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D M Ojcius

Publications and source records attributed to D M Ojcius.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of a monoclonal antibody reacting with antigen-4 domain of gp900 in Cryptosporidium parvum invasive stages.

Cryptosporidium parvum (Protozoa, Apicomplexa) infects the apical surface of intestinal epithelial cells, where it grows and divides within a membrane-bound parasitophorous vacuole. gp900, an abundant glycoprotein of C. parvum merozoites and sporozoites, is localized in micronemes and at the surface of invasive stages and participates in the invasion process. Here, we describe a new monoclonal antibody (mAb) against gp900. As shown by immunofluorescence of excysted parasites and immunoelectron microscopy of infected tissues, the mAb reacted with micronemes present in the apical pole of invasive stages. In immunoprecipitation experiments, the mAb was shown to react with a high molecular weight antigen co-migrating with gp900. Finally, three reactive clones were selected upon screening of a C. parvum genomic expression library with the mAb; and sequencing of the insert from one of them showed a 596 bp sequence identical to the DNA region encoding a domain of gp900 identified as antigen 4.

Animals↗
PubMed2001

Modulation of P2Z/P2X(7) receptor activity in macrophages infected with Chlamydia psittaci.

Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.

Adenosine Triphosphate↗
PubMed2000

Fluorescent labelling of intracellular bacteria in living host cells.

The fluorescent reagent, CellTracker, labels metabolically-active cells and was used here to label Chlamydia in vivo during their exponential phase of growth in infected cells. HeLa cells infected with C. psittaci were labelled with the CellTracker reagents between 15 and 48 h post-infection. The fluorescent label accumulated in the host-cell membrane compartment (inclusion) within which Chlamydia reside and replicate, and was also incorporated by the bacteria. Labelling with the CellTracker affected neither the growth nor the differentiation of the chlamydiae, and labelled chlamydiae isolated from infected cells were infectious. Our results demonstrate that the CellTracker could become a valuable tool for in vivo labelling of obligate intracellular parasites for which no genetic tools exist.

Bacterial Adhesion↗
PubMed2000

'Infectious web'.

Infections by Helicobacter pylori are responsible for duodenal and gastric ulcers and are a significant risk factor for the development of gastric adenocarcinoma. H. pylori was discovered in 1983, but many institutes in Canada, Europe, and the United States are already involved in programs to understand and treat the infections, as reflected by the growing number of internet sites devoted to this bacterium. Most AIDS patients and about 20% of children with acute lymphoblastic leukemia develop Pneumocystis carinii pneumoniae. Information on clinical symptoms and treatment, as well as the P. carinii genome sequencing project, are described at several web sites. Students and researchers wishing to understand the correlation between telomere length and AIDS may turn to web sites of the University of Colorado and Washington University School of Medicine for the latest on telomeres and telomerase, and their function in aging and cancer.

Helicobacter Infections↗
PubMed2000

'Infectious web'.

Exhaustive information on the Epstein-Barr virus, a member of the herpes family, is described at the International Herpes Management Forum web-site. Cervical cancer associations, AIDS treatment projects, and the Los Alamos National Laboratories provide useful information on papillomavirus infections, as well as hyperlinks to recent international papillomavirus conferences. A private pharmaceutical company, in collaboration with the National Institutes of Health, has launched a lively web-site covering different aspects of microbial infections for the general public.

Herpesvirus 4, Human↗
PubMed2000

'Infectious web'.

A comprehensive list of all known bacterial pathogens of humans is now available at various web-sites on the internet. The sites contain hyperlinks to original scientific literature, along with general information on laboratory testing, antibiotic resistance and clinical treatment. More specific sites highlight the fungus Pneumocystic carinii, arguably the main cause of pneumonia in immunosuppressed individuals.

Bacteria↗
PubMed2000

Effect of Chlamydia trachomatis infection and subsequent tumor necrosis factor alpha secretion on apoptosis in the murine genital tract.

The pathology observed during Chlamydia infection is due initially to localized tissue damage caused by the infection itself, followed by deleterious host inflammatory responses that lead to permanent scarring. We have recently reported that the infection by Chlamydia in vitro results in apoptosis of epithelial cells and macrophages and that infected monocytes secrete the proinflammatory cytokine interleukin-1beta. At the same time, proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) can also trigger apoptosis of susceptible cells. To study the possible relationship between Chlamydia trachomatis infection and apoptosis in vivo, we used the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling technique to determine whether infection may cause apoptosis in the genital tract of mice and, conversely, whether cytokines produced during the inflammatory response may modulate the level of apoptosis. Our results demonstrate that infected cells in the endocervix at day 2 or 7 after infection are sometimes apoptotic, although there was not a statistically significant change in the number of apoptotic cells in the endocervix. However, large clumps of apoptotic infected cells were observed in the lumen, suggesting that apoptotic cells may be shed from the endocervix. Moreover, there was a large increase in the number of apoptotic cells in the uterine horns and oviducts after 2 or 7 days of infection, which was accompanied by obvious signs of upper tract pathology. Interestingly, depletion of TNF-alpha led to a decrease in the level of apoptosis in the uterine horns and oviducts of animals infected for 7 days, suggesting that the inflammatory cytokines may exert part of their pathological effect via apoptosis in infected tissues.

Animals↗
PubMed2000

'Infectious web'.

This issue of 'Infectious Web' includes web-sites related to AIDS/HIV, pathogenic characteristics and resistance to Staphylococcus spp., diagnostic and clinical aspects of arthritis, and comprehensive information resources on malaria, cystic fibrosis and biological weapons.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Caspase-dependent apoptosis during infection with Cryptosporidium parvum.

The protozoan parasite Cryptosporidium parvum causes persistent diarrhea and malnutrition in children and the diarrhea-wasting syndrome in AIDS. No therapy exists for eliminating the parasite in the absence of a healthy immune response. Although it had been reported that infection of intestinal cell lines with C. parvum leads to host cell death, the mechanisms of cytolysis have not been characterized. We show here that infection with C. parvum leads to typical apoptotic nuclear condensation and DNA fragmentation in host cells. Both nuclear condensation and DNA fragmentation are inhibited by a caspase inhibitor, showing that caspases are involved in this type of apoptosis. Finally, blocking apoptosis with the caspase inhibitor increases the percentage of infected cells, suggesting that parasites may use apoptosis to exit from the infected cell or that the infected cells may eliminate the parasite through apoptosis. These results suggest that apoptosis could be involved in the pathogenesis of C. parvum infections in vivo, and raise the possibility that therapeutic interference with host cell death could alter the course of the pathology in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Enhancement of endocytosis due to aminophospholipid transport across the plasma membrane of living cells.

Formation of intracellular vesicles is initiated by membrane budding. Here we test the hypothesis that the plasma membrane surface area asymmetry could be a driving force for vesicle formation during endocytosis. The inner layer phospholipid number was therefore increased by adding exogenous aminophospholipids to living cells, which were then translocated from the outer to the inner layer of the membrane by the ubiquitous flippase. Addition of either phosphatidylserine or phosphatidylethanolamine led to an enhancement of endocytosis, showing that the observed acceleration does not depend on the lipid polar head group. Conversely, a closely related aminophospholipid that is not recognized by the flippase, lyso-alpha-phosphatidylserine, inhibited endocytosis, and similar results were obtained with a cholesterol derivative that also remains in the plasma membrane outer layer. Thus an increase of lipid concentration in the inner layer enhanced internalization, whereas an increase of the lipid concentration in the outer layer inhibited internalization. These experiments suggest that transient asymmetries in lipid concentration might contribute to the formation of endocytic vesicles.

Biological Transport↗
PubMed1999

P2Z/P2X7 receptor-dependent apoptosis of dendritic cells.

Macrophages and thymocytes express P2Z/P2X7 nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7 receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Z receptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7 receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis.

Adenosine Diphosphate↗
PubMed1999

Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth.

Chlamydiae enter epithelial cells via a mechanism that still remains to be fully elucidated. In this study we investigated the pathway of entry of C. psittaci GPIC and C. trachomatis LGV/L2 into HeLa cells and demonstrated that it does not depend on clathrin coated vesicle formation. We used mutant cell lines defective in clathrin-mediated endocytosis due to overexpression of dominant negative mutants of either dynamin I or Eps15 proteins. When clathrin-dependent endocytosis was inhibited by overexpression of the dynK44A mutant of dynamin I (defective in GTPase activity), Chlamydia entry was not affected. However, in these cells there was a dramatic inhibition in the proliferation of Chlamydia and the growth of the chlamydia vacuole (inclusion). When clathrin-dependent endocytosis was inhibited by overexpression of an Eps15 dominant negative mutant, the entry and growth of Chlamydia was unaltered. These results indicate that the effect on the growth of Chlamydia in the dynK44A cells was not simply due to a deprivation of nutrients taken up by endocytosis. Instead, the dominant-negative mutant of dynamin most likely affects the vesicular traffic between the Chlamydia inclusion and intracellular membrane compartments. In addition, cytochalasin D inhibited Chlamydia entry by more than 90%, indicating that chlamydiae enter epithelial cells by an actin-dependent mechanism resembling phagocytosis. Finally, dynamin is apparently not involved in the formation of phagocytic vesicles containing Chlamydia.

Actins↗
PubMed1999

'Infectious web'.

The infection-related web-sites discussed in this issue include sites on the Campylobacter jejuni genome, Toxoplasma-expressed sequence tags (ESTs), Salmonella infections, the history and biology of tuberculosis, both DNA and RNA vaccines, and interactions between cytomegalovirus and antibodies.

Animals↗
PubMed1999

'Infectious web'.

Explore the source record for details and available documents.

Communicable Diseases↗
PubMed1998

Apoptosis of epithelial cells and macrophages due to infection with the obligate intracellular pathogen Chlamydia psittaci.

We have characterized the cytotoxic activity of the obligate intracellular bacterium Chlamydia psittaci, which resides within a membrane-bound vacuole during the 2-day infection cycle. We have established that infected epithelial cells and macrophages die through apoptosis, which is measurable within 1 day of infection and requires productive infection by the bacteria. Inhibition of host cell protein synthesis has no effect on cell death, but blocking bacterial entry or bacterial protein synthesis prevents apoptosis, implying that bacterial growth is required for death of the host cell. Apoptosis was confirmed through the use of electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, gel agarose electrophoresis of fragmented DNA, and propidium-iodide labeling of host cell nuclei. Although infected cells died preferentially, both infected and uninfected cells became apoptotic, suggesting that the infected cells may secrete proapoptotic factors. Inhibition of either of two proapoptotic enzymes, caspase-1 or caspase-3, did not significantly affect Chlamydia-induced apoptosis. These results suggest that, as in the case of apoptosis due to Bax expression or oncogene dysregulation, which initiate the apoptotic program within the cell interior, the Chlamydia infection may trigger an apoptotic pathway that is independent of known caspases. As apoptotic cells secrete proinflammatory cytokines, Chlamydia-induced apoptosis may contribute to the inflammatory response of the host.

Apoptosis↗
PubMed1998

Enhancement of ATP levels and glucose metabolism during an infection by Chlamydia. NMR studies of living cells.

The Chlamydia species are obligate intracellular bacteria that proliferate only within the infected cell. Since the extracellular bacteria are metabolically inert and there are no cell-free systems for characterizing Chlamydia metabolism, we studied metabolic changes related to ATP synthesis and glycolysis in HeLa cells infected with Chlamydia psittaci during the course of the 2-day infection cycle using noninvasive 31P and 13C NMR methods. We find that the infection stimulates ATP synthesis in the infected cell, with a peak of ATP levels occurring midway through the infection cycle, when most of the metabolically active bacteria are proliferating. The infection also stimulates synthesis of glutamate with a similar time course as for ATP. The stimulation is apparently due to an enhancement in glucose consumption by the infected cell, which also results in an increased rate of lactate production and glutamate synthesis as well as higher glycogen accumulation during the infection. Concurrently, infection leads to an increase in the expression of the glucose transporter, GLUT-1, on HeLa cells, which may account for the enhanced glucose consumption. The chlamydiae are thus able to stimulate glucose transport in the host cell sufficiently to compensate for the extra energy load on the cell represented by the infection.

Adenosine Triphosphate↗
PubMed1998

Internalization of Chlamydia by dendritic cells and stimulation of Chlamydia-specific T cells.

Chlamydia species are the causative agents of trachoma, various forms of pneumonia, and the most common sexually transmitted diseases. Although the infection cycle has been extensively characterized in epithelial cells, where the Chlamydia entry-vacuoles avoid fusion with host-cell lysosomes, the cellular immune response has received less attention. Moreover, despite the abundant presence of dendritic cells (DC) in the sites of infection, the interaction between Chlamydia and DC has never been studied. We observe that DC kill Chlamydia trachomatis and Chlamydia psittaci. The chlamydiae are internalized by the DC in a nonspecific manner through macropinocytosis, and the macropinosomes fuse subsequently with DC lysosomes expressing MHC class II molecules. The interaction induces maturation of the DC, since presentation of an exogenous Ag is severely inhibited after a 1-day incubation, although chlamydial Ags are still presented and recognized by Chlamydia-specific CD4+ T cells. Thus, DC most likely play a role in initiating the T cell response in vivo and could potentially be used in adoptive transfer therapies to vaccinate against Chlamydia.

Animals↗