1,721,128 research outputs found
Protein microarrays: from serodiagnosis to whole proteome scale analysis of the immune response against pathogenic microorganisms.
No full textA radiolabel release microassay for phagocytic killing of candida albicans.
No full textThe chromium release technique for quantifying intracellular killing of radiolabelled Candida albicans particles was exploited in a microassay in which murine and human phagocytes acted as effectors under peculiarly simple conditions. At appropriate effector: target ratios and with a 4 h incubation, up to 50% specific chromium release could be detected in the supernatant with no need for opsonization or lysis of phagocytes. This simple microassay permits easy-to-perform, simultaneous testing of a variety of different phagocytes even if only available in limited amounts, and provides an objective measurement of intracellular killing of Candida albicans. © 1982
Critical observations on computerized analysis of banding patterns with commercial software packages
No full textTetanus toxin-sensitive VAMP-related proteins are present in murine macrophages
No full textThe light chain of tetanus neurotoxin (TeTx) is a zinc endopeptidase specific for VAMP/synaptobrevin (VAMP), a 120-amino-acid integral protein previously described in the small synaptic vesicles of neuronal cells. TeTx has been shown to be active also on nonneuronal cells. By SDS-PAGE and quantitative immunoblotting on proteins derived from murine macrophages (M phi) exposed to TeTx, we have shown that: (1) VAMP-related proteins are also present in M phi and (2) such proteins are sensitive to TeTx proteolytic cleavage. The demonstration that TeTx acts on VAMP-related proteins also in M phi offers a new and useful tool for molecular studies on M phi exocytosis
Anticryptococcal Resistance in the Mouse Brain: Beneficial Effects ofLocal Administration of Heat-Inactivated Yeast Cells
No full textUsing a murine model, we have previously shown that brain resistance to local infection with opportunistic fungi is affected by manipulation of the host myelomonocytic compartment. Here, we demonstrate thatintracerebral administration of heat-inactivated Cryptococcus neoformans (H-CN) yeast cells results in a consistent enhancement of mouse survival to subsequent local challenge with lethal doses of C. neoformans. The phenomenon, more pronounced upon double H-CN treatment, is associated with (i) massive local inflammatoryresponse, (ii) reduced growth of the fungus within the brain, and (iii) induction of delayed-type hypersensitivity. Moreover, H-CN treatment confers protection against local heterologous challenges. Our data provide initial evidence that intracerebral administration of H-CN results in the establishment of aspecific and specific immune responses; the mechanisms of elicitation and relative contributions to host antimicrobial resistance remain to be elucidated.Using a murine model, we have previously shown that brain resistance to local infection with opportunistic fungi is affected by manipulation of the host myelomonocytic compartment. Here, we demonstrate that intracerebral administration of heat-inactivated Cryptococcus neoformans (H-CN) yeast cells results in a consistent enhancement of mouse survival to subsequent local challenge with lethal doses of C. neoformans. The phenomenon, more pronounced upon double H-CN treatment, is associated with (i) massive local inflammatory response, (ii) reduced growth of the fungus within the brain, and (iii) induction of delayed-type hypersensitivity. Moreover, H-CN treatment confers protection against local heterologous challenges. Our data provide initial evidence that intracerebral administration of H-CN results in the establishment of a specific and specific immune responses; the mechanisms of elicitation and relative contributions to host antimicrobial resistance remain to be elucidated
Augmentation of GG2EE macrophage cell line-mediated anti-Candida activity by gamma interferon, tumor necrosis factor and interleukin 1.
No full textThe expression of anti-Candida activity in the GG2EE macrophage cell line, generated by immortalization of fresh bone marrow with v-raf and v-myc oncogenes, was studied. GG2EE cells spontaneously inhibited the growth of an agerminative mutant of Candida albicans in vitro. The anti-Candida activity was maximal after 8 h of coculture and was proportional to the effector-to-target ratio. Gamma interferon (IFN-gamma), interleukin-1 (IL-1), and tumor necrosis factor (TNF) all significantly enhanced the anti-Candida activity of GG2EE cells. In contrast, IL-3, IL-4, and colony-stimulating factor 1 were ineffective. The augmentation of anti-Candida activity was not always concomitant with enhancement of phagocytosis, since IFN-gamma and colony-stimulating factor 1, but not IL-1 or TNF, augmented the phagocytic ability of GG2EE cells. Furthermore, the augmentation of anti-Candida activity in GG2EE cells did not correlate with the acquisition of antitumor activity. In fact, none of the cytokines alone were able to induce antitumor activity in GG2EE cells, which, however, could be activated to a tumoricidal stage by IFN-gamma plus heat-killed Listeria monocytogenes. These findings demonstrate that GG2EE cells exhibit spontaneous anti-Candida activity and that such activity is enhanced by TNF, IL-1, and IFN-gamma
A rapid Candida albicans hyphal‐form growth inhibition assay: determination of myelomonocytic‐mediated antifungal activity: Schnellverfahren zur Messung der Wachstumshemmung von Candida albicans durch Myelomonozyten‐vermittelte antifungale Aktivität
No full textSummary. An in vitro microassay for the measurement of Candida albicans hyphal‐form growth inhibition by myelomonocytic cells is described. The assay is rapid, easy‐to‐perform and objective. A Candida strain capable of in vitro dimorphic transition from yeast to hyphal form has been employed. The assay is based on the incorporation of 3H‐glu‐cose by the fungus, the effect being dependent upon the time of pulse, size of the inoculum and concentration of radiolabelled metabolite. In particular, C. albicans hyphal form, obtained by a 3 h incubation in vitro in the presence of 10% fetal calf serum, is co‐incubated with the effector cells. A pulse with 3H‐glucose in water is then performed and the radioactivity incorporated by the residual Candida is taken as an indication of hyphal growth. We found that polymorphonuclear cells, peritoneal macrophages and the cloned GG2EE macrophage cell line significantly inhibited hyphal growth, the effects being time and effector‐to‐target cell ratio dependent. Zusammenfassung. Es wird ein Mikroverfahren in vitro zur Messung der Wachstumshemmung von Candida albicans in der Myzelphase durch Mye‐lomonozyten beschrieben. Der Ansatz ist schnell, leicht in der Ausführung und liefert objektive Re‐sultate. Hierzu wurde ein Candida‐Stamm verwen‐det mit der Fähigkeit, in vitro den dimorphen Pha‐senwechsel von der Hefe‐ zur Myzelphase zu durch‐laufen. Der Ansatz basiert auf dem 3H‐Glucose‐Ein‐bau durch den Pilz. Dieser Einbau ist abhängig von der Expositionszeit, der Inoculum‐Größe und der Konzentration des radioaktiv markierten Metabo‐liten. Hierzu wird die C. albicans‐Myzelphase, er‐halten nach 3 h Bebrütung in vitro in der Gegen‐wart von 10%igem fötalem Kälberserum, mit den Effektorzellen ko‐inkubiert. Dann werden die Hefen der 3H‐Glucose in Wasser ausgesetzt, und die ein‐gebaute Radioaktivität in den Candida‐Zellen wird als Indikator des Hyphenwachstums angesehen. Unsere Ergebnisse zeigen, daß polymorphonukleäre Zellen, Peritonealmakrophagen und die geklonte GG2EE‐Makrophagen‐Zellinie das Hyphenwachs‐tum in Abhängigkeit von der Expositionszeit und dem Effektor/Zielzell‐Verhältnis signifikant hem‐men. Copyright © 1991, Wiley Blackwell. All rights reserve
Protective effect of picolinic acid on mice intracerebrally infected with lethal doses of Candida albicans
No full textWe have studied the effects of picolinic acid (PLA), a product of tryptophan degradation, on mouse susceptibility to intracerebral infection with Candida albicans. We show that intraperitoneal administration of PLA significantly enhances the median survival time of mice inoculated with the lethal challenge. Furthermore, intracerebral administration of this agent induces a protective state against the local lethal infection, the phenomenon depending upon the administration schedule and doses of PLA employed. According to survival data, yeast growth in the brain as well as yeast colonization of the kidneys are drastically reduced in PLA-treated mice compared with those for untreated controls. Northern (RNA) blot analysis of brain tissues demonstrates that mRNA levels specific for tumor necrosis factor and interleukin 1 are augmented and induced, respectively, after inoculation of PLA. These results indicate that PLA has a protective effect likely involving elicitation of a cytokine response in vivo against fungal infections
Role of the capsule in microglial cell-Cryptococcus neoformans interaction: impairment of antifungal activity but not of secretory functions
No full textUsing two isogenic strains of Cryptococcus neoformans, we studied the influence of the capsule in C. neoformans microglial-cell interaction. We demonstrate that the acapsular mutant yeasts (CAP67) are more susceptible to phagocytosis and killing than encapsulated yeasts (B3501) by the murine microglial cells, BV-2. RT-PCR analysis showed that the pattern of gene transcripts for tumour necrosis factor alpha (TNF-a), interleukin (IL)-1 beta, IL-6, IL-12p40 and granulocyte macrophage colony stimulating factor re:mains unchanged following BV-2 cell infection with CAP67 or B3501 yeasts. Moreover, no induction of TNF-alpha secretion occurs in BV-2 cells infected with either B3501 or CAP67 yeasts or exposed to glucuronoxylomannan (GXM) or galactoxylomannan (GalXM). Nevertheless, lipopolysaccharide-induced TNF-alpha secretion is downregulated by cell infection with B3501 or CAP67 yeasts or exposure to GXM or GalXM. Overall, by means of a continuous cell line, it appears that the C. neoformans capsule is detrimental to microglial cell antifungal activity, while no effect can be attributed to the capsule as trend of cytokine gene expression and TNF-alpha secretion
Microglial cell-mediated anti-Candida activity: temperature, ions, protein kinase C as crucial elements.
Full text linkAn in vitro established microglial cell line, BV-2, constitutively exhibits high levels of anti-Candida activity. To elucidate the cascade of events leading to the accomplishment of such activity, we studied its dependence on temperature and ion availability. The role of protein kinases has also been studied by the specific inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and N-(2-guanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA 1004). We found that (a) the BV-2 cell/Candida conjugate formation is a discrete step, temperature-, ion- and protein kinase-independent; (b) the phagocytic event, which is protein kinase-independent, is significantly impaired by temperature decrease and ion deprivation; (c) the fulfillment of anti-Candida effects is strictly dependent upon temperature, ion availability and functional protein kinase. Functional protein kinase C, but not other kinases, is required for the accomplishment of anti-Candida activity, which, in fact, is selectively abrogated by H7 but not HA. Furthermore, protein kinase C activators, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1-oleoyl-2-acetyl glycerol (OAG), consistently potentiate BV-2 cell-mediated anti-Candida activity, the phenomena being dose-dependent. These results indicate that the multistep events leading a microglial cell to express anti-Candida activity can be dissected and differentiated for biochemical and biological demands, the latest along the cascade being the most demanding steps
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