could have originated from the activation of virus from the latent reservoir. In patients with incomplete suppression by chemotherapy, however, the viral rebound is likely triggered by ongoing, low-level replication of HIV-1, perhaps occurring in lymphoid tissues.

The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy.

Replication-competent HIV-1 can be isolated from infected patients despite prolonged plasma virus suppression by anti-retroviral treatment. Recent studies have identified resting, memory CD4+ T lymphocytes as a long-lived latent reservoir of HIV-1 (refs. 4,5). Cross-sectional analyses indicate that the reservoir is rather small, between 103 and 107 cells per patient. In individuals whose plasma viremia levels are well suppressed by anti-retroviral therapy, peripheral blood mononuclear cells containing replication-competent HIV-1 were found to decay with a mean half-life of approximately 6 months, close to the decay characteristics of memory lymphocytes in humans and monkeys. In contrast, little decay was found in a less-selective patient population. We undertook this study to address this apparent discrepancy. Using a quantitative micro-culture assay, we demonstrate here that the latent reservoir decays with a mean half-life of 6.3 months in patients who consistently maintain plasma HIV-1 RNA levels of fewer than 50 copies/ml. Slower decay rates occur in individuals who experience intermittent episodes of plasma viremia. Our findings indicate that the persistence of the latent reservoir of HIV-1 despite prolonged treatment is due not only to its slow intrinsic decay characteristics but also to the inability of current drug regimens to completely block HIV-1 replication.

Rapid production and clearance of HIV-1 and hepatitis C virus assessed by large volume plasma apheresis.

BACKGROUND: In chronic HIV-1 infection, dynamic equilibrium exists between viral production and clearance. The half-life of free virions can be estimated by inhibiting virion production with antiretroviral agents and modelling the resulting decline in plasma HIV-1 RNA. To define HIV-1 and hepatitis C virus (HCV) dynamics, we used plasma apheresis to increase virion clearance temporarily while leaving virion production unaffected. METHODS: Plasma virus loads were measured frequently before, during, and after apheresis in four HIV-1-infected patients, two of whom were also co-infected with HCV. Rates of virion clearance were derived by non-linear least-square fitting of plasma virus load to a model of viral dynamics. FINDINGS: Virion clearance rate constants were 0.0063/min (9.1/day) to 0.025/min (36.0/day; half-life 28-110 min) for HIV-1 and 0.0038/min (5.5/day) to 0.0069/min (9.9/day; half-life 100-182 min) for HCV. These values provided estimates of daily particle production of 9.3 log10-10.2 log10 particles for HIV-1 and 11.6 log10-13.0 log10 particles for HCV. INTERPRETATION: Our findings confirm that HIV-1 and HCV are produced and cleared extremely rapidly. New estimates for HIV-1 clearance are up to ten times higher than previous ones, whereas HCV clearance is similar to previous estimates.

A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.

OBJECTIVES: HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase. RESULTS: In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays. CONCLUSIONS: Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.

Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy.

BACKGROUND: In patients infected with human immunodeficiency virus type 1 (HIV-1), combination antiretroviral therapy can result in sustained suppression of plasma levels of the virus. However, replication-competent virus can still be recovered from latently infected resting memory CD4 lymphocytes; this finding raises serious doubts about whether antiviral treatment can eradicate HIV-1. METHODS: We looked for evidence of residual HIV-1 replication in eight patients who began treatment soon after infection and in whom plasma levels of HIV-1 RNA were undetectable after two to three years of antiretroviral therapy. We examined whether there had been changes over time in HIV-1 proviral sequences in peripheral-blood mononuclear cells, which would indicate residual viral replication. We also performed in situ hybridization studies on tissues from one patient to identify cells actively expressing HIV-1 RNA. We estimated the rate of decrease of latent, replication-competent HIV-1 in resting CD4 lymphocytes on the basis of the decrease in the numbers of proviral sequences identified during primary infection and direct sequential measurements of the size of the latent reservoir. RESULTS: Six of the eight patients had no significant variations in proviral sequences during treatment. However, in two patients there was sequence evolution but no evidence of drug-resistant viral genotypes. In one patient, extensive in situ studies provided additional evidence of persistent viral replication in lymphoid tissues. Using two independent approaches, we estimated that the half-life of the latent, replication-competent virus in resting CD4 lymphocytes was approximately six months. CONCLUSIONS: These findings suggest that combination antiretroviral regimens suppress HIV-1 replication in some but not all patients. Given the half-life of latently infected CD4 lymphocytes of about six months, it may require many years of effective antiretroviral treatment to eliminate this reservoir of HIV-1.

Determinants of case selection at morning report.

OBJECTIVE: To determine why residents present certain cases and not others at morning report (MR) in an institution that permits residents the free choice of cases. DESIGN/PARTICIPANTS: Prospective survey of 10 second- and third-year residents assigned to the medical service. SETTING: A 241-bed teaching hospital with 55 categorical internal medicine residents. MEASUREMENTS AND MAIN RESULTS: Over a 4-week period, there were 194 admissions to the medical service on 18 call days preceding MR. Of these admissions, 30 (15%) were presented at MR. Cases were more likely to be presented if they were considered unusual or rare in presentation or incidence (P = .001), involved significant management issues (p = .001), or were associated with remarkable imaging studies or other visual material (p = .006). Residents were more likely to present cases in which they disagreed with attending physicians on management plans (p = .005). Overall, residents rated few admissions as having notable physical examination findings (29/194) or ethical or cost issues (6/194). Of the seven most common admitting diagnoses, representing 44% of admissions, residents did not present cases involving four of these diagnoses. CONCLUSIONS: Residents presented cases at MR that they felt were unique or rare in presentation or incidence for purposes of discussing management issues. Complete resident freedom in choosing MR cases may narrow the scope of MR and exclude common diagnoses and other issues of import such as medical ethics or economics.

Cryptosporidiosis in persons with HIV infection.

Cryptosporidium parvum is an ubiquitous protozoan parasite that is a major cause of diarrhoea in individuals infected with human immunodeficiency virus. The hallmarks of infection include profuse watery diarrhoea which may become chronic in the severely immunosuppressed individual. No uniformly effective therapy exists. Current treatment relies upon a trial of anti-retroviral and specific anti-cryptosporidial medications, adequate fluid and nutritional support, and anti-motility agents.

Management of persistent B19 parvovirus infection in AIDS.

An HIV+ 26-year-old white man with a CD4 count of 0.06 x 10(9)/l was found to have red blood cell aplasia secondary to B19 parvovirus infection. Regular infusions of intravenous immunoglobulin (IVIG) were begun and resulted in marked reticulocytosis and correction of anaemia. The patient has been followed for over 4 years and has become anaemic and reticulocytopenic whenever IVIG was interrupted. Serial dot blot analysis of the patient's sera for B19 parvovirus DNA showed absence of DNA immediately following IVIG treatments but reappearance within 3-6 weeks. Regular IVIG was effective in controlling but not eradicating B19 parvovirus infection in this HIV+ patient.