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PubMed2006

Therapeutic strategies towards HIV-1 infection in macrophages.

It is widely recognized that macrophages (M/M) represent a crucial target of HIV-1 in the body and play a pivotal role in the pathogenic progression of HIV-1 infection. This strongly supports the clinical relevance of therapeutic strategies able to interfere with HIV-1 replication in M/M. In vitro studies showed that nucleoside analogue inhibitors of HIV-1 reverse transcriptase have potent antiviral activity in M/M, although the limited penetration of these compounds in sequestered body compartments and low phosphorylation ability of M/M, suggest that a phosphonate group linked to NRTIs may confer greater anti-HIV-1 activity in M/M. Differently, the antiviral activity of non-nucleoside reverse transcriptase inhibitors in M/M is similar to that found in CD4+ lymphocytes. Interestingly, protease inhibitors, acting at a post-integrational stage of HIV-1 life-cycle are the only drugs active in chronically infected M/M. A careful analysis of the distribution of antiviral drugs, and the assessment of their activity in M/M, represent key factors in the development of therapeutic strategies aimed to the treatment of HIV-1-infected patients. Moreover, testing new and promising antiviral compounds in such cells may provide crucial hints about their efficacy in patients infected by HIV.

Anti-HIV Agents↗
PubMedDate not supplied

Impact of tuberculosis on HIV-1 replication, diversity, and disease progression.

HIV and Mycobacterium tuberculosis not only co-circulate throughout the developing world but each has contributed to prevalence and mortality caused by the other. Several reports have described how HIV-1 increases the incidence of new M. tuberculosis infections, exacerbates the severity of tuberculosis (TB), and re-activates latent M. tuberculosis. However, the converse relationship is more difficult to understand considering TB can emerge in asymptomatic individuals and as an opportunistic infection during AIDS. Development of TB in HIV infected individuals with higher CD4 cell counts (> 200/mm3) appears to increase the rate of disease progression and mortality. Higher viral loads, increased HIV-1 diversity, and changes in cytokine/chemokine levels in HIV-infected individuals with TB appear to be related to a localized immune stimulation. Specifically, increased levels of TNF alpha and MCP-1, induced by TB, may activate HIV replication in lymphocytes, monocytes, and macrophages that are resident or have migrated to M. tuberculosis infected organs (e.g. pleura or lung). The HIV-1 found in blood following this TB-mediated burst in load and diversity appear to be phylogenetically-related to HIV-1 clones that have evolved independently in the lung or pleural compartments, now infected by M. tuberculosis.

AIDS-Related Opportunistic Infections↗
PubMed2000

A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children.

HIV-1 persists in a latent state in resting CD4(+) T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4(+) T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4(+) T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1-3 years on HAART, latent replication-competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4(+) T cells will be a major obstacle to HIV-1 eradication in children.

Adolescent↗
PubMed1988

Nuclear matrix associated poly(ADP-ribose) metabolism in regenerating rat liver.

We have examined a possible role for protein poly(ADP-ribosylation) during in vivo DNA replication by studying the metabolism of poly(ADP-ribose) in the nuclear matrix fraction from normal and regenerating rat liver. This fraction contains the newly replicated DNA and thus allows for the examination of the events closely associated with the replication process. It was found that 55% of the total nuclear protein-bound poly(ADP-ribose) and 15-35% of the total nuclear poly(ADP-ribose)-polymerase activity were tightly associated with this subnuclear compartment in normal liver. Surgical removal of two-thirds of the liver initiated a time-dependent decrease in nuclear matrix associated polymers of ADP-ribose and poly(ADP-ribose) polymerase activity which reached a minimum of 40% of control livers after 24 h, before returning to normal levels at 41 h post-partial hepatectomy. In contrast, the total levels of poly(ADP-ribose) in intact liver and the total polymerase activity of isolated nuclei exhibited a 2-fold increase over basal levels. These results are consistent with the conclusion that the nuclear matrix is a major poly(ADP-ribosylation) site within the nucleus and that this metabolic reaction may be closely connected with the events modulating DNA replication in this fraction.

Animals↗
PubMed1996

Acidification of phagosomes containing Salmonella typhimurium in murine macrophages.

Salmonella species are facultative intracellular pathogens. Following entry into mammalian host cells, they reside in membrane-bound vacuoles, resist killing, and replicate. In this work, we investigated the importance of phagosomal pH in the ability of Salmonella typhimurium to survive and replicate within macrophages. Intraphagosomal pH was measured in situ by recording the fluorescence intensity of a pH-sensitive probe, DM-NERF dextran. The majority of vacuoles containing S. typhimurium (live, heat killed, or formalin fixed) acidified from pH > or = 6.0 to between pH 4.0 and 5.0 within 60 min after formation. In contrast, Mycobacterium avium-containing vacuoles failed to acidify even at later time points. Acidification of S. typhimurium-containing vacuoles was completely blocked by treatment of host cells with bafilomycin A, a specific inhibitor of vacuolar proton-ATPases. Bafilomycin inhibition of vacuolar acidification from the onset of infection significantly decreased the survival of S. typhimurium in macrophages. Furthermore, bafilomycin treatment at 2, 4, 8, or even 12 h postinfection decreased the percentage of recoverable bacteria by up to 20-fold. Loss of bacterial viability was seen with several other reagents which, like bafilomycin, raise the pH of phagosomal compartments but are not directly lethal to the bacteria or host cells. Thus, we conclude that Salmonella-containing phagosomes acidify soon after formation and hypothesize that an acidic environment is necessary for survival and replication of the bacteria within the macrophage.

Animals↗
PubMed2006

Administration of fludarabine-loaded autologous red blood cells in simian immunodeficiency virus-infected sooty mangabeys depletes pSTAT-1-expressing macrophages and delays the rebound of viremia after suspension of antiretroviral therapy.

A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4+ T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4+ or CD8+ T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.

Adenine↗
PubMed2001

Live-cell microscopy of single nuclear chromosomes and genome compartments: evaluation of labeling procedure and imaging conditions.

BACKGROUND: Single chromosomes and genome compartments in nuclei of living mammalian cells can be analyzed microscopically after specific labeling with fluorescent dyes. This is achieved by incorporating fluorescent nucleotides into the chromosomal DNA during replication (Zink et al.: Hum Genet 102:241-251, 1998; Manders et al.: J Cell Biol 144:813-821, 1999; Sadoni et al.: J Cell Biol 146:1211-1226, 1999). We characterized the potential artificial impact of this approach on chromosome structure and dynamics. We also evaluated potential sources of artifacts in corresponding live-cell imaging. MATERIALS AND METHODS: The subchromosomal distribution of labeled DNA was analyzed, and the fate of labeled nucleotides within cell nuclei was studied. Cell-cycle parameters were used to analyze cell function after incorporation of fluorescent nucleotides. The influences of phototoxic effects on cell division and morphology were studied. RESULTS: Fluorescent nucleotides were only incorporated for a restricted time period during S-phase, and a uniform labeling of chromosomal DNA could not be achieved. Fluorescent nucleotides incorporated into the DNA showed no or only mild effects on cell growth. Cell-cycle parameters and cellular morphology were valuable indicators for proper cell function during live-cell imaging. CONCLUSIONS: There is no indication for a substantial impairment of cellular functions if fluorochromes are covalently linked to chromosomal DNA. The controls we present for proper cell function during the imaging period are of general importance, as appropriate controls for live cell microscopy have not yet been well-defined.

Artifacts↗
PubMed1987

Cell proliferation studies in rat prostate. I. The proliferative role of basal and secretory epithelial cells during normal growth.

In the prostates of rats ranging from 10 days to 8 months of age, the proliferative activity of basal and secretory epithelial cells was studied. No clear evidence was found that basal cells alone represented the proliferative compartment, or that they were responsible for the replacement of secretory cells during normal turnover. Cell kinetic and morphological evidence indicated that basal and secretory cells were self-replicating cell types with discrete functions.

Animals↗
PubMedDate not supplied

The Nramp1 protein and its role in resistance to infection and macrophage function.

Susceptibility to infectious diseases is under genetic control in humans. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. The Nramp1 gene was isolated by positional cloning the host resistance locus Bcg/Ity/Lsh, and mutations at this locus impair the resistance of mice to infections with intracellular parasites, such as Salmonella, Leishmania, and Mycobacterium. Allelic variants at the human Nramp1 homologue have recently been found to be associated with susceptibility to tuberculosis and leprosy in humans. The Nramp1 protein is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication. Although the biochemical mechanism of action of Nramp1 at that site remains unknown, Nramp homologues have been identified in many other animal species and actually define a protein family conserved from bacteria to humans. Some of these homologues have been shown to be divalent cation transporters. Recently, a second member of the mammalian Nramp family, Nramp2, was discovered and shown to be mutated in animal models of iron deficiency. The Nramp2 protein was subsequently shown to be the major transferrin-independent iron uptake system of the intestine. Together, these results suggest that Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space.

Animals↗
PubMed1980

Subcompartments of the G1 phase of cell cycle detected by flow cytometry.

Cellular DNA and RNA were simultaneously quantitated in individual cells of cycling and quiescent populations by flow cytometry. Based on differences in RNA content, two distinct subcompartments, G1A and G1B, were recognized in the G1 phase of exponentially growing cells. After mitosis, cells reside in the low-RNA, G1A compartment from which they cannot enter the S phase directly. An increase in RNA above a critical level is required for G1 cells to be able to initiate DNA replication; G1 cells with RNA values above this level are classified as G1B. Cell transition from G1A to G1B and from G1B to S was analyzed in a stathmokinetic experiment on L1210 cells with a doubling time of 10 hr. The half-time of cell residence in the indeterminate state of G1 was found to be 1 hr, and the duration of the deterministic portion of G1 phase was 2 hr. The indeterminate state, although not identical with G1A, is most likely located in G1A. Cell quiescence induced by serum deprivation (3T3 cells) or by addition of n-butyrate (L1210) results in cell arrest at a state which, judged by RNA content, is similar to that of G1A of exponentially growing cells. The exit from this state, however, is much slower after stimulation of these blocked cells than the transition of G1A to G1B in cells growing exponentially. Thymidine or hydroxyurea arrest cells in G1B or perhaps at the border between G1B and S. Prolonged incubation of 3T3 cells to confluence results in a marked loss of cellular RNA below the level of the G1A state. This deep quiescent state (G1Q) is distinctly different from the G1A state of cycling cells or cells blocked in G1A by serum deprivation.

Acridine Orange↗
PubMed2002

Human immunodeficiency virus type 1 (HIV-1) quasispecies at the sites of Mycobacterium tuberculosis infection contribute to systemic HIV-1 heterogeneity.

We have recently reported an increased heterogeneity in the human immunodeficiency virus type 1 (HIV-1) envelope gene (env) in HIV-1-infected patients with pulmonary tuberculosis (TB) compared to patients with HIV-1 alone. This increase may be a result of dissemination of lung-derived HIV-1 isolates from sites of Mycobacterium tuberculosis infection and/or the systemic activation of the immune system in response to TB. To distinguish between these two mechanisms, blood and pleural fluid samples were obtained from HIV-1-infected patients with active pleural TB in Kampala, Uganda (CD4 cell counts of 34 to 705 cells/microl, HIV-1 plasma loads of 2,400 to 280,000 RNA copies/ml, and HIV-1 pleural loads of 7,600 to 4,500,000 RNA copies/ml). The C2-C3 coding region of HIV-1 env was PCR amplified from lysed peripheral blood mononuclear cells and pleural fluid mononuclear cells and reverse transcriptase-PCR amplified from plasma and pleural fluid HIV-1 virions of eight HIV-1 patients with pleural TB. Phylogenetic and phenetic analyses revealed a compartmentalization of HIV-1 quasispecies between blood and pleural space in four of eight patients, with migration events between the compartments. There was a trend for a greater genetic heterogeneity in the pleural space, which may be the result of an M. tuberculosis-mediated increase in HIV-1 replication and/or selection pressure at the site of infection. Collectively, these findings suggest that HIV-1 quasispecies in the M. tuberculosis-infected pleural space may leak into the systemic circulation and lead to increased systemic HIV-1 heterogeneity during TB.

AIDS-Related Opportunistic Infections↗
PubMed1998

Peripheral blood-derived CD34+ progenitor cells: CXC chemokine receptor 4 and CC chemokine receptor 5 expression and infection by HIV.

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.

Cells, Cultured↗
PubMed2001

An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic alpha-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.

Amino Acid Sequence↗
PubMed1996

What drives codon choices in human genes?

Synonymous codon usage is based and the bias seems to be different in different organisms. Factors with proposed roles in causing codon bias include degree and timing of gene expression, codon-anticodon interactions, transcription and translation rate and fidelity, codon context, and global and local G + C content. We offer a new perspective and new methods for elucidating codon choices applied especially to the human genome. We present data supporting the thesis that codon choices for human genes are largely a consequence of two factors: (1) amino acid constraints, (2) maintaining DNA structures dependent on base-step conformational tendencies consistent with the organism's genome signature that is determined by genome-wide processes of DNA modification, replication and repair. The related codon signature defined as the dinucleotide relative abundances at the distinct codon positions (1,2), (2,3), and (3,4) (4 = 1 of the next codon) accommodates both the global genome signature and amino acid constraints. In human genes, codon positions (2,3) and (3,4) containing the silent site have similar codon signatures reflecting DNA symmetry. Strong CG and TA dinucleotide underrepresentation is observed at all codon positions as well as in non-coding regions. Estimates of synonymous codon usage based on codon signatures are in excellent agreement with the actual codon usage in human and general vertebrate genes. These properties are largely independent of the isochore compartment (G + C content), gene size, and transcriptional and translational constraints. We hypothesize that major influences on codon usage in human genes result from residue preferences and diresidue associations in proteins coupled to biases on the DNA level, related to replication and repair processes and/or DNA structural requirements.

Codon↗
PubMed1996

Bacterial lipopolysaccharide confers resistance to G418, doxorubicin, and taxol in the murine macrophage cell line, RAW264.

Many bacterial pathogens including Salmonella and Listeria replicate within macrophages. The susceptibility of these organisms to various antibiotics is dependent on the ability of macrophages to take up, retain, and deliver the antibiotic to the correct intracellular compartment. In this context, macrophages are known to express proteins that are involved in efflux of antibiotics and cytotoxic drugs, thereby reducing intracellular accumulation of such compounds. In our studies on the action of bacterial lipopolysaccharide (LPS) on the macrophage-like cell line, RAW264 we found that LPS treatment of these cells conferred resistance to the neomycin-related aminoglycoside G418 (geneticin). This phenotype was stable and was specific to LPS since colony-stimulating factor 1 and phorbol myristate acetate had no effect on G418 resistance. We have extended this observation to show that LPS induces transient resistance to the cytotoxic drugs taxol and doxorubicin. Macrophage resistance to cytotoxic drugs and antibiotics may have a number of important clinical consequences.

Animals↗
PubMed2001

Reducing the genome size of organelles favours gene transfer to the nucleus.

Endosymbiotic organelles exhibit strong genetic erosion during their evolution as a result of the loss of unnecessary genes and of gene transfer to the nucleus. The reasons for this erosion are much debated. Unidirectionality of DNA exchange between cell compartments could favour biased gene transfer, but selection might also act to favour nuclear localization of genes, for example, because organelles accumulate more mutations than do nuclei. Selection for rapid replication might be a general cause of organelle genome reduction. This selection also accounts for the compactness of organelle genomes.

Journal Article↗
PubMedDate not supplied

Reliability and validity of an algorithm for fuzzy tissue segmentation of MRI.

PURPOSE: A new multistep, volumetric-based tissue segmentation algorithm that results in fuzzy (or probabilistic) voxel description is described. This algorithm is designed to accurately segment gray matter, white matter, and CSF and can be applied to both single channel high resolution and multispectral (multiecho) MR images. METHOD: The reliability and validity of this method are evaluated by assessing (a) the stability of the algorithm across time, rater, and pulse sequence; (b) the accuracy of the method when applied to both real and synthetic image datasets; and (c) differences in specific tissue volumes between individuals with a specific genetic condition (fragile X syndrome) and normal control subjects. RESULTS: The algorithm was found to have high reliability, accuracy, and validity. The finding of increased caudate gray matter volume associated with the fragile X syndrome is replicated in this sample. CONCLUSION: Since this segmentation approach incorporates "fuzzy" or probabilistic methods, it has the potential to more accurately address partial volume effects, anatomical variation within "pure" tissue compartments, and more subtle changes in tissue volumes as a result of disease and treatment. The method is a component of software that is available in the public domain and has been implemented on an inexpensive personal computer thus offering an attractive and promising method for determining the status and progression of both normal development and pathology of the CNS.

Adolescent↗
PubMed2001

HIV-1 induction-maintenance at the lymph node level: the "Apollo-97" Study.

OBJECTIVE: To assess the effects of five-drug combination therapy on HIV-1 load in lymph nodes and subsequent maintenance with four and three drugs. METHODS: Ten pharmacotherapeutically naive patients received a combination of zidovudine, lamivudine, didanosine, ritonavir, and saquinavir for 24 weeks, then zidovudine, lamivudine, didanosine, and saquinavir for the next 24 weeks, and finally zidovudine, lamivudine, and saquinavir for the last 24 weeks. HIV-1 RNA in lymph nodes was measured using quantitative polymerase chain reaction (PCR) at baseline, after 12, 24, 48, and 78 weeks. Plasma HIV-1 RNA, proviral DNA in peripheral blood mononuclear cells (PBMCs), circulating lymphocyte subsets, and protease inhibitor levels in blood were also regularly measured. Genotypic resistance was assessed in the different compartments in 2 patients who were failed by therapy. RESULTS: HIV-1 RNA decreased in lymph nodes in 9 patients and was stable in 1 despite initial control of plasma replication <20 copies/ml in each patient. Lymph node levels rebounded in 1 patient at week 72 as a result of lack of adherence and remained stable in the 8 others despite maintenance regimens. This represents a mean drop of -3.17 log in lymph nodes for the 8 patients maintaining undetectable viremia at 72 weeks. In the patient with stable lymph node viral RNA, selection of the M184V mutation was demonstrated at this level before detection in plasma and low blood saquinavir levels were found throughout the study. Continuous improvements in immune parameters were observed in all cases, although PBMC proviral DNA levels either showed a continuous decrease or stabilized to a plateau. CONCLUSIONS: More complex regimens do not perform better in lymph nodes than classic triple therapy. The persistence of HIV-1 RNA in lymph nodes could be related with cellular resistance mechanisms rather than an insufficient potency of the regimens.

Adult↗