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PubMed2000

Interaction of Mycobacterium avium-containing phagosomes with the antigen presentation pathway.

Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomal characteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC class II molecules. Indeed, in resting macrophages MHC class II levels decreased strongly in phagosomes containing M. avium during a 4-day infection. Phagosomal MHC class II of early (4 h) infections was partly surface-derived and associated with peptide. Activation of host macrophages led to the appearance of H2-M, a chaperon of Ag loading, and to a strong increase in MHC class II molecules in phagosomes of acute (1 day) infections. Comparison with the kinetics of MHC class II acquisition by IgG-coated bead-containing phagosomes suggests that the arrest in phagosome maturation by mycobacteria limits the intersection of mycobacteria-containing phagosomes with the intracellular trafficking pathways of Ag-presenting molecules.

Animals↗
PubMed2003

Persistence of HIV-1 drug resistance mutations and emergence during antiretroviral treatment interruption: considerations from a clinical case.

BACKGROUND: HIV drug resistance (HDR) represents a crucial problem in antiretroviral therapy today. HDR mutations can accumulate, persist in the proviral genome and re-emerge, promoting further failure of rescue regimens. Resistance testing on the proviral genome has been suggested in order to detect the presence of archived resistance mutations and to guide drug prescription. CASE REPORT: We report the persistence of HDR mutations to protease inhibitors (PIs) during effective highly active antiretroviral therapy (HAART) not including PIs and their transient re-emergence after treatment interruption. Further follow-up has demonstrated a progressive reversion toward the wild type. At that point in time, proviral DNA analysis was not more sensitive in detecting mutations than tests of the plasma viral load. CONCLUSIONS: This case demonstrates that HDR mutations can persist even though plasma viral load is undetectable, implying the ability of HIV to escape from treatment recycling and the use of drugs to which HIV is cross-resistant. Also, this case suggests that plasmatic and intracellular HIV compartments find a balance under conditions of active viral replication, which could hamper detection of resistance mutations in the proviral DNA. Therefore, in the case of active viral replication, proviral analysis may not actually provide more information than testing the plasma viral load. The clinical utility and specific indications for testing proviral DNA require further investigation.

Anti-HIV Agents↗
PubMedDate not supplied

Cell renewal and cancer risk of the stomach: analysis of cell proliferation kinetics in atrophic gastritis.

We examined cell proliferation kinetics of gastric mucosa in 28 patients affected by chronic atrophic gastritis by means of autohistoradiography of biopsies incubated with tritiated thymidine. The results have been compared with those obtained from 12 patients with normal gastric mucosa. In chronic atrophic gastritis, patients showed a proliferative pattern similar to controls. The remaining patients had an increased number of replicating cells together with an expansion of the proliferative compartment towards the surface of the mucosa. These results suggest that in chronic atrophic gastritis, as far as cell proliferation is concerned, two subgroups of patients with two different levels of risk of developing gastric cancer exist. The first one, showing an expansion of the proliferating area, probably is at higher risk. As a matter of fact, such an abnormality expresses an alteration of cell growth control similar to that observed in preneoplastic conditions of the colon and in gastric mucosa of animals treated with carcinogenic substances.

Adult↗
PubMed1986

Analysis of growth fractions and stem cell compartments in transformed rat tracheal epithelial cell colonies.

The purpose of the studies described here was to define the biological behavior of the various clonally transformed colonies observed in cultures of carcinogen-exposed rat tracheal epithelial cells. As described in the preceding paper (H. Kitamura et al., Cancer Res., 46: 4631-4641, 1986), these colonies fall into four morphologically distinct categories. In the studies reported here we found that type I colonies had the smallest growth fraction (7%) and contained the lowest frequency of clonogenic cells (approximately 10(-3)). Colonies of types II to IV had mean growth fractions of 21 to 28%, and the frequency of clonogenic cells was 2 to 5 X 10(-2) when measured under growth-permissive conditions (3T3 feeders). When the clonogenic cell assays were performed under selective conditions to identify cell variants which can grow without feeder support, the average frequency of such clonogenic cells in type I colonies was less than 4 X 10(-5) and in colonies of types II to IV, between 5 X 10(-4) and 10(-2). In type IV colonies, the total number of cells per colony increased 8-fold between 5 and 12 wk postcarcinogen, but the clonogenic cell compartment increased 42-fold; the compartment of variant clonogenic cells, which are able to replicate on plastic, increased 139-fold during the same period of time. This indicated that major changes in the self-renewal capacity of the clonogenic cells were taking place during this early stage of transformation. Examination of the daughter colonies produced by replating colonies of types I to IV revealed that clonogenic cells with different growth potential existed within the same parent colony. Comparison of transformed colonies of the same type showed a marked degree of heterogeneity in the sizes of growth fractions and clonogenic cell fractions. These studies further indicated that, within all colonies, including the most advanced transformants, the majority of the cells were nonreplicating, terminal cells, suggesting that, at least during early stages of transformation, the transformed characteristics were not transferred from parent to daughter cells. With the exception of type I colonies, most of the colonies recognizable at 5 wk after carcinogen exposure progressed with time and acquired the morphological characteristics of type IV colonies, which were the most transformed phenotype. We conclude that transformation of rat tracheal epithelial cells is an asynchronous process and that the morphologically distinct types of rat tracheal epithelial cell colonies represent different stages of the clonal evolution of transformants.

Animals↗
PubMed1998

Cells transfected with a non-neutralizing antibody gene are resistant to HIV infection: targeting the endoplasmic reticulum and trans-Golgi network.

Plasmids containing single chain Fv (scFv) non-neutralizing human anti-HIV-1 gp41 Ab cDNA, with or without endoplasmic reticulum (ER) or trans-Golgi network (TGN) retention signals, were constructed. Stable transfectants expressing these scFvs then were generated from COS-7 cells and HIV-1-susceptible CD4+ human T cells (Jurkat). scFv without a retention signal was secreted from cells, whereas scFv with an ER or TGN retention signal remained primarily within targeted intracellular compartments. The expression of scFv, scFv-ER, and scFv-TGN did not adversely affect the appearance of uninfected cells, as measured by growth rate or CD4 expression. Pulse-chase experiments revealed that the t(1/2) of scFv-ER and scFv-TGN within cells was greater than 24 h and less than 9 h, respectively. The scFv-ER and scFv-TGN bound HIV gp160, and the scFv-ER-gp160 and the scFv-TGN-gp160 complexes were stable within HIV-infected transfectants. Further studies revealed that the maturation processing of gp160 into gp120 and gp41 was blocked in the scFv-ER transfectants, but not in the scFv-TGN transfectants. Moreover, HIV replication, as measured by p24, was inhibited by up to 99% in cells transfected with scFv-ER or scFv-TGN, but was not inhibited in cells transfected with the secretory form of scFv. It is concluded that the targeting of non-neutralizing anti-HIV-1 Abs to specific intracellular compartments blocks HIV replication and represents a potential therapeutic strategy for protecting uninfected lymphopoietic stem cells from HIV-1-infected patients.

Animals↗
PubMed2006

Mechanisms of pathogen entry through the endosomal compartments.

Several pathogens - bacteria, viruses and parasites - must enter mammalian cells for survival, replication and immune-system evasion. These pathogens generally make use of existing cellular pathways that are designed for nutrient uptake, receptor downregulation and signalling. Because most of these pathways end in lysosomes, an organelle that is capable of killing microorganisms, pathogens have developed remarkable means to avoid interactions with this lytic organelle.

Animals↗
PubMed1995

Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell.

We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus-producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle.

Cell Line↗
PubMed2006

Histatin 5-derived peptide with improved fungicidal properties enhances human immunodeficiency virus type 1 replication by promoting viral entry.

Antimicrobial peptides are found in a number of body compartments and are secreted at mucosal surfaces, where they form part of the innate immune system. Many of these small peptides have a broad spectrum of inhibitory activity against bacteria, fungi, parasites, and viruses. Generally, the peptide's mode of action is binding and disruption of membranes due to its amphipathic properties. Histatin 5 is a salivary peptide that inhibits Candida albicans, an opportunistic fungus that causes oropharyngeal candidiasis in a majority of human immunodeficiency virus type 1 (HIV-1)-infected patients progressing towards AIDS. Previously, we increased the fungicidal properties of histatin 5 by replacing amino acids in the active domain of histatin 5 (Dh-5) (A. L. Ruissen, J. Groenink, E. J. Helmerhorst, E. Walgreen-Weterings, W. van't Hof, E. C. Veerman, and A. V. Nieuw Amerongen, Biochem. J. 356:361-368, 2001). In the current study, we tested the anti-HIV-1 activity of Dh-5 and its derivatives. Although Dh-5 inhibited HIV-1 replication, none of the peptide variants were more effective in this respect. In contrast, one of the derivatives, Dhvar2, significantly increased HIV-1 replication by promoting the envelope-mediated cell entry process. Most likely, Dhvar2 affects membranes, thereby facilitating fusion of viral and cellular membranes. This study shows that modification of antimicrobial peptides in order to improve their activity against a pathogen may have unpredictable and unwanted side effects on other pathogens.

Amino Acid Sequence↗
PubMed2006

[Virus persistence in hepatitis C: lifelong infection despite therapy?].

BACKGROUND: Chronic hepatitis C infection still represents a clinical and scientific challenge. Exciting progress has been achieved by the use of combined therapy regimens with pegylated interferon and ribavirin resulting in sustained virological response rates of 60-80%, depending on the genotype. VIRUS PERSISTENCE DESPITE SUCCESSFUL THERAPY: Despite favorable longterm data with regard to viremia, liver histology and serum liver enzymes in treated patients who comply with the criteria of sustained virological response, a complete elimination of the hepatitis C virus (HCV) is rarely observed. Besides liver tissue, peripheral blood mononuclear cells (PBMCs) could be proven as locations of HCV persistence. It is assumed that there are further extrahepatic compartments in the host organism in which virus particles capable of replication remain, in spite of a seemingly successful therapy. OCCULT HEPATITIS C: The problem of the existence of small amounts of potentially replicative viruses becomes apparent even in occult hepatitis C, a constellation in which anti-HCV antibodies are missing, but HCV RNA in liver tissue and mostly also in PBMCs exist. CONCLUSION: The precise significance of the HCV persistence in the host organism is still inconclusive; according to first research results, however, it can lead to a deterioration of the liver histology. At present, it is also unclear if patients with occult hepatitis C as well as with evidence of HCV RNA in the liver and/or extrahepatic compartments after seemingly successful antiviral treatment are to be regarded as infectious.

Antiviral Agents↗
PubMed2000

Analysis of the chicken DNA fragments that contain structural sites of attachment to the nuclear matrix: DNA-matrix interactions and replication.

Ten short DNA fragments have been selected from a library of the nuclear matrix-attached DNA (nmDNA) from chicken erythrocytes by their ability to hybridize with the fraction of chicken replication origins isolated by nascent DNA strand extrusion. The primary structure of these fragments has been determined. Five of the sequences contained a topoisomerase II recognition site. Most of the studied DNA fragments also have a common eight-nucleotide motif, GCAGACCG/A. A sequence-specific DNA-binding protein with a MW of 55 kDa that interacted with this motif has been identified. Some of the cloned DNA fragments promoted an increased level of transient plasmid replication in transfected chicken cells. The ability of plasmid bearing nmDNA fragments to replicate correlated directly with their ability to target plasmids to the nuclear matrix compartment.

Animals↗
PubMed2000

Compartmentalization in environmental science and the perversion of multiple thresholds.

Nature and living organisms are separated into compartments. The self-assembly of phospholipid micelles was as fundamental to the emergence of life and evolution as the formation of DNA precursors and their self-replication. Also, modern science owes much of its success to the study of single compartments, the dissection of complex structures and event chains into smaller study objects which can be manipulated with a set of more and more sophisticated equipment. However, in environmental science, these insights are obtained at a price: firstly, it is difficult to recognize, let alone to take into account what is lost during fragmentation and dissection; and secondly, artificial compartments such as scientific disciplines become self-sustaining, leading to new and unnecessary boundaries, subtly framing scientific culture and impeding progress in holistic understanding. The long-standing but fruitless quest to define dose-effect relationships and thresholds for single toxic agents in our environment is a central part of the problem. Debating single-agent toxicity in splendid isolation is deeply flawed in view of a modern world where people are exposed to low levels of a multitude of genotoxic and non-genotoxic agents. Its potential danger lies in the unwarranted postulation of separate thresholds for agents with similar action. A unifying concept involving toxicology and radiation biology is needed for a full mechanistic assessment of environmental health risks. The threat of synergism may be less than expected, but this may also hold for the safety margin commonly thought to be a consequence of linear no-threshold dose-effect relationship assumptions.

Dose-Response Relationship, Drug↗
PubMed2003

Clinical implications of HIV-1 drug resistance in the neurological compartment.

The tropism of human immunodeficiency virus type 1 (HIV-1) for the central nervous system (CNS) develops early during the course of the infection. Potent antiretroviral therapy has been demonstrated to be effective in controlling the replication of HIV-1 in cerebrospinal fluid (CSF), even though a variable response in this compartment compared with that in plasma has been observed. Different concentrations of antiretroviral drugs are found in CSF and the use of antiretroviral drugs penetrating across the blood-brain barrier is considered to be required for controlling CNS infection in advanced patients, particularly in those with neurological disorders. The compartmentalization of HIV-1 infection in the CNS may affect the treatment response, which may cause a different evolution of viral drug resistance in the 2 compartments. Although HIV-1 resistance testing in CSF is not recommended for the routine management of patients with virological failure, treatment decisions in patients with neurological disorders may require knowledge of the resistance profile of the virus in the CSF.

AIDS Dementia Complex↗
PubMed2001

The replication timing program of the Chinese hamster beta-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase.

We have examined the dynamics of nuclear repositioning and the establishment of a replication timing program for the actively transcribed dihydrofolate reductase (DHFR) locus and the silent beta-globin gene locus in Chinese hamster ovary cells. The DHFR locus was internally localized and replicated early, whereas the beta-globin locus was localized adjacent to the nuclear periphery and replicated during the middle of S phase, coincident with replication of peripheral heterochromatin. Nuclei were prepared from cells synchronized at various times during early G1 phase and stimulated to enter S phase by introduction into Xenopus egg extracts, and the timing of DHFR and beta-globin replication was evaluated in vitro. With nuclei isolated 1 h after mitosis, neither locus was preferentially replicated before the other. However, with nuclei isolated 2 or 3 h after mitosis, there was a strong preference for replication of DHFR before beta-globin. Measurements of the distance of DHFR and beta-globin to the nuclear periphery revealed that the repositioning of the beta-globin locus adjacent to peripheral heterochromatin also took place between 1 and 2 h after mitosis. These results suggest that the CHO beta-globin locus acquires the replication timing program of peripheral heterochromatin upon association with the peripheral subnuclear compartment during early G1 phase.

Animals↗
PubMed2004

IcmF and DotU are required for optimal effector translocation and trafficking of the Legionella pneumophila vacuole.

The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages. Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system. To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery. The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth. Two of these genes, icmF and dotU, are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons. DotU protein is degraded in the early postexponential phase in wild-type L. pneumophila and at all growth phases in an icmF mutant. IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent. In the absence of either gene, L. pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC. Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.

Animals↗
PubMed2026

Maintenance of nucleosome organization through replication and transcription counteracts aberrant coalescence of active chromatin.

Nucleosomes with their associated modifications organize and regulate the genome. It is unclear how this is integrated with the requirement of replication and transcription to access the DNA template without jeopardizing chromatin function. Here, we reveal a unified requirement for the histone chaperone FACT in mediating nucleosome disruption and reassembly during mammalian replication and transcription. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome wide, and chromatin structure in their wake collapses, with reduced nucleosome occupancy, irregular spacing, and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. Similarly, aberrant compartments form in cells failing to maintain chromatin fiber structure through replication. Nucleosome organization therefore dynamically regulates genome architecture, guarding against spurious chromatin aggregation.

Nucleosomes↗
PubMed1995

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells.

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.

Base Sequence↗
PubMed2003

HIV and cells of macrophage/dendritic lineage and other non-T cell reservoirs: new answers yield new questions.

Defining how human immunodeficiency virus (HIV) interacts with macrophages, dendritic cells (DC), and other non-T cell reservoirs remains a critical area of research despite widespread use in the developed world of highly active antiretroviral therapy. In fact, as highlighted at the Fifth International Workshop on HIV and Cells of Macrophage/Dendritic Lineage and Other Reservoirs, as viral suppression in T cells becomes increasingly effective, these alternative reservoirs may take on even greater relative importance as sites for viral persistence and as a target for purging. These cells may be especially important reservoirs in several critical settings of clinical relevance, and there are major differences in the molecular mechanisms that regulate HIV replication in these cells compared with T cells. Dysfunction of these cells may also play a major role in particular aspects of pathogenesis. Three broad themes emerged from the workshop regarding areas of recent progress, which also serve to identify current research challenges of (i). determining the role played by macrophages, DC, and other non-T cell viral targets in transmission and dissemination and as viral reservoirs at various stages of disease and in different compartments in vivo; (ii). identifying the molecular mechanisms by which virus-cell interactions affect the inflammatory, immune, and other functions of these cells; and (iii). defining the unique pathways that regulate infection and replication in these cellular compartments. This issue of JLB contains several reviews and original reports resulting from the workshop that address recent progress and highlight the current research questions regarding these cell types.

Dendritic Cells↗
PubMed1991

Epstein-Barr virus infection in oral hairy leukoplakia: virus replication in the absence of a detectable latent phase.

Epstein-Barr virus (EBV) infects both B lymphocytes and oropharyngeal epithelium, and it has been argued that the true reservoir of virus persistence in vivo is the self-renewing basal epithelial compartment. The identification of oral hairy leukoplakia (HL) of AIDS patients as a clinically apparent focus of EBV replication in lingual epithelium therefore provides a means of studying the EBV-epithelial cell interaction in situ. Replicative EBV DNA and productive cycle antigens are restricted to the upper, more differentiated epithelial layers in HL, and here we have applied highly sensitive in situ hybridization and immunohistological methods to examine the lower basal/suprabasal layers for evidence of latent EBV infection. We could not detect EBV DNA in these layers using an in situ DNA hybridization protocol which, on reference B cell lines, detected 1 viral genome/cell. Likewise, using sensitive in situ RNA hybridization for both the small non-polyadenylated EBER RNAs (abundant transcripts seen in all known forms of EBV latency) and the latent membrane protein (LMP) mRNA (the most abundant viral mRNA in B lymphoblastoid cell lines), the basal/suprabasal cells in HL were consistently negative; immunohistological staining with specific monoclonal antibodies also gave no evidence of latently infected LMP-positive cells. When the biopsy extracts were analysed by immunoblotting with selected human antisera, in addition to abundant productive cycle antigens, a band of constant size (66K) was observed which also reacted with immunopurified antibodies monospecific for one of the latency-associated nuclear antigens, EBNA 1; the cellular origin of this EBNA 1 could not be ascertained, but it is possible that in HL the protein is expressed during the productive cycle. The absence of demonstrable EBV latency in the basal/suprabasal cells of HL suggests that this is purely a virus replicative lesion which is sustained by continual re-infection of the maturing epithelium, not by the maturation of latently infected cells from the basal compartment.

Antigens, Viral↗