Morphine effects on HTLV-I infection in the presence or absence of concurrent HIV-1 infection.

Human T-cell leukemia virus type I (HTLV-I) infection is emerging as an important complication in HIV infection and AIDS in injecting drug users. HIV-1 and HTLV-I share a common host in CD4+ T lymphocytes. However, the result of HIV-1 infection is the decimation of this cell population, whereas a hallmark of HTLV-I infection is the inappropriate proliferation of infected cells. Combined epidemiologic data suggest that HTLV-I infection is enhanced during concurrent HIV-1/HTLV-I infection; however, there are currently no in vitro studies focusing on the effects of drugs of abuse on retrovirus coinfection. We have found that in an in vitro coinfection system (HIV-1 + HTLV-I), morphine treatment further enhanced the levels of HTLV-I p19. In addition, indicators of in vitro infection by cell-free HIV-1 were reduced by morphine treatment in both single and dual in vitro infection experiments. Interleukin 2 levels in the affected cultures were found to increase with combined HTLV-I infection and morphine treatment. These in vitro results indicate the need to further explore the activity of HTLV-I within opiate-treated cells, as this oncoretrovirus appears to be especially sensitive to morphine-induced alterations to its host cell environment.

Enhancement of cellular immune response in HIV-1 seropositive individuals: A DNA-based trial.

A DNA-based vaccine containing HIV-1 Env and Rev genes was tested for safety and host immune response in 15 HIV-infected asymptomatic patients with CD4-positive lymphocyte counts >/=500/microl of blood and receiving no antiviral therapy. Successive groups of patients received three doses of vaccine at 30, 100, or 300 microg at 10-week intervals in a dose-escalation trial. Some changes were noted in cytotoxic T-lymphocyte activity against gp160-bearing targets. Importantly, enhanced specific lymphocyte proliferative activity against HIV-1 envelope was observed in multiple patients. Three of three patients in the 300-microg dose group also developed increased MIP-1alpha levels which were detectable in their serum. Interestingly patients in the lowest dose group showed no overall changes in the immune parameters measured. The majority of patients who exhibited increases in any immune parameters were contained within the 300 microg, which was the highest dose group. These studies support further investigation of this technology for the production of antigen-specific immune responses in humans.

DNA vaccination with HIV-1 expressing constructs elicits immune responses in humans.

Humoral and cellular immune responses have been produced by intramuscular vaccination with DNA plasmids expressing HIV-1 genes, suggesting possible immunotherapeutic and prophylactic value for these constructs. Vaccination with these constructs has decreased HIV-1 viral load in HIV-1-infected chimpanzees. In addition, naive (i.e. non-HIV-1-infected) chimpanzees were protected against a heterologous challenge with HIV-1. Ongoing phase I clinical trials show that therapeutic vaccinations indeed boost anti-HIV-1 immune responses in humans. A therapeutic phase I trial on humans with these constructs induced a good safety profile and also demonstrated an immunological potentiation. These findings indicate that further studies with these constructs in humans are warranted.

Opiate effects on in vitro human retroviral infection.

The transmission and progression of the human retroviruses HIV-1 and HTLV-1/2 can be most likely influenced by a variety of "lifestyle cofactors" which includes the use of certain injected pharmaceuticals. Some investigations have suggested that HIV-1 infected individuals who are injecting drug users (IDUs) may undergo an accelerated rate of progression to AIDS. It is known that opioid receptors exist on cells pertinent to immune function, and that the activation or inhibition of these receptors may enhance or down-regulate some cell activities. The mechanisms for these effects have not yet been elucidated, nor have the effects of opioids on retroviral infection models been fully determined. While some work has been performed on the effects of opiates on infection by HIV-1 and SIV virtually no work has been done on the potential effects of this class of drugs on HTLV-1 and 2 infection. The potential effects of opiates on these retroviruses are important because of the higher incidence of infection in IDUs. Because IDUs compose one of the emerging high risk populations for infection with HIV-1 and more recently HTLV it is relevant to analyze the direct and indirect effects of opioids on the progression of retroviral infections. Our preliminary results from in vitro syncytia formation studies suggest a modulation by opioid-selective receptor agonists of in vitro infection by both HIV-1 and HTLV-I. These initial results underscore the necessity for further studies to define and elucidate the role of opiate abuse in the infection by human retroviruses as well as the associated pathogenesis.

Cannabinoid receptor agonists enhance syncytia formation in MT-2 cells infected with cell free HIV-1MN.

Marijuana and other drugs have been suggested to act as cofactors for HIV infection. Interestingly, delta 9-THC has been shown to upregulate NF kappa B, a transcription factor utilized by HIV. Therefore, it was of interest to investigate whether cannabinoids can modulate HIV infection and replication. Initially, we tested for evidence of receptor expression by examining for receptor mRNA in various cell lines used to study HIV infection and replication. Cellular RNA was isolated from SupT, and H9, H9MN, and MT-2 cells and RT-PCR was performed. Results showed that, although all of the cell lines tested were positive for CB2 mRNA, only the MT-2 cells also expressed CBI mRNA. Since the MT-2 cells expressed both CBI and CB2 receptor mRNA, we next wanted to determine whether different cannabinoid receptor agonists such as CP-55,940, delta 9-THC, WIN-55,212-2, and WIN-55,212-3 influenced infection of these cells by cell free HIV-1MN. Infectivity assays were performed where MT-2 cells were incubated with drug and cell free virus for 90 min, the free virus washed off, and the cells incubated further, and checked for virus growth by syncytia formation. It was found that the drugs significantly increased syncytia formation when MT-2 cells were cultured in the presence of both drug and cell free HIV-1MN. In conclusion, of the cell lines tested, only the MT-2 cells were positive for both CB1 and CB2 mRNA. In addition, since syncytia formation is an indication of virus infection and cytopathicity it was concluded that cannabimimetic drugs may enhance HIV-1 infection of susceptible cells.

Nucleic acid immunization of chimpanzees as a prophylactic/immunotherapeutic vaccination model for HIV-1: prelude to a clinical trial.

Vaccine development strategies have often utilized recombinant envelope glycoproteins which usually generate strong humoral immune responses but which do not generate strong cytotoxic T lymphocytes (CTL). A recent novel experimental vaccination approach involves the technology known as nucleic acid immunization in which DNA plasmids expressing a gene of interest is injected intramuscularly in experimental animals. These expressed proteins then are presented to the immune system with the subsequent development of strong antibody and cellular (particularly CTL) immune responses. These types of immune responses have been elicited in rodents as well as nonhuman primates including chimpanzees. Results from studies on nucleic acid immunization of HIV-1 infected chimpanzees with envelope glycoprotein expressing constructs indicated that this method was able to decrease substantially HIV-1 viral load in these chimpanzees. These data are useful for the development and implementation of human phase I clinical trials with HIV constructs expressing various genes from the HIV-1 genome.

Multiple determinants controlling activation of yeast replication origins late in S phase.

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.