Clonal analysis of T-cell responses to the HIV-1 envelope proteins in AIDS vaccine recipients.

Both CD4+ and CD8+ CTL responses specific for the HIV-1 envelope proteins can be elicited in seronegative humans by candidate AIDS vaccines. The phenotype of the responding CTL depends upon the nature of the vaccine, with CD8+ CTL being found exclusively in recipients of live virus vaccines. Both types of CTL are active against HIV-1-infected cells in vitro. However, the potential efficacy of vaccine-induced CTL in preventing infection in vaccinated individuals exposed to HIV-1 is unknown and is likely to be dependent upon complex factors including lytic activity against divergent strains, cytokines produced, and the lysis of noninfected CD4+ T cells.

Hepatitis A.

Hepatitis A remains an important cause of community-acquired hepatitis in the United States and in the world. In recent years, improvements in personal hygiene and environmental sanitation have led to declines in overall hepatitis A infection rates in developed countries, although sporadic outbreaks still occur with similar rates of hospitalization and loss of work. Therapy remains supportive and prevention holds the key to elimination of widespread infection. Acute infection can be prevented or attenuated with IG or with inactivated, highly immunogenic vaccines. Elderly persons and those with advanced liver disease are at higher risk of the consequences of acute HAV, and they represent target populations for immediate vaccination. Challenges for the future include strategies for broad-based population vaccination, including cost-effective approaches.

Intrasphincteric injection of botulinum toxin for suspected sphincter of Oddi dysfunction.

Botulinum toxin is a potent inhibitor of the release of acetylcholine from nerve endings. It has previously been shown that it can effectively reduce lower oesophageal sphincter pressures both in animals and humans with achalasia. This study examined the hypothesis that locally injected botulinum toxin could also reduce sphincter of Oddi pressure in patients with sphincter of Oddi dysfunction. Two patients with postcholecystectomy pain syndrome were diagnosed with sphincter of Oddi dysfunction (by biliary manometry in one patient and by hepatobiliary scanning criteria in the other). Botulinum toxin was injected into the sphincter of Oddi, by a sclerotherapy needle passed through a duodenoscope. In the first patient, intrasphincteric injection of botulinum toxin reduced sphincter pressure by about 50%, an effect that was sustained for at least four months. In the second patient, intrasphincteric injection caused about a 50% improvement in bile flow, with normalisation of scintigraphy. Neither patient showed any sustained improvement in pain despite these objective findings. Both patients eventually had endoscopic sphincterotomy, which also did not result in symptomatic improvement in either patient. No side effects were seen. Intrasphincteric botulinum toxin is a simple and effective means of lowering sphincter of Oddi pressure. This technique has potential for being useful clinically.

Production of transmembrane and secreted forms of tumor necrosis factor (TNF)-alpha by HIV-1-specific CD4+ cytolytic T lymphocyte clones. Evidence for a TNF-alpha-independent cytolytic mechanism.

Candidate AIDS vaccines consisting of recombinant forms of the HIV-1 envelope glycoprotein induce, in seronegative human volunteers, an env-specific T cell response that includes CD4+, MHC class II-restricted CTL capable of lysing HIV-1-infected target cells. In this study, we have analyzed the production of the cytokines TNF-alpha and lymphotoxin (LT) by a set of env-specific CD4+ human CTL clones. TNF-alpha and LT are of interest because of their potential role in target cell destruction by CD4+ CTL. Our studies focused on the possibility that a cell surface form of TNF-alpha expressed by CTL after physiologic activation with target APC might participate in the cytolytic reactions mediated by these clones. We found that, upon interaction with target cells expressing env epitopes in the context of the appropriate MHC class II molecules, CD4+ CTL released TNF-alpha with kinetics that were rapid, compared with other cytokines, and that were generally similar to the kinetics of target cell destruction. LT secretion was not detected during the time course of the cytolytic reactions. A novel flow cytometric assay was used to show that physiologic activation of CD4+ CTL with target APC induced expression by the CTL of cell surface forms of TNF-alpha. Immunoprecipitations from activated, surface-iodinated CTL clones revealed two forms of surface TNF-alpha, a 26-kDa form, representing the transmembrane precursor of secreted TNF-alpha, as well as the 17-kDa secreted form bound to the cell surface. For a subset of CD4+ CTL, we found that treatment of CTL with cyclosporin A inhibited Ag-induced production of both transmembrane and secreted forms of TNF-alpha but had no effect on cytolysis. Thus, although transmembrane and secreted TNF-alpha produced by HIV-1-specific CD4+ CTL may have important effects in vivo, the rapid destruction of target APC by the set of CD4+ CTL clones described here occurs through a TNF-alpha-independent mechanism.

Clinical characterization of a competitive PCR assay for quantitative testing of hepatitis C virus.

Rational clinical application of quantitative assessments of hepatitis C virus (HCV) RNA depends on an understanding of factors affecting the assay and its intrinsic variability. The effects of three types of blood collection tubes, two storage temperatures, five processing times, and two laboratories on a commercially available quantitative reverse transcriptase PCR assay (AMPLICOR HCV MONITOR) were evaluated. HCV RNA concentrations were assessed in 356 specimens representing 178 aliquots from nine patients. In a multivariate generalized linear model, HCV RNA concentrations decreased when centrifugation was delayed more than 6 h (P = 0.005) and were marginally different between laboratories (P = 0.06), but precentrifugation storage temperature (P = 1.00) and anticoagulation (P = 0.22) had no effect. After adjusting for other factors, the HCV concentration of 95% of a subject's samples were within 0.44 log. Specimens procured for reverse transcriptase PCR-based quantitative HCV testing should be centrifuged within 6 h of collection. Serial assessments should ideally be performed in the same laboratory, and changes in HCV RNA concentration of less than 0.44 log may not be biologically important.

Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines.

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.