Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells.

The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection.

Cellular protein kinase C isoform zeta regulates human parainfluenza virus type 3 replication.

Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.

CSF-1 as a regulator of macrophage activation and immune responses

Macrophage activation is a key determinant of susceptibility and pathology in a variety of inflammatory diseases. The extent of macrophage activation is tightly regulated by a number of pro-inflammatory cytokines (e.g. IFN-gamma, IL-2, GM-CSF, IL-3) and anti-inflammatory cytokines (e.g. IL-4, IL-10, TGF-beta). Macrophage colony-stimulating factor (CSF-1/M-CSF) is a key differentiation, growth and survival factor for monocytes/macrophages and osteoclasts. The role of this factor in regulating macrophage activation is often overlooked. This review will summarize our current understanding of the effects of CSF-1 on the activation state of mature macrophages and its role in regulating immune responses.

Antiretrovirals as antimalarial agents

Recent studies have indicated that antiretroviral protease inhibitors may affect outcome in malarial disease. We have investigated the antimalarial activities of 6 commonly used antiretroviral agents. Our data indicate that, in addition to the previously published effects on cytoadherence and phagocytosis, the human immunodeficiency virus (HIV)-1 protease inhibitors saquinavir, ritonavir, and indinavir directly inhibit the growth of Plasmodium falciparum in vitro at clinically relevant concentrations. These findings are particularly important in light of both the high rate of malaria and HIV-1 coinfection in sub-Saharan Africa and the effort to employ highly active antiretroviral therapy in these regions.

The impact of changes to heroin supply on blood-borne virus notifications and injecting related harms in New South Wales, Australia

Background: In early 2001 Australia experienced a sudden and unexpected disruption to heroin availability, know as the 'heroin shortage'. This 'shortage has been linked to a decrease in needle and syringe output and therefore possibly a reduction in injecting drug use. We aimed to examine changes, if any, in blood-borne viral infections and presentations for injecting related problems related to injecting drug use following the reduction heroin availability in Australia, in the context of widespread harm reduction measures. Methods: Time series analysis of State level databases on HIV, hepatitis B, hepatitis C notifications and hospital and emergency department data. Examination of changes in HIV, hepatitis B, hepatitis C notifications and hospital and emergency department admissions for injection-related problems following the onset of the heroin shortage; non-parametric curve-fitting of number of hepatitis C notifications among those aged 15 - 19 years. Results: There were no changes observed in hospital visits for injection-related problems. There was no change related to the onset heroin shortage in the number of hepatitis C notifications among persons aged 15 - 19 years, but HCV notifications have subsequently decreased in this group. No change occurred in HIV and hepatitis B notifications. Conclusion: A marked reduction in heroin supply resulted in no increase in injection-related harm at the community level. However, a delayed decrease in HCV notifications among young people may be related. These changes occurred in a setting with widespread, publicly funded harm reduction initiatives.

Oral viral infections and the therapeutic use of antiviral agents in dentistry

This paper reviews the current concepts of viral classification, infection and replication. The clinical presentation of common oral viral infections encountered in the dental practice are discussed, including: herpes simplex virus types 1 and 2; Epstein-Barr virus; varicella-zoster virus; Coxsackie virus; human papilloma virus; and human immunodeficiency virus. The diagnosis, principles of management and pharmacological agents available for the treatment of oral viral infections are also discussed.

Using cohort studies to estimate mortality among injecting drug users that is not attributable to AIDS

Background: Injecting drug use (IDU) and associated mortality appear to be increasing in many parts of the world. IDU is an important factor in HIV transmission. In estimating AIDS mortality attributable to IDU, it is important to take account of premature mortality rates from other causes to ensure that AIDS related mortality among injecting drug users (IDUs) is not overestimated. The current review provides estimates of the excess non-AIDS mortality among IDUs. Method: Searches were conducted with Medline, PsycINFO, and the Web of Science. The authors also searched reference lists of identified papers and an earlier literature review by English et al (1995). Crude. mortality rates (CMRs) were derived from data on the number of deaths, period of follow UP, and number of participants. In estimating the all-cause mortality, two rates were calculated: one that included all cohort studies identified in the search, and one that only included studies that reported on AIDS deaths in their cohort. This provided lower and upper mortality rates, respectively. Results: The current paper derived weighted mortality rates based upon cohort studies that included 179 885 participants, 1 219 422 person-years of observation, and 16 593 deaths. The weighted crude AIDS mortality rate from studies that reported AIDS deaths was approximately 0.78% per annum. The median estimated non-AIDS mortality rate was 1.08% per annum. Conclusions: Illicit drug users have a greatly increased risk of premature death and mortality due to AIDS forms a significant part of that increased risk; it is, however, only part of that risk. Future work needs to examine mortality rates among IDUs in developing countries, and collect data on the relation between HIV and increased mortality due to all causes among this group.

theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Greater CD8+ TCR heterogeneity and functional flexibility in HIV-2 compared to HIV-1 infection

Virus-specific CD8+ T cells are known to play an important role in the control of HIV infection. In this study we investigated whether there may be qualitative differences in the CD8+ T cell response in HIV-1- and HIV-2-infected individuals that contribute to the relatively efficient control of the latter infection. A molecular comparison of global TCR heterogeneity showed a more oligoclonal pattern of CD8 cells in HIV-1- than HIV-2-infected patients. This was reflected in restricted and conserved TCR usage by CD8+ T cells recognizing individual HLA-A2- and HLA-B57-restricted viral epitopes in HIV-1, with limited plasticity in their response to amino acid substitutions within these epitopes. The more diverse TCR usage observed for HIV-2-specific CD8 T cells was associated with an enhanced potential for CD8+ expansion and IFN- production on cross-recognition of variant epitopes. Our data suggest a mechanism that could account for any possible cross-protection that may be mediated by HIV-2-specific CD8+ T cells against HIV-1 infection. Furthermore, they have implications for HIV vaccine development, demonstrating an association between a polyclonal, virus-specific CD8+ T cell response and an enhanced capacity to tolerate substitutions within T cell epitopes.

Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5' -untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3-'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

Genomic variation of HIV-1 and its effects on drug resistance and molecular epidemiology analyses

In the first part of this study human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences derived from 201 clones of the C2-V3 env region and the first exon of the tat gene were obtained from six MV-1 infected heterosexual couples. These molecular data were used to confirm the epidemiological relationships. The ability of the molecular data to draw such conclusions was also tested with multiple phylogenetic analyses. The tat region was much more useful in establishing epidemiological relationships than the commonly used C2-V3.^ Subsequently, using nucleotide sequences from the first exon of the Tat gene, we tested the hypothesis that a Florida dentist (a common source) infected five of his patients in the course of dental procedures, against the null hypothesis that the dentist and each individual of the dental group independently acquired the virus within the local community. Multiple phylogenetic analyses demonstrated that the sequences of the five patients were significantly more related to each other than to sequences of the controls. Our results using Tat sequences, combined with envelope sequence data, strongly support a common phylogenetic epidemiological relationship among these five patients.^ A third study is presented, which deals with the effects of genomic variations in drug resistance. HIV-1 reverse transcriptase (RT) mutations were detected in DNA from peripheral blood mononuclear cells from 11 of 12 HIV-infected children after 11-20 months of zidovudine monotherapy. The codon 41/215 mutant combination was associated with general decline in health status. Patients developing the codon 70 mutation tended to have a better health status. ^

Impact of nutrition education on knowledge and eating patterns in HIV-infected individuals

Acquired Immune Deficiency Syndrome (AIDS) and impaired or threatened nutritional status seem to be closely related. It is now known that AIDS results in many nutritional disorders including anorexia, vomiting, protein-energy malnutrition (PEM), nutrient deficiencies, and gastrointestinal, renal, and hepatic dysfunction (1-7, 8). Reversibly, nutritional status may also have an impact on the development of AIDS among HIV-infected people. Not all individuals who have tested antibody positive for the Human Immunodeficiency Virus (HIV) have developed AIDS or have even shown clinical symptoms (9, 10). A poor nutritional status, especially PEM, has a depressing effect on immunity which may predispose an individual to infection (11). It has been proposed that a qualitatively or quantitatively deficient diet could be among the factors precipitating the transition from HIV-positive to AIDS (12, 13). The interrelationship between nutrition and AIDS reveals the importance of having a multidisciplinary health care team approach to treatment (11), including having a registered dietitian on the medical team. With regards to alimentation, the main responsibility of a dietitian is to inform the public concerning sound nutritional practices and encourage healthy food habits (14). In individuals with inadequate nutritional behavior, a positive, long-term change has been seen when nutrition education tailored to specific physiological and emotional needs was provided along with psychological support through counseling (14). This has been the case for patients with various illnesses and may also be true in AIDS patients as well. Nutritional education specifically tailored for each AIDS patient could benefit the patient by improving the quality of life and preventing or minimizing weight loss and malnutrition (15-17). Also, it may influence the progression of the disease by delaying the onset of the most severe symptoms and increasing the efficacy of medical treatment (18, 19). Several studies have contributed to a dietary rationale for nutritional intervention in HIV-infected and AIDS patients (2, 4, 20-25). Prospective, randomized clinical research in AIDS patients have not yet been published to support this dietary rationale; however, isolated case reports show its suitability (3). Furthermore, only nutrition intervention as applied by a medical team in an institution or hospital has been evaluated. Research is lacking concerning the evaluation of nutritional education of either non-institutionalized or hospitalized groups of persons who are managing their own food choice and intake. This study compares nutrition knowledge and food intakes in HIV-infected individuals prior to and following nutrition education. It was anticipated that education would increase the knowledge of nutritional care of AIDS patients and lead to better implementation of nutrition education programs.

The Immunomodulatory Role of Alcohol on HIV-Infected Monocyte-Derived Dendritic Cells

Alcohol is known to induce inflammation in the presence of the human immunodeficiency virus (HIV). In our previous studies, we revealed that alcohol induces cannabinoid receptors which play a role in the regulation of inflammatory cytokine production in monocyte-derived dendritic cells (MDDC). However, the ability of alcohol to alter MDDC function during HIV infection has not been clearly elucidated yet. To study the potential impact of alcohol on HIV-infected MDDC (confirmed by p24 ELISA), monocytes were isolated from commercially available buffy coats and cultured for 7 days with GM-CSF and IL-4. MDDC were infected with HIV- 1Ba-L and treated with different concentrations of alcohol (0.1% band 0.2%) for 4-7 days. MDDC phenotype, endocytosis, cytokine production, and ability to transmit HIV to T cells were analyzed. Uninfected CD4+ T cells were co-cultured for 7 days with either infected/treated MDDC or the supernatants from infected/treated MDDC. Inflammatory cytokine arrays were performed using supernatants from HIV-infected MDDC treated with alcohol. Results showed that HIV positive MDDC treated with alcohol had higher levels of infection compared to untreated HIV positive controls. CD4+ T cells exposed to HIV-infected MDDC acquired 100-fold higher levels of p24 compared to CD4+ T cells exposed to only supernatants. CD4+ T cells exposed to HIV-infected and alcohol-treated MDDC had higher levels of infection compared to controls. Cytokine array data show dysregulation of cytokine production by alcohol. In addition, MDDC phenotype and endocytic capacity were altered in the alcohol treated MDDC. Our results indicate a crucial role of MDDC in HIV transmission to T cells and provide insights into the inflammatory role alcohol exerts on dendritic cell function in the context of HIV infection. Supported by the National Institute on Alcohol Abuse and Alcoholism award R00AA021264, the National Institute on Drug Abuse award R01DA034547, and the Institute on NeuroImmune Pharmacology at FIU.