vendredi 24 avril 2009
par   G. Grégori

Christersson CE, Fornalik MS, Baier RE, Glantz PO (1987) In vitro attachment of oral microorganisms to solid surfaces : evaluation of a controlled flow method. Scand J Dent Res 95 :151-158


A flow cell method was modified to provide shear-controlled experimental conditions for monitoring initial attachment and detachment of oral microorganisms to solid surfaces. Whole unstimulated human saliva was collected and circulated at a flow rate of 1 ml/min, through a cell composed of two parallel test plates. Infrared-transparent plates of medium surface energy served as test substrata in these initial calibration experiments. The plates presented a similar distribution of polar forces and dispersion forces at the surface as that of human tooth enamel and some restorative dental materials. Internal reflection infrared spectroscopy verified the presence of deposited organic material. After saliva had been circulated through the flow cells for 15 min at 37 degrees C, sterilized distilled water was introduced at the same flow rate and time of exposure to remove unattached microorganisms. Morphologic characterizations and counts of adherent Gram-stainable microorganisms were performed using incident light microscopy. Three different surface zones corresponding to the inlet area, the middle area and the outlet area of the flow cell were analyzed, and compared with enumerations of microorganisms in the whole saliva samples. Numbers of attached microorganisms in the three zones followed predictions from the laminar flow conditions, with a positive correlation shown between total numbers of microorganisms in saliva and total numbers of microorganisms attached. Cocci and rods were the only morphotypes observed on the plates, and no significant difference could be detected between the percentage of cocci and rods attached in the three different zones.(ABSTRACT TRUNCATED AT 250 WORDS)

Frommel TO, Balber AE (1987) Flow cytofluorimetric analysis of drug accumulation by multidrug-resistant Trypanosoma brucei brucei and T. b. rhodesiense. Mol Biochem Parasitol 26 :183-191


Dual laser flow cytofluorimetry has been used to compare accumulation of compounds representing three major classes of trypanocidal drugs by drug sensitive and drug resistant clones of Trypanosoma brucei brucei and T. b. rhodesiense. Clones selected for resistance to melarsoprol were shown to be cross-resistant in vivo to two diamidines, pentamidine and Berenil, but not to suramin. At 35 degrees C, bloodstream forms of these multidrug-resistant clones accumulated lower intracellular concentrations of the diamidines 4’,6-diamidino-2-phenyl-indole (DAPI) and Hoechst 33342, the phenanthridine ethidium bromide, and the acridine acriflavine than drug sensitive parasites. Accumulation of all four drugs was saturable. Drug concentrations giving half-maximal rates of accumulation were increased in the resistant clones relative to the sensitive parent clones. The rate of DAPI accumulation by both resistant and sensitive parasites was strongly temperature dependent and increased sharply above 27 degrees C. Two distinct populations were resolved in mixtures of sensitive and resistant clones exposed to DAPI. Resistant and sensitive cells accumulated identical intracellular concentrations of DAPI following brief treatment with the detergent Triton X-100. The results suggest that alterations in the surface membrane of multidrug-resistant trypanosomes reduce accumulation of several drugs relative to drug sensitive parasites.

Kozel TR, Pfrommer GS, Redelman D (1987) Activated neutrophils exhibit enhanced phagocytosis of Cryptococcus neoformans opsonized with normal human serum. Clin Exp Immunol 70 :238-246


We studied the effect of agents that activate neutrophils on phagocytosis of C. neoformans. The amount of CR3 on the surface of neutrophils was used as a marker for neutrophil activation. Surface CR3 was estimated by flow cytometry using phycoerythrin-labelled anti-CR3 (anti-Leu-15) monoclonal antibody. Phagocytosis was determined by incubation of neutrophils with cryptococci that had been preincubated with normal human serum. We found that treatment of neutrophils with (i) the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, (ii) zymosan activated serum, (iii) supernatant fluid from a mixed leucocyte culture, or (iv) supernatant fluid from human leucocytes cultured with phytohaemagglutinin produced a dose-dependent increase in CR3 density. These agents also markedly enhanced phagocytosis of opsonized cryptococci in a parallel dose-dependent fashion. These results indicate that phagocytosis of cryptococci opsonized with normal human serum is markedly enhanced by treatment of neutrophils with reagents that stimulate neutrophils. Our results demonstrate that neutrophils activated in an appropriate manner are capable of efficient phagocytosis of encapsulated cryptococci. This potential phagocyte activity may account in part for the high natural resistance to cryptococcosis.

Owen-Schaub LB, Hemstreet GP, 3rd, Hemingway LL, Abraham SR, DeBault LE (1987) Population dynamics of the murine lymphokine activated killer system : precursor frequency and kinetics of maturation and renewal. Cell Tissue Kinet 20 :591-602


The proliferation kinetics and population renewal of recombinant interleukin-2 (rIL-2)-induced murine lymphokine activated killers (LAK) arising from splenic precursors was studied. Extensive proliferation has been shown to accompany the de novo generation of LAK cytotoxicity. In this report, a thymidine ’hot pulse’ suicide technique was employed to examine the sensitivity of LAK progenitors during various time periods following culture initiation. Hot pulse during the first 24 hr of culture resulted in a 30-35% reduction in lytic activity when assayed on day 5. Pulse periods between days 1 and 4 resulted in almost complete inhibition (90-95%) of lytic function when assayed on day 5. Proliferation of LAK progenitors was documented by limiting dilution analysis comparison of splenic precursors and functionally mature LAK cultures. These studies showed a 75- to 80-fold enrichment of LAK progenitors after 3 days culture in rIL-2. By flow cytometric cell cycle analysis, we demonstrated that the number of cells in the S/G2/M phase increased with the length of rIL-2 culture and represented approximately 40% of the cells by day 4. Finally, we used the rate of decay of lytic activity following irradiation as a factor to define the mean life span of a cytotoxic effector in the absence of cellular input. An exponential decrease to approximately 50% of controls was observed within 8-9 hr after irradiation. Taken together, these results suggest that the LAK system is highly dynamic and requires continuous cellular proliferation for its maintenance.

Tertti R, Eerola E, Lehtonen OP, Stahlberg TH, Viander M, Toivanen A (1987) Virulence-plasmid is associated with the inhibition of opsonization in Yersinia enterocolitica and Yersinia pseudotuberculosis. Clin Exp Immunol 68 :266-274


Plasmid-cured variants of virulent strains of Yersinia enterocolitica and Y. pseudotuberculosis were obtained by selection after growth in calcium-deficient medium. To obtain antigen preparations consisting of whole bacteria the original plasmid-containing strains and the plasmid-cured variants were grown in conditions favouring expression of the temperature-inducible outer membrane proteins of Yersinia (YOP) (37 degrees C, calcium-deficient culture medium). The presence or absence of the YOP on the bacteria was verified by immunoblotting. Opsonophagocytosis of YOP-negative Yersinia preparations (YOP-) was compared to that of YOP-containing ones (YOP+) in human polymorphonuclear leukocyte (PMN) chemiluminescence (CL) assay. The attachment of complement C3b on the surface of the bacteria after opsonization with normal human serum was determined by using a fluorescent anti-C3c-antibody and flow cytometry. YOP+ bacteria resisted opsonization in the absence of specific antibodies, as indicated by diminished C3b-fixation on bacteria and weaker CL response. This implies that virulence-plasmid-coded structures provide Y. enterocolitica and Y. pseudotuberculosis with an ability to avoid complement-mediated opsonization and phagocytosis.

Waldman FM, Hadley WK, Fulwyler MJ, Schachter J (1987) Flow cytometric analysis of Chlamydia trachomatis interaction with L cells. Cytometry 8 :55-59


Immunofluorescent staining and flow cytometric analysis have been investigated as means of studying the early stages of in vitro infection of Chlamydia trachomatis. The lymphogranuloma venereum strain of C. trachomatis was grown in vitro in L cells, fixed in p-formaldehyde, stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to the chlamydial major outer membrane protein, and analyzed flow cytometrically. Infected cells stained 50-100 times more intensely than uninfected cells, and they could easily be discriminated by flow analysis. The number of infected cells and the fluorescence intensity of individual cells were proportional to the multiplicity of infection. The attachment of purified elementary bodies to L cells could be analyzed by immunofluorescence and flow cytometry. Cells exposed to 0.26 inclusion-forming units/cell could be discriminated from an unexposed population. Flow analysis of purified elementary bodies was possible after fluorescent staining with the aid of a laser-based cytometer and gating on low volume.

Wang A, Wang CC, Alderete JF (1987) Trichomonas vaginalis phenotypic variation occurs only among trichomonads infected with the double-stranded RNA virus. J Exp Med 166 :142-150


Trichomonas vaginalis isolates were examined for the presence of viral double-stranded RNA (dsRNA) and the property of phenotypic variation. Only the heterogeneous isolates composed of mAb-reactive and -nonreactive organisms, as determined by indirect immunofluorescence and flow cytofluorometry, and capable of phenotypic variation possessed the dsRNA. Both the positive and negative phenotype subpopulations separated from the heterogeneous parent contained equal amounts of the dsRNA. Loss of the dsRNA upon prolonged in vitro cultivation always correlated with the lack of expression of the major immunogen. The data indicate a relationship between the presence of the dsRNA and the ability of the pathogenic human trichomonads to express immunogens on their surfaces and to undergo phenotypic variation.