vendredi 24 avril 2009
par   G. Grégori

Abad FX, Pinto RM, Bosch A (1998) Flow cytometry detection of infectious rotaviruses in environmental and clinical samples. Appl Environ Microbiol 64 :2392-2396


A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) has been developed. The technique was validated by performing a seminested reverse transcription-PCR assay with sorted cell populations. The efficiency of the procedure has been compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF-OM) and with that of infection of MA104 cells and detection by IIF-FC. The limit of sensitivity for the detection of the cell-adapted strain Ito(r) P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to be 1/2 x 10(6) and 1/2 x 10(4) for IIF-OM with MA104 cells and IIF-FC with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells was tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection.

al-Majali A, Asem E, Lamar C, Robinson JP, Freeman MJ, Saeed M (1998) Effect of dietary insulin on the response of suckling mice enterocytes to Escherichia coli heat-stable enterotoxin. Vet Res 29 :527-536


Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of 2-day-old Swiss Webster suckling mice were used. For this study, 5, 10, 25 and 50 micrograms of insulin was given orally to half the mice in each group for 7 days. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, a suckling mouse assay in which 1 microgram of STa was given to all mice in insulin-treated and control groups was performed. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin-treated groups compared to control mice (P < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an up-regulatory effect on the STa-receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction, which is a major cause of diarrhea in newborn animals and human infants.

Bagley J, Aboody-Guterman K, Breakefield X, Iacomini J (1998) Long-term expression of the gene encoding green fluorescent protein in murine hematopoietic cells using retroviral gene transfer. Transplantation 65 :1233-1240


BACKGROUND : A major goal in retroviral-based gene therapy is to establish methods that allow for selection and tracking of transduced cell populations. Green fluorescent protein (GFP) may be useful for gene therapy applications because it is a naturally fluorescent protein that can be detected using conventional flow cytometers facilitating rapid analysis and purification of transduced cell populations. However, it is unknown whether GFP can be stably expressed in vivo, particularly in multiple bone marrow-derived cell lineages. METHODS : A murine retrovirus carrying the gene encoding GFP was used to infect murine bone marrow cells (BMCs). These studies were conducted to (1) directly determine whether GFP could be used as a marker of BMC transduction, (2) determine whether GFP is capable of being expressed in multiple bone marrow-derived hematopoietic cell lineages, and (3) determine whether GFP could be used to follow the fate of transduced cells in vivo. RESULTS : Infection of BMCs with retroviruses carrying the gene encoding GFP resulted in a fluorescent signal in viable transduced cells that was detectable by flow cytometry. Expression of GFP was detected in multiple bone marrow-derived cell lineages after transduction, including stem cell antigen-positive (Sca-1+), lineage marker-negative (Lin-) cells. Using GFP as a selectable marker, we were able to enrich for transduced cells by cell sorting. Mice reconstituted with enriched populations of GFP+ cells showed a significant increase in the percentage of cells expressing GFP in the periphery when compared with mice reconstituted with unenriched transduced bone marrow. CONCLUSIONS : These data indicate that GFP can be used to select for transduced BMCs in vitro, expressed in multiple bone marrow-derived cell lineages, used to select transduced cells, and follow the fate of transduced cells long-term in vivo.

Berk SG, Ting RS, Turner GW, Ashburn RJ (1998) Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl Environ Microbiol 64 :279-286


Two Acanthamoeba species, fed at three temperatures, expelled vesicles containing living Legionella pneumophila cells. Vesicles ranged from 2.1 to 6.4 microns in diameter and theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.

Boel E, Bootsma H, de Kruif J, Jansze M, Klingman KL, van Dijk H, Logtenberg T (1998) Phage antibodies obtained by competitive selection on complement-resistant Moraxella (Branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein. Infect Immun 66 :83-88


We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitive M. catarrhalis strains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.

Borth N, Mitterbauer R, Mattanovich D, Kramer W, Bayer K, Katinger H (1998) Flow cytometric analysis of bacterial physiology during induction of foreign protein synthesis in recombinant Escherichia coli cells. Cytometry 31 :125-129


The production of foreign proteins at high yields represents a severe metabolic stress for Escherichia coli cells. In many cases, induction of protein synthesis results in rapid exhaustion of the cellular energy and metabolic precursors and thus in cell death. Therefore, sustained production of foreign proteins requires some fine tuning of the specific production rate to meet the capabilities of the cell. This has stimulated us to analyze by flow cytometry the physiological behaviour of recombinant E. coli cells producing human superoxide dismutase (SOD). Two strains that produce SOD under the control of either a combined T7/lac promoter or the phi10 promoter were compared by using the following parameters : (a) total DNA content as an indicator of cell division, (b) total RNA content as a measure for protein synthesis activity, (c) total protein content representing cell size, and (d) intracellular SOD content as a measure for productivity. Results show that those cells that continue to increase their biomass after induction of foreign protein synthesis also have the highest specific production rate. Cells, however, do not divide to a measureable degree but rather increase their size. The results confirm the importance of fine-tuning expression systems to prolong the lifetime of cells after induction. This will result in an increased yield.

Botello E, Nordstrom K (1998) Effects of chromosome underreplication on cell division in Escherichia coli. J Bacteriol 180 :6364-6374


The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth. We have studied the coordination between replication and cell division by using a temperature-controlled Escherichia coli intR1 strain. In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study the response of cell division to a delayed initiation of replication. The bacteria were grown exponentially at 39 degreesC (normal DNA/mass ratio) and shifted to 38 and 37 degreesC. In the last two cases, new, stable, lower DNA/mass ratios were obtained. The rate of replication elongation was not affected under these conditions. At increasing degrees of underreplication, increasing proportions of the cells became elongated. Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big. The elongated cells often produced one daughter cell lacking a chromosome ; this was always the smallest daughter cells, and it was the size of a normal newborn cell. These results favor a model in which cell division takes place at only distinct cell sizes. Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids. This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value. Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion.

Bowden RA, Cloeckaert A, Zygmunt MS, Dubray G (1998) Evaluation of immunogenicity and protective activity in BALB/c mice of the 25-kDa major outer-membrane protein of Brucella melitensis (Omp25) expressed in Escherichia coli. J Med Microbiol 47 :39-48


The antibody response specific to the 25-kDa major outer-membrane protein (Omp25) of Brucella melitensis expressed in Escherichia coli was assessed in BALB/c mice. Groups of mice were immunised and boosted either with sonicated E. coli carrying plasmid pAC2533-E. coli (pAC2533)-expressing the gene coding for Omp25 (omp25 gene) of B. melitensis, or with E. coli carrying plasmid pUC19-E. coli (pUC19). One control group received saline. The evolution of antibody responses was investigated by indirect ELISA with whole rough (R) B. melitensis H38 cells as antigen. Serum antibody titres of mice immunised with E. coli (pAC2533) were appreciably higher than those of mice immunised with E. coli (pUC19). The specificity to Omp25 of murine antibodies induced by E. coli (pAC2533) was demonstrated by SDS-PAGE and immunoblotting of five B. melitensis strains. Binding of antibody in E. coli (pAC2533) immune sera to the surface of B. melitensis strains differing in their smooth lipopolysaccharide (S-LPS) expression was also studied by whole-cell ELISA and by flow cytometry. Antibody reactivity to R and smooth-rough (S-R) was much stronger than that to smooth (S) B. melitensis strains, indicating a much better accessibility of Omp25 to antibody on strains lacking or expressing less O-polysaccharide on their surface. The antibodies to Omp25 were predominantly of IgG2a isotype. The capacity of E. coli (pAC2533) to induce protective immune responses against four challenge strains of B. melitensis was further evaluated in mice. Significant reductions in splenic infections, in comparison with mice immunised with E. coli (pUC19) and unimmunised (saline injection) mice, were observed in R B. melitensis B115, S-R B. melitensis EP and S B. melitensis H38 infected mice. Protection against S B. melitensis 16M was not significant. The data from the present study, together with previous results, suggest that humoral immunity against probably conformational, well-exposed epitopes of the Omp25 could contribute to protective mechanisms against B. melitensis infection in mice.

Braunagel SC, Parr R, Belyavskyi M, Summers MD (1998) Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase. Virology 244 :195-211


Baculovirus infection results in the induction of membrane structures within the nucleoplasm of the host cells. The source of these membranes is unclear ; however, using the normal dynamics of cellular membranes and the nuclear envelope as a model, it is possible that the cell cycle might play a role in the regulation of formation of these intranuclear membranes. Therefore, one goal of this study was to investigate the effect of baculovirus infection on the cell cycle of Sf9 host cells. Since few data are available on the cell cycle of insect cells, the first task was to define Sf9 cell cycle kinetics. The cell cycle phase distribution of Sf9 cells grown in suspension culture was determined to be evenly distributed (29% of the cells in G1, 33% in S, and 36% in G2/M phase), with the duration of G1 and S phases both being about 6 h and the combined duration of G2/M phase being about 8 h. When Sf9 cells were infected with AcMNPV (Autographa californica nuclear polyhedrosis virus), approximately 84% of the cells were arrested in G2/M phase by 18-24 h p.i. Concomitant with the viral-induced arrest in G2/M phase, high levels of both cdc2-associated histone H1 kinase activity and cyclin B protein were detected. By 24 h p.i. cyclin B was no longer detected ; however, cdc2-associated histone H1 kinase activity remained throughout the infection. These data suggested that early in infection, cyclin B/cdc2 complex may be used to regulate the transition from G2 to M phase, but prolonged arrest may be due to a protein(s) encoded by AcMNPV. DNA hybridization analysis showed that the maximal rate of viral DNA replication occurred before G2/M arrest. We noted that viral DNA replication still occurred late in infection, when the majority of the cells were arrested in G2/M phase. Since cellular DNA replication normally does not occur during G2 or M phase, experiments were designed to determine if viral DNA replication could occur even when host cell DNA replication was arrested. Sf9 cells were arrested and "frozen" at the boundary of G1/S phase using 5-fluoro-2’deoxyuridine (FdUrd) treatment and then infected with AcMNPV In the blocked, infected cells, viral DNA replication was detected ; however, cellular DNA remained at steady-state levels. These results suggested that cellular DNA replication was not necessary for viral DNA replication and show that viral DNA replication was not significantly inhibited by FdUrd treatment. It was a surprise to detect viral DNA replication when the host cells were "frozen" at G1/S phase. We wanted to determine if the viral infection was progressing to the stage of progeny virus production. Our data showed that progeny budded virus (BV) and virus-induced intranuclear microvesicles were produced in the frozen, infected cells ; however, the intranuclear microvesicles had an unusual structure. They were irregular in shape and thickened compared to those observed in a normal infection. Very few enveloped nucleocapsids were visible in the nucleus of the frozen, infected cells and the occluded-derived virus envelope proteins, ODV-E66 and ODV-EC27, were not detected by Western blot analyses. Since the cells were sustained at the boundary of G1 and S phases for the duration of this experiment, the decreased amount of enveloped ODV in the nucleus could be due to several factors, including decreased levels of proteins expressed from late genes, aberrant microvesicles, or the necessity of G2/M phasing of the infected cell for efficient production and maturation of intranuclear microvesicles. These data indicate that AcMNPV infection results in cell cycle arrest in G2/M phase and this arrest may be due to a viral-encoded protein(s) that has cdc2-associated kinase activity. (ABSTRACT TRUNCATED)

Chang WL, van der Heyde HC, Klein BS (1998) Flow cytometric quantitation of yeast a novel technique for use in animal model work and in vitro immunologic assays. J Immunol Methods 211 :51-63


Animal models of fungal and other infectious diseases often require that the number of organisms in tissue be quantified, traditionally by grinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficiency. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum yeast were labelled with specific monoclonal or polyclonal antibodies to stain surface determinants or with Calcofluor to stain cell-wall chitin. A defined number of fluorescently labelled beads were added prior to acquisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side scatter and intensity of fluorescence. Cultured yeast were enumerated by both standard CFU determination and flow cytometry in a range of 10(2) to 10(7) cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytometric method was applied to murine models of histoplasmosis and blastomycosis to quantify the burden of fungi in the lungs of infected mice. Labelling yeast with Calcofluor alone resulted in unacceptably high levels of nonspecific binding to mouse cell debris. In contrast, labelling H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accurate quantitation. We conclude that this flow cytometry technique is rapid, efficient and reliable for quantifying the burden of infection in animal models of fungal disease. The technique also should lend itself to performing cytotoxicity assays that require discrimination of live and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms.

Clarke RG, Pinder AC (1998) Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers. J Appl Microbiol 84 :577-584


A proprietary fluorogenic marker for cell viability (Chemchrome) was investigated for the detection of bacteria using flow cytometry. This marker was used in combination with fluorescently labelled monoclonal antibodies (against Salmonella typhimurium). Owing to the former’s broad band emission spectrum, it was necessary to use the novel dye RED613 for the antibodies. This combined protocol, being sensitive only to the live Salm. typhimurium cells, reduced errors due to intrinsic fluorescence and non-specific binding. Detection of the order of 100 cells ml-1 was achieved in 30 min. This level was achieved even in the presence of large numbers of non-target or dead organisms.

Cleare W, Casadevall A (1998) The different binding patterns of two immunoglobulin M monoclonal antibodies to Cryptococcus neoformans serotype A and D strains correlate with serotype classification and differences in functional assays. Clin Diagn Lab Immunol 5 :125-129


Cryptococcus neoformans var. neoformans strains have historically been divided into serotypes A and D on the basis of reactivity with rabbit sera. Previously, we noted that two murine immunoglobulin M monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan produced different indirect immunofluorescence (IF) patterns, described as annular and punctate, when bound to C. neoformans cells from different strains. In this study, we examined the reactivity of these two MAbs, known as 12A1 and 13F1, with 20 C. neoformans var. neoformans strains, of which 13 were serotype A and 7 were serotype D. For all strains, MAb binding was studied by IF and agglutination assays. In addition, we blindly tested the IF patterns of 22 C. neoformans var. neoformans strains. For selected strains, MAb binding was studied by flow cytometry (FACScan) and phagocytosis assays. The epitopes recognized by MAbs 12A1 and 13F1 were found in all of the strains. MAb 12A1 binding produced an annular IF pattern with all of the strains, irrespective of the serotype classification. MAb 13F1 binding produced annular binding with all of the serotype A strains and punctate binding with 19 of 20 serotype D strains. In general, the punctate IF pattern was associated with lower fluorescence intensity, a requirement for higher antibody concentrations to produce yeast cell agglutination, and lower opsonic efficacy. Our results provide strong support for the existing classification of two serological types for strains assigned to variety neoformans and indicate qualitative and quantitative antigenic differences among serotype A and D strains.

Cloeckaert A, Weynants V, Godfroid J, Verger JM, Grayon M, Zygmunt MS (1998) O-Polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry. Clin Diagn Lab Immunol 5 :862-870


Smooth Brucella strains are classified into three serotypes, i.e., A+M-, A-M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported : A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities : C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

Codon AC, Benitez T, Korhola M (1998) Chromosomal polymorphism and adaptation to specific industrial environments of Saccharomyces strains. Appl Microbiol Biotechnol 49 :154-163


Several industrial Saccharomyces strains, including bakers’, wine, brewers’ and distillers’ yeasts, have been characterized with regards to their DNA content, chromosomal polymorphism and homologies with the DNA of laboratory strains. Measurement of the DNA contents of cells suggested that most of the industrial yeasts were aneuploids. Polymorphisms in the electrophoretic chromosomal pattern were so large that each strain could be individually identified. However, no specific changes relating to a particular group were observed. Hybridization using different probes from laboratory strains was very strong in all cases, indicating that all industrial strains possess a high degree of DNA homology with laboratory yeasts. Probes URA3, CUP1, LEU2, TRP1, GAL4 or ADC1 demonstrated the presence of one or two bands, two especially in bakers’ strains. Also, results indicate that all hybridized genes are located on the same chromosomes both in laboratory and industrial strains. Translocation from chromosome VIII to XVI seems to have occurred in a distillers’ strain, judging by the location of the CUP1 probe. Finally, when the SUC2 probe is used, results indicate a very widespread presence of the SUC genes in only bakers’ and molasses alcohol distillers’ strains. This clearly suggests that amplification of SUC genes is an adaptive mechanism conferring better fitness upon the strains in their specific industrial conditions. The widespread presence of Ty1 and Ty2 elements as well as Y’ subtelomeric sequences could account for the inter- and intrachromosomal changes detected.

Dehio M, Knorre A, Lanz C, Dehio C (1998) Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker. Gene 215 :223-229


Expression vectors suitable for directing high levels of protein synthesis in Bartonella henselae have been constructed based on the mobilisable broad-host-range (IncQ) plasmidpMMB206. They confer kanamycin resistance and feature the taclac (tac-lacUV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. While expression of genes fused to the taclac promoter is constitutive in one vector, the lacIq gene carried by the other vector allows a controlled, IPTG-inducible gene expression. These vectors were tested by subcloning a mutated gfp gene coding for the green fluorescent protein (GFP) from Aequorea victoria into the multiple cloning site and introducing the resulting plasmids into Escherichia coli and B. henselae. GFP expression was determined by measuring fluorescence via flow cytometry or directly by immunoblotting. Compared to E. coli, expression of GFP in B. henselae was more tightly controlled by lacIq and resulted in much higher levels of both IPTG-induced and constitutive gene expression. In vitro infection of endothelial cells indicated that GFP expression does not adversely affect the interaction of B. henselae with host cells. These data demonstrate that (i) the established expression vectors are useful for directing controlled or constitutive high-level protein synthesis in B. henselae and (ii) that GFP is a valuable expression marker which may has important applications in studying the bacterial genetics and cellular interactions of this emerging human pathogen.

Densmore CL, Smith SA, Holladay SD (1998) In vitro effects of the extracellular protein of Renibacterium salmoninarum on phagocyte function in brook trout (Salvelinus fontinalis). Vet Immunol Immunopathol 62 :349-357


Renibacterium salmoninarum is a facultative intracellular pathogen often found in host phagocytes where it appears to successfully avoid the host fish’s immunological defenses. The objective of this investigation was to determine if soluble extracellular protein (ECP) produced by R. salmoninarum may contribute to the immunomodulation in bacterial kidney disease (BKD) via inhibition of phagocyte respiratory burst and/or phagocytosis mechanisms. Splenic cells from adult brook trout (Salvelinus fontinalis) were incubated with two different concentrations of ECP (0.1 mg/ml and 1.0 mg/ml) and viable R. salmoninarum. Splenic cell cultures were evaluated for respiratory burst activity via flow cytometry with the dichlorofluorescin diacetate (DCF-DA) assay and for phagocytosis via light microscopic assessment of microsphere engulfment. Respiratory burst activity was significantly inhibited in all treatment groups as compared to untreated fish, while no differences were noted in phagocytic activity.

Fenili D, Pirovano B (1998) The automation of sediment urinalysis using a new urine flow cytometer (UF-100). Clin Chem Lab Med 36 :909-917


The UF-100 analyser is a fully automated instrument that stains the DNA and the membranes of the formed elements in native urine. The sample then passes as a laminar flow through a laser beam and light scattering, fluorescence and impedance are measured. The main purpose of the present work was to assess the analytical performance and the accuracy of the measurements of the UF-100 analyser. No carryover was observed, while the linearity was higher then the upper limit (40000 total particles microl(-1)) suggested by the manufacturer. The within-run imprecision was low, ranging from 17.7 % to 2.4% and was up to threefold better than manual microscopy. Comparison of results obtained by sediment microscopy (performed according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations) and by the UF-100 analyser showed a linear correlation with r = 0.833 for erythrocytes, r = 0.934 for leukocytes, r = 0.880 for epithelial cells and r = 0.40 for casts. To evaluate the reliability of the UF-100 analyser in detecting bacteria we compared the results with the microbial culture (n = 608). Using a cut-off value of bacterial count above 1800 degrees l(-1) and at leukocyte count above 45 microl(-1), the analyser detected positive cultures with a sensitivity of 87 % and a specificity of 80 %. In conclusion, the UF-100 analyser can improve the work flow, increasing the output of urinalysis by reducing the number of specimens submitted for microscopy. Also the method provides reliable information for the identification of urinary tract inflammation and bacterial infection.

Fernandez-Alonso M, Lorenzo G, Perez L, Bullido R, Estepa A, Lorenzen N, Coll JM (1998) Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus. Dis Aquat Organ 34 :167-176


Antibody linear epitopes of the glycoprotein G (gpG) of the viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus of salmonids, were mapped by pepscan using overlapping 15-mer peptides covering the entire gpG sequence and ELISA with polyclonal and monoclonal murine and polyclonal trout antibodies. Among the regions recognized in the pepscan by the polyclonal antibodies (PAbs) were the previously identified phosphatidylserine binding heptad-repeats (Estepa & Coll 1996 ; Virology 216:60-70) and leucocyte stimulating peptides (Lorenzo et al. 1995 ; Virology 212:348-355). Among 17 monoclonal antibodies (MAbs), only 2 non-neutralizing MAbs, 110 (aa 139-153) and IP1H3 (aa 399-413), could be mapped to specific peptides in the pepscan of the gpG. Mapping of these MAbs was confirmed by immunoblotting with recombinant proteins and/or other synthetic peptides covering those sequences. None of the neutralizing MAbs tested reacted with any of the gpG peptides. Previously mapped MAb resistant mutants in the gpG did not coincide with any of the linear epitopes defined by the pepscan strategy, suggesting the complementarity of the 2 methods for the identification of antibody recognition sites.

Friedrich TD, Okubo E, Laffin J, Lehman JM (1998) Okadaic acid induces appearance of the mitotic epitope MPM-2 in SV40-infected CV-1 cells with a >G2-phase DNA content. Cytometry 31 :260-264


Simian virus 40 (SV40) infection of quiescent monkey kidney cells stimulates two successive rounds of cellular DNA synthesis without an intervening mitosis. This uncoupling of S phase and mitosis indicates that SV40 modulates pathways regulating the G2-to-M phase transition. To examine the integrity of mitotic initiation pathways in infected cells that have bypassed mitosis, SV40-infected CV-1 cells were treated with okadaic acid (OA), a known inducer of premature mitosis in other cell types. OA treatment triggered the appearance of the mitotic marker MPM-2 in SV40-infected CV-1 cells progressing through either the first (diploid) or second (tetraploid) S phases. These results demonstrate that a subset of mitotic pathways are intact but inactive in SV40-infected cells that have bypassed mitosis and initiated tetraploid S phase.

Galdiero M, Palomba E, De L, Vitiello M, Pagnini P (1998) Effects of the major Pasteurella multocida porin on bovine neutrophils. Am J Vet Res 59 :1270-1274


OBJECTIVE : To evaluate in vitro effect of the major fraction of outer membrane proteins of Pasteurella multocida with porin-like activities on some biological functions of bovine neutrophils. ANIMALS : Neutrophils from 5 adult cattle. PROCEDURE : Variations in such biological processes as actin polymerization and chemotaxis and evaluation of hydrogen peroxide attributable to variable concentrations of P multocida were recorded and compared. Data were obtained, using the porin and lipopolysaccharide (LPS) isolated from a strain of P multocida cultivated in brain-heart infusion (BHI) broth. Various concentrations of porin and LPS were analyzed to evaluate changes in functional activation and microbicidal activity of bovine neutrophils. RESULTS : The 37.5-kd major polypeptide of the outer membrane of P multocida was isolated. Presence of this porin was significantly correlated with variations of some biological functions of bovine neutrophils. These immunocompetent cells had a concentration-dependent increase in actin polymerization and chemotactic activity. A concentration-dependent variation in the oxidative burst also was observed. CONCLUSIONS : The porins of gram-negative bacteria affect several biological functions of cells involved in the immune response as well as in inflammation. Significant correlation of results of in vitro experiments also was identified between porin and LPS effect. Pretreatment of bovine neutrophils with various concentrations of porin always caused a concentration-dependent increase in examined biological activities.

Gibellini D, Panaya R, Rumpianesi F (1998) Induction of apoptosis by Chlamydia psittaci and Chlamydia trachomatis infection in tissue culture cells. Zentralbl Bakteriol 288 :35-43


The role of programmed cell death (apoptosis) in LLC-MK2 cells infected with Chlamydia trachomatis LGV2 serotype and Chlamydia psittaci 6BC strain was investigated using flow cytometry and TUNEL procedures. The number of apoptotic cells was significantly higher at 72 and 96 hours post infection in the Chlamydia infected cell cultures in comparison with mock-infected cells. We postulate the apoptotic process to be a mechanism induced by C. trachomatis and C. psittaci infection in LLC-MK2 cells.

Hansen GH, Delmas B, Besnardeau L, Vogel LK, Laude H, Sjostrom H, Noren O (1998) The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment. J Virol 72 :527-534


Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.

Harder TC, Harder M, de Swart RL, Osterhaus AD, Liess B (1998) Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses. Vet Q 20 :50-55


The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

Hautala T, Grunst T, Fabrega A, Freimuth P, Welsh MJ (1998) An interaction between penton base and alpha v integrins plays a minimal role in adenovirus-mediated gene transfer to hepatocytes in vitro and in vivo. Gene Ther 5 :1259-1264


Studies in cultured cell lines have shown that adenovirus infection involves binding of adenovirus fiber to its cell surface receptor and binding of penton base to alpha v integrins. However, much less is known about the role of these interactions in cells that are targets for adenovirus-mediated gene transfer. Earlier work showed that hepatocytes are readily infected by adenovirus, making them an attractive target for gene therapy in several diseases. We found that addition of fiber protein blocked adenovirus infection of primary cultures of hepatocytes. This suggests an important role for fiber and its receptor. However, mutation of the integrin-binding motif in penton base did not inhibit infection of hepatocytes, even though the mutation impaired infection of HeLa cells. Hepatocytes had undetectable amounts of alpha v integrins on their cell surface and showed no specific adherence to vitronectin, the natural substrate of alpha v integrins. Adenovirus with an intact penton base enhanced infection of liver following intravenous injection, but only by three-fold as compared with virus in which the integrin-binding motif was disrupted. These studies suggest that interactions between cell surface integrins and penton base are not required for adenovirus infection of hepatocytes in vitro, but the interaction enhances infection to a small degree in vivo.

Hep A, Zaloudik J, Janakova J, Habanec B, Prasek J, Dolina J, Dite P (1998) [Effect of H. pylori eradication regimes on the proliferation index of gastric mucosa]. Vnitr Lek 44 :447-450


Eradication regimes with the blocking agent of the proton pump and without it do not influence the activity of cell division after treatment of Helicobacter pylori (H.p.) when using cytoflowmetric evaluation. The non-significant difference in proliferation activity of the gastric mucosa after treatment of H.p. can be also a sign of more rapid repair of the gastric mucosa after elimination of the inflammatory elements.

Hirayama K, Kobayashi M, Kondoh M, Muro K, Iwabuchi S, Yoh K, Ishizu T, Kikuchi S, Yamaguchi N, Nagase S, Koyama A (1998) Henoch-Schonlein purpura nephritis associated with methicillin-resistant Staphylococcus aureus infection. Nephrol Dial Transplant 13 :2703-2704


Jansen WT, Gootjes J, Zelle M, Madore DV, Verhoef J, Snippe H, Verheul AF (1998) Use of highly encapsulated Streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies. Clin Diagn Lab Immunol 5 :703-710


A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 microliter per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

Just T, Burgwald H, Broe MK (1998) Flow cytometric detection of EBV (EBER snRNA) using peptide nucleic acid probes. J Virol Methods 73 :163-174


The application of peptide nucleic acid (PNA) probes for detection of Epstein-Barr Virus (EBV) snRNA in fixed cells is described. Fluorescein labelled PNA probes were used to detect EBER1 and EBER2 snRNA in Raji, Daudi and HS-Sultan cells. The fixation and permeabilization of cells were optimized. The optimal fixation was found to be 5% acetic acid plus 4% paraformaldehyde in PBS and the optimal permeabilization 0.5% Tween 20 in PBS whereas no proteolytic digestion was needed. The hybridization time needed with the PNA probes was only 1 h. When running mixed samples of Ramos (EBV neg.) Raji, Daudi and HS-Sultan (EBV pos.) cells in flow cytometry a strong fluorescence signal was seen in Raji, Daudi and HS-Sultan cells whereas no fluorescence signal was seen in the Ramos cells. In total 0.5% EBER positive Raji cells could easily be identified in a mixture of Raji and Ramos cells. The results were verified by fluorescence microscopy. It is concluded that PNA probes can be used for in situ hybridization in solution and the analysis can be done using flow cytometry or fluorescence microscopy. PNA probes therefore may facilitate and enhance the potential use of the in situ hybridization/flow cytometry combination.

Kalinin NL, Pronin AV (1998) [The visualization of streptococcal cells by the use of fluorescein-bond dextran]. Zh Mikrobiol Epidemiol Immunobiol :6-9


The method for the visualization of streptococcal cells, based on the phenomenon of binding between fluorescein isothiocyanate-labeled dextran and the surface structures of streptococci, is proposed. S.cricetus, S.sobrinus and S.faecalis strains were studied. The data obtained in this study were compared with the results of flow cytofluorimetry of these microbial cells. The stability of binding dextrans by microbes and the influence of ionic force on this process were determined at room temperature, +4 degrees C and -20 degrees C.

Karpf AR, Brown DT (1998) Comparison of Sindbis virus-induced pathology in mosquito and vertebrate cell cultures. Virology 240 :193-201


We have compared Sindbis virus-induced cytopathology in vertebrate and mosquito (Aedes albopictus) cell cultures. It has been shown that vertebrate cells undergo apoptosis when infected by Sindbis virus and this was confirmed here using hamster cells (BHK). The occurrence of cell death in Sindbis virus-infected A. albopictus cells is a cell clone-specific phenomenon and, unlike in BHK cell cultures, mosquito cell death does not correlate with a large induction of apoptosis, as determined by assays testing for DNA fragmentation or reduced cellular DNA content. Cell cycle distribution changes were observed in Sindbis virus-infected BHK and C7-10 cell cultures, and the changes are distinct, both in the time of induction and the types of perturbations. In Sindbis virus-infected BHK cells, the major cell cycle profile change is the early accumulation of cells with sub-G1 DNA content and a corresponding reduction in the proportion of cells in G1 and G2/M. For Sindbis virus-infected C7-10 cells, the major perturbations are an increased proportion of cells showing G2/M or polyploid DNA content and a reduction in the proportion of G1 and S phase cells. These data suggest that the pathology induced in mosquito cell cultures by Sindbis virus infection may be distinct from the pathology which appears in vertebrate cell cultures.

Kirk SM, Schell RF, Moore AV, Callister SM, Mazurek GH (1998) Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours. J Clin Microbiol 36 :1568-1573


Susceptibility testing of Mycobacterium tuberculosis is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.

Kusunoki H, Hu D, Piyankarage RH, Sugii S, Uemura T (1998) Flow cytometric analysis for enterotoxin exposed on Clostridium perfringens spores. J Vet Med Sci 60 :1357-1359


Flow cytometric method (FCM) with fluorescent-labeled anti-CPE antibody was applied to develop a rapid, specific, and convenient method to detect enterotoxin (CPE) exposed on the surface of spores of Clostridium perfringens. The results obtained indicate that FCM can specifically detect CPE exposed on C. perfringens spores for a short time. Thus, FCM is found to be a rapid, specific, and convenient assay method for detection of CPE exposed on C. perfringens spores, suggesting that it will be hopefully useful to diagnose food poisoning caused by C. perfringens.

Kyd JM, Cripps AW, Murphy TF (1998) Outer-membrane antigen expression by Moraxella (Branhamella) catarrhalis influences pulmonary clearance. J Med Microbiol 47 :159-168


Moraxella (Branhamella) catarrhalis is a common respiratory tract pathogen in man. The bacterium shows a strong tendency to form aggregates in vitro. A variant strain of M. catarrhalis that showed a reduced tendency to form aggregates was selected by successive in-vitro passage in broth culture from which aggregates had settled. The non-clumping variant strain showed alteration in expression of outer-membrane antigens, including the HMW-OMP, an outer-membrane protein of c. 200 kDa, outer-membrane protein CD and lipo-oligosaccharide. A mouse model for pulmonary challenge with M. catarrhalis revealed significant differences in the rate of clearance of the isogenic variant strains from the lung. The parent strain caused enhanced recruitment of neutrophils to the lung and more rapid clearance of bacteria from the lungs in comparison to the non-clumping variant. It is concluded that alteration of expression of surface molecules by M. catarrhalis has a significant impact in an in-vivo model of pulmonary clearance.

Lebaron P, Parthuisot N, Catala P (1998) Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl Environ Microbiol 64 :1725-1730


Seven blue nucleic acid dyes from Molecular Probes Inc. (SYTO-9, SYTO-11, SYTO-13, SYTO-16, SYTO-BC, SYBR-I and SYBR-II) were compared with the DAPI (4’,6-diamidino-2-phenylindole) method for flow cytometric enumeration of live and fixed bacteria in aquatic systems. It was shown that SYBR-II and SYTO-9 are the most appropriate dyes for bacterial enumeration in nonsaline waters and can be applied to both live and dead bacteria. The fluorescence signal/noise ratio was improved when SYTO-9 was used to stain living bacteria in nonsaline waters. Inversely, SYBR-II is more appropriate than SYTO dyes for bacterial enumeration of unfixed and fixed seawater samples.

Lee EK, Kehrli ME, Jr. (1998) Expression of adhesion molecules on neutrophils of periparturient cows and neonatal calves. Am J Vet Res 59 :37-43


OBJECTIVE : To determine expression of the beta 2-integrin (CD18) family of adhesion molecules and L-selectin (CD62L) on neutrophils from periparturient cows and calves. ANIMALS : 8 periparturient Holstein cows and 9 Holstein calves. PROCEDURE : Constitutive CD18 and CD62L expression on neutrophils was determined by flow cytometry, using specific monoclonal antibodies. Platelet-activating factor was used to activate neutrophils in vitro to measure down-regulation of CD62L and up-regulation of CD18 on activated neutrophils. RESULTS : Mean values for constitutive and platelet-activating factor-stimulated CD18 expression on neutrophils from cows and calves were highest at parturition, then decreased during the first 24 hours after parturition on calf neutrophils, whereas CD62L expression decreased markedly by 9 to 24 hours after parturition on cow and calf neutrophils. Constitutive amounts of CD18 and CD62L on cow neutrophils returned to prepartum values by day 3 after parturition. Amounts of CD18 expression on calves’ neutrophils recovered by 1 week of age, but did not reach original birth values, whereas CD62L expression exceeded birth values by day 3 after parturition. Cows had leukocytosis (neutrophilia), with a doubling of circulating neutrophils 9 hours after calving that was inversely correlated with CD62L expression on neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE : Low amounts of CD62L and CD18 on calf neutrophils and of CD62L on neutrophils from cows for several days after parturition may result in impaired inflammatory response. Low CD62L expression may contribute to increased susceptibility to disease.

Lehman JM, Jacobberger JW (1998) Virus-cell interactions. Introduction. Cytometry 31 :233-234


Levy L, Broad S, Zhu AJ, Carroll JM, Khazaal I, Peault B, Watt FM (1998) Optimised retroviral infection of human epidermal keratinocytes : long-term expression of transduced integrin gene following grafting on to SCID mice. Gene Ther 5 :913-922


Previous attempts to achieve long-term gene expression in retrovirally transduced human epidermal keratinocytes in vivo have been largely unsuccessful. This has been variously attributed to a failure to target epidermal stem cells, suboptimal grafting conditions or inactivation of the retroviral vector. In an attempt to overcome these problems we expressed the chick beta 1 integrin subunit in primary human epidermal keratinocytes, which allowed us to monitor retroviral gene expression on a cell-by-cell basis. We describe optimised methods for selecting high-titre amphotropic packaging cells and for infecting keratinocytes in culture. When transduced cells were grafted into mice, graft survival was comparable in nude and SCID mice, but it was essential to combine the keratinocytes with a dermal substrate. Using these methods the majority of keratinocytes expressed the chick beta 1 integrin subunit for at least 16 weeks after grafting. We conclude that epidermal keratinocytes are attractive recipient cells for gene therapy.

Limaye MS, Coakley WT (1998) Clarification of small volume microbial suspensions in an ultrasonic standing wave. J Appl Microbiol 84 :1035-1042


The removal of Saccharomyces cerevisiae and Escherichia coli from 2.5 ml suspensions in ultrasonic standing wave formed at 1 or 3 MHz has been characterized. The standing wave was set up by a plane transducer and reflector mounted in the vertical plane. Cells in the ultrasonic field first concentrated in vertical planes at half wavelength separations. The ultrasound was then pulsed to allow clumps of concentrated cells to sediment in a controlled way during the short ’off’ intervals. Yeast removal from suspension at a concentration of 3 x 10(9) ml-1 (14% volume v/v) was 99.5% in a total time of 4.5 min. Almost total (99.5%) clarification of prokaryote (E. coli) suspension was achieved here for the first time in a standing wave field. The clarification of a 1.3 x 10(11) ml-1 (16% v/v) E. coli suspension occurred over 11.5 min. The period decreased to 7 min in the presence of a polycationic flocculant, polyethyleneimine. The implications of the results for design of systems to further reduce clarification times are discussed. Removal efficiency for both S. cerevisiae and E. coli decreased with decrease in cell concentration. This concentration dependence is shown not to be simply a consequence of acoustic interaction between single cells. Flow cytometry of stained cells detected no loss of cell viability arising from the ultrasonic procedure.

Logan RP, Robins A, Turner GA, Cockayne A, Borriello SP, Hawkey CJ (1998) A novel flow cytometric assay for quantitating adherence of Helicobacter pylori to gastric epithelial cells. J Immunol Methods 213 :19-30


Adherence may be an important virulence factor for Helicobacter pylori. Current methods available for quantitation of adherence are time consuming and liable to observer error. A new direct technique for fluorescent labelling of bacteria has been developed to quantitate adherence of H. pylori to epithelial cells by fluorescence activated cell sorting (FACS). Type strains of H. pylori, H. mustelae, H. cinaedi and H. fennelliae were grown microaerobically in broth culture for 24 h and fluorescently labelled by incubation with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37 degrees C. After washing to remove excess CFDA-SE, bacteria were co-incubated (ratio 10:1) with gastric epithelial cells at 37 degrees C for up to 24 h. After washing to remove non-adherent bacteria, epithelial cells were detached with EDTA (2 mM) and fixed with formaldehyde for flow cytometry. Adherence was quantitated both in terms of the proportion of cells with adherent H. pylori and as the mean number of adherent bacteria per cell. All H. pylori strains adhered to gastric-type epithelial cells. The proportion of cells with bound bacteria varied from 40-99% and the number of bacteria per cell from 1-50, both of which correlated with microscopy (r = 0.6, and r = 0.8 respectively, n = 35). Time course studies demonstrated saturation of binding by H. pylori within 90 min. For H. mustelae, H. cinaedi and H. fennelliae the proportion of cells with bound bacteria varied from 5-15% and the mean number of bacteria per cell was < 4. Binding of H. pylori to epithelial cells could be partly blocked by pre-incubation with polyclonal anti-sera or using oligosaccharides against potential binding epitopes of gastric mucus. Fluorescent labelling of H. pylori with CFDA-SE in combination with flow cytometry provides a quick, specific, and sensitive method to quantitate in vitro the adherence of H. pylori.

Lybarger L, Dempsey D, Patterson GH, Piston DW, Kain SR, Chervenak R (1998) Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation. Cytometry 31 :147-152


The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm ; > 90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm ; approximately 35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

Maisnier-Patin S, Dasgupta S, Krabbe M, Nordstrom K (1998) Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli. Mol Microbiol 30 :1067-1079


The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild-type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni- to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC. Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi- or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.

Makristathis A, Rokita E, Labigne A, Willinger B, Rotter ML, Hirschl AM (1998) Highly significant role of Helicobacter pylori urease in phagocytosis and production of oxygen metabolites by human granulocytes. J Infect Dis 177 :803-806


The contribution of Helicobacter pylori urease, the vacuolating cytotoxin VacA, and the 128-kDa protein CagA to the stimulation of human granulocytes in terms of phagocytosis and oxidative burst was evaluated. Blood was incubated with H. pylori strains and corresponding isogenic mutants lacking either the large urease subunit (UreB) or an accessory urease protein (UreG) or VacA or CagA. Phagocytosis and oxidative burst were monitored by flow cytometry. The UreB-lacking mutant was phagocytosed more efficiently (P < .001) and induced significantly less oxidative burst (P < .001) than its parental strain or the UreG-lacking mutant, which produces an enzymatically inactive urease. Values of the other mutants did not differ greatly from those of their parental strain. These data indicate inflammatory effects of H. pylori urease causing inhibition of phagocytosis and stimulation of oxidative burst by a pathway being largely independent of ammonia production.

Matsumoto K, Shimmura S, Goto E, Saito K, Takeuchi T, Miyajima S, Negi A, Tsubota K (1998) Lecithin-bound superoxide dismutase in the prevention of neutrophil-induced damage of corneal tissue. Invest Ophthalmol Vis Sci 39 :30-35


PURPOSE : To evaluate the effects of a lipophilic analog of superoxide dismutase (SOD) in the prevention of polymorphonuclear leukocyte (PMN)-induced damage to corneal epithelial cells in vitro and in bacterial corneal ulcers in vivo. METHODS : Immortalized human corneal epithelial cells (T-HCEC) were cocultured with human PMNs activated with N-formyl-methionyl-leucyl-phenylalanine for 18 hours, after which lactate dehydrogenase (LDH) activity of the supernatant was measured as a marker of cellular damage. The inhibitory effects of lecithin-bound SOD (PC-SOD) and unmodified SOD, as well as PMNs pretreated with anti-CD 18 monoclonal antibody, were compared with untreated control. The retention of each drug on the ocular surface of healthy volunteers was measured by flow cytometry using brush cytology samples. The protective effects of a 0.1% solution of PC-SOD on Pseudomonas aeruginosa corneal infection in guinea pigs were assessed by inflammatory grading scores and histology. RESULTS : Both PC-SOD and SOD effectively suppressed PMN-induced LDH release in T-HCEC in a dose-dependent manner. LDH release was also attenuated when PMNs were pretreated with anti-CD 18 antibodies, suggesting that adhesion molecules were involved in the process. Brush cytology of conjunctival samples showed that PC-SOD was retained longer on the ocular surface compared with unmodified SOD. PC-SOD significantly prevented excessive tissue damage by infiltrating PMNs in P. aeruginosa corneal infection, whereas in control eyes, perforation of the cornea occurred by 6 days. CONCLUSIONS : PC-SOD was effective in attenuating PMN-related tissue damage to corneal tissue both in vitro and in P. aeruginosa infection in guinea pigs.

McVay LD, Jaswal SS, Kennedy C, Hayday A, Carding SR (1998) The generation of human gammadelta T cell repertoires during fetal development. J Immunol 160 :5851-5860


The nature of how human gammadelta T cells are normally generated is not clear. We have used an RT-PCR assay and DNA sequencing to identify and compare delta-encoded TCRs (TCRDs) that are generated de novo in the fetal gut, liver, and thymus and to determine when, where, and how the TCRD repertoire is established during normal embryonic development. Rearranged TCRDV genes are first expressed outside of the thymus in the liver and primitive gut between 6 and 9 wk gestation. Although DV1Rs and/or DV2Rs predominated, differences in the pattern of TCRDV gene rearrangement and transcription in each tissue during ontogeny were identified. Specific, DV2-encoded TCRs are highly conserved throughout ontogeny in the tissues from the same and between genetically distinct donors. Although the thymic and intestinal gammadelta T cell repertoires partially overlap early in development, they diverge and become nonoverlapping during the second trimester, and the generation of the intestinal gammadelta T cell repertoire is characterized by differences in the processing of DV1Rs and DV2Rs. Whereas the structural diversity of DV1Rs progressively increases during gut development up to birth, DV2Rs have limited structural diversity throughout ontogeny. Together, our findings provide evidence for the ability of different fetal tissues to support the development of gammadelta T cells.

Morris AC, Riley JL, Fleming WH, Boss JM (1998) MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression. Am J Reprod Immunol 40 :385-394


PROBLEM : Major histocompatibility complex (MHC) class II molecule expression is specifically suppressed on fetal trophoblasts, even in response to interferon (IFN)-gamma, a potent inducer of MHC class II genes. The suppression of class II induction has been suggested to play a role in preventing rejection of the fetal allograft. The mechanism of this suppression is unknown. METHOD OF STUDY : Human trophoblast cell lines were examined for expression of MHC class II transcription factors and for activity of the IFN-gamma signaling pathway. Additionally, trophoblast cells were transfected with a vector expressing the class II transactivator, CIITA, and assayed for class II expression. RESULTS : The MHC class II transcription factors RFX and X2BP and the IFN-gamma signaling pathway components are expressed constitutively and are functional in trophoblasts. However, CIITA expression was absent in trophoblasts and could not be induced by IFN-gamma. Transfection of CIITA into trophoblast cells resulted in derepression of class II gene expression. CONCLUSIONS : The lack of induction of MHC class II genes in response to IFN-gamma in trophoblast cells is caused neither by the absence of factors that bind class II promoters, nor by a lesion in the IFN-gamma signaling pathway, but results from a specific inhibition of the CIITA gene.

Murrell-Bussell S, Nguyen D, Schober WD, Scott J, Simpson JL, Elias S, Bischoff FZ, Lewis DE (1998) Optimized fixation and storage conditions for FISH analysis of single-cell suspensions. J Histochem Cytochem 46 :971-974


In our protocol to isolate and identify fetal cells in maternal peripheral blood, antibody (Ab)-stained cells are preserved with paraformaldehyde (PF) before batch flow cytometric sorting. However, PF fixation compromises the quality of subsequent interphase fluorescence in situ hybridization (FISH). We therefore examined the effect of PF concentrations and storage time in phosphate-buffered saline (PBS) on the quality of FISH signals. Cells were analyzed for changes in light scatter, morphology, and accessibility of target cell DNA. Fixation in 3% PF for 1 hr was ideal for both flow cytometry and subsequent FISH detection. However, beyond 10 days of storage, FISH quality deteriorated. (J Histochem Cytochem 46:971-973, 1998)

Navarro-Garcia F, Alonso-Monge R, Rico H, Pla J, Sentandreu R, Nombela C (1998) A role for the MAP kinase gene MKC1 in cell wall construction and morphological transitions in Candida albicans. Microbiology 144 ( Pt 2) :411-424


The Candida albicans MKC1 gene encodes a mitogen-activated protein (MAP) kinase, which has been cloned by complementation of the lytic phenotype associated with Saccharomyces cerevisiae slt2 (mpk1) mutants. In this work, the physiological role of this MAP kinase in the pathogenic fungus C. albicans was characterized and a role for MKC1 in the biogenesis of the cell wall suggested based on the following criteria. First, C. albicans mkc1 delta/mkc1 delta strains displayed alterations in their cell surfaces under specific conditions as evidenced by scanning electron microscopy. Second, an increase in specific cell wall epitopes (O-glycosylated mannoprotein) was shown by confocal microscopy in mkc1 delta/mkc1 delta mutants. Third, the sensitivity to antifungals which inhibit (1,3)-beta-glucan and chitin synthesis was increased in these mutants. In addition, evidence for a role for the MKC1 gene in morphological transitions in C. albicans is presented based on the impairment of pseudohyphal formation of mkc1 delta/mkc1 delta strains on Spider medium and on the effect of its overexpression on Sacch. cerevisiae colony morphology on SLADH medium. Using the two-hybrid system, it was also demonstrated that MKC1 is able to interact specifically with Sacch. cerevisiae Mkk1p and Mkk2p, the MAP-kinase kinases of the PKC1-mediated route of Sacch. cerevisiae, and to activate transcription in Sacch. cerevisiae when bound to a DNA-binding element. These results suggest a role for this MAP kinase in the construction of the cell wall of C. albicans and indicate its potential relevance for the development of novel antifungals.

Osaki T, Taguchi H, Yamaguchi H, Kamiya S (1998) Detection of Helicobacter pylori in fecal samples of gnotobiotic mice infected with H. pylori by an immunomagnetic-bead separation technique. J Clin Microbiol 36 :321-323


By an immunomagnetic-bead (IMB) separation technique, isolation of Helicobacter pylori from gastrointestinal and fecal samples of gnotobiotic mice infected with the microorganism was tried. The isolation rate of H. pylori from stomach samples after IMB separation was not higher than that of direct culture of the samples. After IMB separation of feces, H. pylori was detectable by PCR, although H. pylori was not culturable.

Osaki T, Yamaguchi H, Taguchi H, Fukuda M, Kawakami H, Hirano H, Watanabe S, Takagi A, Kamiya S (1998) Establishment and characterisation of a monoclonal antibody to inhibit adhesion of Helicobacter pylori to gastric epithelial cells. J Med Microbiol 47 :505-512


Monoclonal antibodies (MAbs) that inhibit adhesion of Helicobacter pylori to human gastric cancer (MKN45) cells were established to clarify the mechanism of adhesion of H. pylori. Of 53 hybridoma clones screened by the primary inhibition assay for adhesion, MAb A20 of IgM class was selected on the basis of both its reactivity to whole cells of H. pylori by ELISA and its inhibitory effect on adhesion of H. pylori. The adhesion of H. pylori strain TK1029 to MKN45 cells was inhibited by MAb A20, depending on the concentration of the MAb. The MAb recognised the surface antigen, lipopolysaccharide (LPS) of H. pylori, suggesting that LPS is associated with adhesion of H. pylori to human gastric epithelial cells.

O’Sullivan D, Tosi P, Creusot F, Cooke BM, Phan TH, Dron M, Langin T (1998) Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum. Curr Genet 33 :291-298


The genome structure of Colletotrichum lindemuthianum in a set of diverse isolates was investigated using a combination of physical and molecular approaches. Flow cytometric measurement of genome size revealed significant variation between strains, with the smallest genome representing 59% of the largest. Southern-blot profiles of a cloned fungal telomere revealed a total chromosome number varying from 9 to 12. Chromosome separations using pulsed-field gel electrophoresis (PFGE) showed that these chromosomes belong to two distinct size classes : a variable number of small (< 2.5 Mb) polymorphic chromosomes and a set of unresolved chromosomes larger than 7 Mb. Two dispersed repeat elements were shown to cluster on distinct polymorphic minichromosomes. Single-copy flanking sequences from these repeat-containing clones specifically marked distinct small chromosomes. These markers were absent in some strains, indicating that part of the observed variability in genome organization may be explained by the presence or absence, in a given strain, of dispensable genomic regions and/or chromosomes.

Ott M, Stockert RJ, Ma Q, Gagandeep S, Gupta S (1998) Simultaneous up-regulation of viral receptor expression and DNA synthesis is required for increasing efficiency of retroviral hepatic gene transfer. J Biol Chem 273 :11954-11961


To understand the relative contribution of viral receptor expression and cell proliferation in retroviral gene transfer, we created human hepatocyte-derived HuH-7.MCAT-1 cell lines. These cells constitutively express the murine ecotropic retroviral receptor MCAT-1 without changes in morphology or proliferation states. The MCAT-1 receptor is also a cationic amino acid transporter, and the HuH-7.MCAT-1.7 cells showed increased Vmax of uptake and steady-state accumulation of the cationic amino acids L-arginine and L-lysine. In HuH-7.MCAT-1 cells, L-arginine uptake was significantly up-regulated by norepinephrine and dexamethasone, and hepatocyte growth factor also increased L-arginine uptake along with cellular DNA synthesis. Gene transfer was also markedly increased in HuH-7. MCAT-1.7 cells incubated with an ecotropic LacZ retrovirus, and this further increased with hormones and hepatocyte growth factor. To define whether viral receptor up-regulation by itself increased gene transfer, cell cycling was inhibited by a recombinant adenovirus expressing the Mad transcription factor (AdMad), which is a dominant-negative c-Myc regulator. This restricted cells in G0/G1, without attenuating MCAT-1 activity, as shown by flow cytometry and L-arginine uptake analysis, respectively. When asynchronously cycling HuH-7.MCAT-1.7 cells were first infected with the AdMad virus and then exposed to the ecotropic LacZ virus, gene transfer was virtually abolished. The data indicate that while up-regulation of viral receptors can greatly enhance retrovirally mediated gene transfer, DNA synthesis remains an absolute requirement for hepatic gene therapy with this approach.

Parida SK, Domann E, Rohde M, Muller S, Darji A, Hain T, Wehland J, Chakraborty T (1998) Internalin B is essential for adhesion and mediates the invasion of Listeria monocytogenes into human endothelial cells. Mol Microbiol 28 :81-93


Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero, causing disseminated sepsis. In both instances, the infection occurs by the crossing of endothelial cells lining a physiological barrier, the blood-brain barrier or the transplacental barrier. In this study, the ability of L. monocytogenes wild-type EGD to invade human umbilical vein endothelial cells (HUVECs) was evaluated using wild-type bacteria and isogenic Listeria mutants. Here, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin B gene product. This was demonstrated in several ways. First, L. monocytogenes strains lacking the inlB gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inlAB was complemented with a plasmid harbouring inlB only, whereas strains expressing inlA did not enter HUVECs. Thirdly, entry of wild-type EGD could be blocked effectively with antibodies to InlB. Fourthly, cell binding assays and flow cytometry with HUVECs showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non-invasive mutants dramatically increased their ability to invade HUVECs. In laser-scanning confocal microscopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry process by scanning electron microscopy revealed that both wild-type EGD and a recombinant strain overexpressing only InlB enter HUVECs in a similar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.

Persons DA, Mehaffey MG, Kaleko M, Nienhuis AW, Vanin EF (1998) An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene. Blood Cells Mol Dis 24 :167-182


Most retroviral vectors used in preclinical and clinical studies contain a selectable marker gene to facilitate the generation of producer clones. However, the expression of such genes in target cells is often undesirable since this may modify cellular phenotype and invoke a host immune response. Unfortunately, the efficient identification of high-titer producer clones for vectors lacking a selectable marker gene continues to be problematic and lacking for a standard methodology. Despite recent improvements in the screening techniques for identifying high-titer producer clones without the aid of a selectable marker, a solution to the fundamental problem of the very low frequency occurrence of high-titer clones within the starting cell population has not emerged. We have developed a strategy which greatly increases the frequency of virus-producing clones, including those with high-titer, within the population of transduced cells to be screened. This approach relies on the use of high-titer vector preparations generated in 293T cells by co-transfection of retroviral packaging and vector plasmids. Viral preparations of a vector lacking a selectable marker were used to repeatedly transduce exponentially growing packaging cells at a high multiplicity of infection (MOI). Each cell in the resulting polyclonal population of producer cells contained multiple copies of the unrearranged vector genome. Greater than 95% of the clones derived from this population produced vector particles as judged by slot blot analysis of viral RNA from conditioned media. Numerous clones with estimated titers of 10(5)-10(6) were identified. These titers were confirmed using a standard vector genome transmission assay. This approach significantly enhances the ability, without large scale screening, to easily identify high-titer clones lacking a selectable marker and should facilitate the routine use of simplified gene marking and therapeutic vectors.

Peterson JW, Boldogh I, Popov VL, Saini SS, Chopra AK (1998) Anti-inflammatory and antisecretory potential of histidine in Salmonella-challenged mouse small intestine. Lab Invest 78 :523-534


Challenge of mouse small intestinal loops with Salmonella typhimurium invoked the accumulation of luminal fluid, acute inflammation, and extensive structural damage to the small intestinal mucosa, as determined by histology and electron microscopy. Intraperitoneal and intestinal luminal injection of L-histidine, a known antioxidant, reduced the amount of fluid accumulating in the intestinal lumen and protected the intestinal tissue from S. typhimurium-induced damage. The reduction in S. typhimurium-induced fluid accumulation by L-histidine was specific for the L-enantiomer because D-histidine had no significant protective effect. Efficacy of L-histidine in protecting the infected intestinal tissue was attributed to the capacity of the imidazole ring to scavenge reactive oxygen species (ROS) generated by cells in the intestine during the acute inflammatory response. Glutathione levels were markedly reduced in S. typhimurium-challenged, inflamed intestinal tissues as a result of ROS generation. Importantly, after dosing the S. typhimurium-challenged mice with L-histidine, the glutathione content of the small intestinal tissue was not significantly different from mock-challenged controls. Further evidence favoring this mechanism included the capacity of L-histidine to scavenge ROS produced as a result of lipopolysaccharide (LPS) exposure of mononuclear cells (U937), as demonstrated with a redox-sensitive fluorescent dye (2’7’-dichlorodihydrofluorescein [DCF]). Addition of L-histidine, and to a lesser extent D-histidine, to the culture media of U937 cells before LPS exposure, resulted in a significant dose-dependent reduction in LPS-induced intracellular DCF fluorescence, as measured quantitatively by flow cytometry. The potential therapeutic value of anti-inflammatory drugs containing an L-histidine-like structure could protect infected mucosal tissues irrespective of microbial etiology.

Rokita E, Makristathis A, Presterl E, Rotter ML, Hirschl AM (1998) Helicobacter pylori urease significantly reduces opsonization by human complement. J Infect Dis 178 :1521-1525


The role of Helicobacter pylori urease in opsonization by human complement was investigated. H. pylori wild type strain N6 and isogenic mutants lacking either the large urease subunit (UreB) or an accessory urease protein (UreG) were incubated with different sera. C3b bound to the bacteria was measured by specific staining and flow cytometry. As compared with opsonization of N6 and the UreG-lacking mutant, opsonization of the UreB-lacking mutant was significantly increased after incubation with sera from both H. pylori uninfected (P<.001) or infected (P<.05) persons. However, when sera from uninfected persons were used, effective opsonization of this mutant proved to be dependent mainly on the classical pathway of complement activation. Irrespective of the serum used, opsonization values were very low after selective inactivation of the classical or the alternative pathway. Reduced opsonization of the urease-expressing strains could, to some extent, result from degradation of bound C3b.

Sato N, Kurotaki H, Watanabe T, Mikami T, Matsumoto T (1998) Use of hemoglobin as an iron source by Bacillus cereus. Biol Pharm Bull 21 :311-314


The hemoglobin binding activity of Bacillus cereus cells was measured with fluoresceinisothiocyanate (FITC)-conjugated hemoglobin using flow cytometry. Growth of B. cereus was markedly inhibited by the addition of apo-transferrin. B. cereus could not use transferrin-bound iron as an iron source in serum. The growth inhibition was reversed by the addition of a FeCl3 solution, erythrocytes or hemoglobin. B. cereus released hemolysin ; these findings suggested that the hemoglobin released from erythrocytes by B. cereus hemolysin binds to B. cereus and is thus used as an iron source.

Schonau A, Stender H, Grauballe PC (1998) A one-step solid phase immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi. J Immunol Methods 218 :9-17


A one-step immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi has been developed. The assay is based on C1q, which binds to immune complexes containing IgG and/or IgM antibodies. Micro-beads pre-coated with antibodies to human C1q are mixed with human serum samples and fluorochrome-labelled B. burgdorferi flagellum antigen. In the presence of serum IgG and/or IgM antibodies to B. burgdorferi, fluorochrome-labelled antigen/antibody complexes are formed. These are then bound by serum C1q and are subsequently captured on the anti-C1q-coated beads. The sample is analysed on a flow cytometer and the presence of fluorescent beads is, thus, indicative of a positive test result. In the present study the sensitivity and specificity of the assay are compared to those of the indirect IDEIA B. burgdorferi IgG and the mu-chain capture IDEIA B. burgdorferi IgM ELISAs for separate determination of IgG and IgM. Detection using a flow cytometer can be performed without separation of the beads from the reaction mixture, which means that in practice, the method is carried out as a one-step assay and it is, thus, very suitable for automation. Other advantages of this kind of assay includes an antibody/antigen reaction which occurs in solution and the potential of using the method for the detection of antibodies against several antigens from the same or different infectious agents (multi-parameter screening).

Seo KH, Brackett RE, Frank JF (1998) Rapid detection of Escherichia coli O157:H7 using immuno-magnetic flow cytometry in ground beef, apple juice, and milk. Int J Food Microbiol 44 :115-123


A rapid method combining flow cytometry and immunomagnetic bead separation (IMFC) was effective for detecting low populations of Escherichia coli O157:H7 inoculated into ground beef, apple juice, and raw milk samples. Modified buffered peptone was most effective for enrichment of E. coli O157:H7 in ground beef. Using IMFC in combination with a 6-h enrichment, as few as four E. coli O157:H7 cells/g of ground beef were detected in as little as a 7-h total analysis time. Our results demonstrate the possibility of using flow cytometry and immunomagnetic bead separation to detect low concentrations of E. coli O157:H7 in ground beef, apple juice and milk.

Seo KH, Brackett RE, Frank JF, Hilliard S (1998) Immunomagnetic separation and flow cytometry for rapid detection of Escherichia coli O157:H7. J Food Prot 61 :812-816


A rapid method for detecting Escherichia coli O157:H7 combining immunomagnetic beads (IMB) and flow cytometry was developed. Labeling antigens separated by IMB with fluorescent antibody enabled the detection of < 10(3) CFU bacteria per ml in pure culture. The optimum concentration of magnetic beads for flow cytometry was lower (ca. 10(5) particles per ml) than that reported for conventional IMB assay (more than 6 x 10(6) to 8 x 10(6) particles per ml). Immunomagnetic separation and flow cytometry (IMFC) were evaluated for detecting E. coli O157:H7 in the presence of a competing microorganism and for detecting antibodies in potassium phosphate buffer. The total assay time from separating antigens with IMB to analyzing with flow cytometry was about 1 h. IMFC detected 10(3) to 10(4) CFU of E. coli O157:H7 per ml in ground beef enrichment broth and could effectively discriminate between E. coli O157:H7 and competing natural flora. The new assay system provides another approach to separation and detection of low populations of pathogens and shows potential for detecting low concentrations of toxins and other soluble antigens directly from food in a short time.

Shi W, Jewett A, Hume WR (1998) Rapid and quantitative detection of Streptococcus mutans with species-specific monoclonal antibodies. Hybridoma 17 :365-371


Streptococcus mutans is known to be a prime etiologic agent for the initiation and progression of human dental caries. Rapid, accurate, and quantitative detection of S. mutans will help us better understand the pathogenic mechanisms of dental caries and will help to develop methods for caries diagnosis and risk assessment. This study describes the development of three highly species-specific monoclonal IgG antibodies against S. mutans. The antibodies were used to develop a number of methods that quantitatively detect S. mutans in less <1> http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9541561 ]

Flow cytometry was used to study the effect of the bacteriocin leucocin B-TA11a on Listeria (L.) monocytogenes. Mixed proportions of dead and live control populations were analyzed by flow cytometry to determine detection limits of the Dead/Live Baclight Bacterial Viability KitTM. High correlations for flow cytometric detection of defined proportions of live or dead cells in mixtures between 10 and 100% of dead (r2 = 0.97) or live (r2 = 0.99) cells were obtained. However, mixtures containing less than 10% of either live or dead control cells gave correlations below 0.72. The growth of L. monocytogenes in the absence and presence of leucocin B-TA11a was analyzed by flow cytometry with Baclight, plate counts, and optical density measurements. Although leucocin B-TA11a initially inhibited listerial growth, the uptake of both Baclight dyes suggested that cells remained viable but became leaky, possibly indicating bacteriocin-induced pore formation in the target membranes.

Takemura A, Matsuda N, Kimura S, Fujiwara T, Nakagawa I, Hamada S (1998) Porphyromonas gingivalis lipopolysaccharide modulates the responsiveness of human periodontal ligament fibroblasts to platelet-derived growth factor. J Periodontal Res 33 :400-407


Lipopolysaccharides (LPS) prepared from periodontopathic bacteria have been known to induce various biological responses which may lead to periodontal tissue breakdown. The purpose of this study was to determine if Porphyromonas gingivalis LPS could affect cellular functions of human periodontal ligament fibroblasts (HPLF). We showed here the responsiveness of cultured HPLF to platelet-derived growth factor (PDGF)-BB, a growth factor for mesenchymal cells, in the presence of P. gingivalis LPS. DNA synthesis of HPLF was enhanced in a dose-dependent manner when LPS were co-incubated for 48 h ; thereafter, it decreased to the baseline level within 24 h incubation. The stimulating effect of PDGF-BB was further enhanced by the pretreatment of HPLF with LPS (10 micrograms/ml) for 48 h. The binding assay of [125I]PDGF-BB and the flow cytometric assay using rabbit antiserum to human PDGF receptor (PDGF-R) beta-type indicated that this enhancement was due to the increase of the number of PDGF-R beta-type on HPLF. Immunoprecipitation using antiserum to human PDGF-R beta-type also showed that the synthesis of PDGF-R beta-type was augmented in the LPS-treated HPLF. These results indicate that P. gingivalis LPS stimulate cellular proliferation and responsiveness to PDGF-BB of cultured HPLF. These cellular reactions may be mediated by PDGF-BB binding, followed by increased synthesis of the receptor protein.

Tombolini R, Jansson JK (1998) Monitoring of GFP-tagged bacterial cells. Methods Mol Biol 102 :285-298


Tortorello ML, Reineke KF, Stewart DS, Raybourne RB (1998) Comparison of methods for determining the presence of Escherichia coli O157:H7 in apple juice. J Food Prot 61 :1425-1430


Six methods were compared for detection of three strains of Escherichia coli O157:H7 in enrichments of inoculated apple juice. Juice was inoculated at levels varying from 0.1 to 100 CFU/ml and centrifuged after overnight storage at 4 degrees C, and pellets were incubated at 37 degrees C in nonselective enrichment broth. At hourly intervals between 5 and 10 h and at 24 h, the enrichments were tested for E. coli O157:H7 by direct fluorescent antibody (DFA), antibody-direct epifluorescent filter technique (Ab-DEFT), direct selective plating on sorbitol MacConkey agar (SMA), immunomagnetic separation coupled to either selective plating (IMS-SMA) or the polymerase chain reaction (IMS-PCR), and flow cytometry (FC). The most consistent detection of 0.1 CFU/ml of the slowest growing strain of the pathogen was provided by the IMS-SMA and IMS-PCR after 8 h of enrichment. The time required for detection at the level of 0.1 CFU/ml for each assay was Ab-DEFT, 11 h ; IMS-PCR, 16 h ; FC, 24 h ; IMS-SMA, 32 h ; and SMA, 48 h. Absolute detection limits (without enrichment) were : IMS-PCR, 10(3) CFU/ml ; Ab-DEFT and IMS-SMA, 10(4) CFU/ml ; SMA, 10(5) CFU/ml ; and DFA, 10(6) CFU/ml. Recovery of the pathogen (10 CFU/ml) in apple juice after 28 days of 4 degrees C storage was possible by means of an 8-h enrichment and Ab-DEFT, IMS-PCR, or IMS-SMA.

Tuomola EM, Salminen SJ (1998) Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. Int J Food Microbiol 41 :45-51


The adhesion of 12 different Lactobacillus strains was studied using Caco-2 cell line as an in vitro model for intestinal epithelium. Some of the strains tested have been used as probiotics, and most of them are used in the dairy and food industry. Human and bovine enterotoxigenic Escherichia coli strains were used as positive and negative control, respectively. Bacterial adhesion to Caco-2 cell cultures was quantitated using radiolabelled bacteria. The adherence of bacteria was also observed microscopically after Gram staining. Viability of bacteria prior to adhesion was verified using flow cytometry. Among the tested strains, L. casei (Fyos) was the most adhesive strain and L. casei var. rhamnosus (Lactophilus) was the least adhesive strain, approximately 14 and 3% of the added bacteria adhered to Caco-2 cell cultures, respectively. The corresponding values for positive and negative control E. coli strains were 14 and 4%, respectively. The Lactobacillus strains tested could not be divided into distinctly adhesive or non-adhesive strains, since there was a continuation of adhesion rates. The four most adhesive strains were L. casei (Fyos), L. acidophilus 1 (LC1), L. rhamnosus LC-705 and Lactobacillus GG (ATCC 53103). No significant differences in the percentage adhesion were observed between these strains. Adhesion of all the strains was dependent on the number of bacteria used, since an approximately constant number of Caco-2 cells was used, indicating that the Caco-2 cell binding sites were not saturated. Viability of bacteria was high since approximately 90% of the bacteria were viable with the exception of L. acidophilus 1 which was 74% viable. Microscopic evaluations agreed with the radiolabelled binding as evidenced by observing more bacteria in Gram-stained preparations of good adhering strains compared to poorly adhering strains.

Vachiery N, Trap I, Totte P, Martinez D, Bensaid A (1998) Inhibition of MHC class I and class II cell surface expression on bovine endothelial cells upon infection with Cowdria ruminantium. Vet Immunol Immunopathol 61 :37-48


Endothelial cells constitute a main target for Cowdria ruminantium (CR) and can potentially play a role as antigen presenting cells (APC). Therefore, we measured, in vitro, the effect of CR infections on the expression of MHC class I and class II molecules on bovine umbilical endothelial cells (BUEC) and on bovine brain endothelial cells (BBEC). A dramatic inhibition of the expression of IFNgamma induced MHC class II molecules was observed on BUEC and to a lesser extent on BBEC upon CR infection. This inhibitory effect was also observed on constitutively expressed MHC class I molecules. Part of the reduction of cell surface MHC molecules could be ascribed to their accumulation in intracellular compartments pinpointing a disruption in the transit of these molecules to the surface of the cells. The exact mechanisms of inhibition are not yet known but, as opposed to what is described in other models, the involvement of prostaglandin E2 can be excluded. The results obtained in this study show that endothelial cells have a decreased capacity to express both MHC class I and class II molecules on their surface upon CR infection, thus favouring the escape of this pathogen from the host immune system.

Valdivia RH, Falkow S (1998) Flow cytometry and bacterial pathogenesis. Curr Opin Microbiol 1 :359-363


Our understanding of microbial adaptations to diverse and threatening environments is limited by the assumption that the behavior of individual bacteria can be accurately determined by measuring the behavior of populations. Recent advances in gene expression reporter systems, fluorescence microscopy and flow cytometry allow microbiologists to explore the complex interactions between bacteria and their environment with single cell resolution. The application of these technologies has been particularly useful in systems, such as host-pathogen interactions, where genetic analysis is often cumbersome. Recently, flow cytometry is increasingly being applied to study host-pathogen interactions.

van Werven T, Burvenich C, Piens K, van den Broek J, Heyneman R, Noordhuizen-Stassen EN, Schukken YH, Brand A (1998) Flow cytometric measurement of neutrophil alkaline phosphatase before and during initiation of an induced Escherichia coli mastitis in cattle. Vet Immunol Immunopathol 62 :235-244


In 12 healthy cows, neutrophil alkaline phosphatase (NAP) activity was measured by flow cytometer before and during an experimentally induced Escherichia coli mastitis, to study the role and increase of NAP in Gram-negative bacterial infections. Percentage of neutrophils containing alkaline phosphatase and intensity of NAP activity were measured. Preinfection percentage of neutrophils with enzyme activity varied between 64.0% and 84.4% and the intensity of enzyme activity was low in all cows. After induction of infection, percentage of neutrophils with enzyme activity showed a significant decrease on day 1 followed by an significant increase on day 3. NAP intensity increased significantly on the second and third day after infection. This increase of intensity was significantly, positively correlated with the severity of infection. From this study we may conclude that variation in susceptibility to E. coli mastitis could not be explained by preinfection NAP levels. The post-infection increase of NAP activity, that was found following an induced infection was more a result of increased enzyme intensity per neutrophil, then from an increase of percentage neutrophils with enzyme activity. Furthermore, a strong correlation was found between NAP intensity and severity of inflammation. There was evidence that the more severely diseased animals showed stronger NAP intensity increase.

Veena P, Traycoff CM, Williams DA, McMahel J, Rice S, Cornetta K, Srour EF (1998) Delayed targeting of cytokine-nonresponsive human bone marrow CD34(+) cells with retrovirus-mediated gene transfer enhances transduction efficiency and long-term expression of transduced genes. Blood 91 :3693-3701


Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34(+) cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol using neoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34(+) cells. BM CD34(+) cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers of neoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transduced neoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

Wang FJ (1998) Analysis of downstream effectors of p53 on cell growth arrest and apoptosis induced by a temperature-sensitive Val138 mutant. J Med Dent Sci 45 :141-149


To elucidate the mechanism for cell growth arrest in relation to apoptosis mediated by the p53 gene, I studied two p53-null human cancer cell lines (K562 and Jurkat) carrying a human p53 temperature sensitive (ts) mutant (p53138val). The cell growth in the newly established K562 cells carrying the ts mutant was arrested in G0/G1 at the permissive temperature (32.5 degrees C) , at least in part, due to induction of p21/waf1. In apoptosis-induced Jurkat cells upon temperature shift-down, a lower level of p21 induction relative to that of MDM2 was observed in comparison with that in K562 cells. Meanwhile, levels of bax and bcl-2 expression were relatively constant upon temperature shift-down in both cell lines. Studies on the phosphorylation pattern of Rb family proteins, and results of gel-shift and antibody super-shift assays showed the accumulation of Rb/E2F complexes in growth-arrested K562 cells, but no such phenomenon was observed in Jurkat cells. Taken together, the results are consistent with the idea that a higher level of p21 expression and its subsequent Rb/E2F complex formation played an important role in the growth arrest of K562 cells.

Weiser JN (1998) Phase variation in colony opacity by Streptococcus pneumoniae. Microb Drug Resist 4 :129-135


Withers HL, Bernander R (1998) Characterization of dnaC2 and dnaC28 mutants by flow cytometry. J Bacteriol 180 :1624-1631


Escherichia coli strains containing thermosensitive dnaC alleles were studied by flow cytometry. Strains containing either the dnaC2 or dnaC28 allele were shifted between different temperatures, and DNA content distributions were gathered. Inhibition of initiation of chromosome replication at nonpermissive temperature, as well as reinitiation of replication at permissive temperature, were found to be affected by a number of parameters. These included the choice of permissive and nonpermissive temperatures, the length of the time of incubation at the nonpermissive temperature, the growth medium, the type of temperature shift used for reinitiation of replication (transient or nontransient), the genetic background of the host cell, and the cell concentration. Reinitiation of replication required neither transcription nor translation, whereas the elongation stage of replication was dependent upon ongoing protein synthesis in the mutants. Efficient use of dnaC mutants for cell cycle studies is discussed.

Withers HL, Nordstrom K (1998) Quorum-sensing acts at initiation of chromosomal replication in Escherichia coli. Proc Natl Acad Sci U S A 95 :15694-15699


Chromosomal replication in Escherichia coli was studied by flow cytometry and was found to be inhibited by an extracellular factor present in conditioned media collected during late exponential and early stationary phase, i.e., via a quorum-sensing mechanism. Our results suggest that the inhibitory activity of the extracellular factor is exerted during initiation of DNA replication rather than during elongation. Furthermore, we present evidence that this interaction may occur directly at each of the replication forks. Unlike other quorum-sensing systems described so far for Gram-negative bacteria, this inhibitory activity does not require transcription or translation to be effective. Implications of quorum-sensing regulation of DNA replication are discussed.

Yang YF, Sylte MJ, Czuprynski CJ (1998) Apoptosis : a possible tactic of Haemophilus somnus for evasion of killing by bovine neutrophils ? Microb Pathog 24 :351-359


Haemophilus somnus is an important veterinary pathogen that causes respiratory disease, arthritis, septicaemia and abortion in cattle and sheep. In the present study we investigated the possibility that H. somnus resists killing by bovine neutrophils, by causing the latter to undergo morphological changes consistent with apoptosis. Both serum-sensitive and serum-resistant strains of H. somnus enhanced bovine neutrophil chromatin condensation and shape change (i.e. zeiosis) in vitro, suggesting that the cells were undergoing apoptosis. Heat-killed or formalin-killed H. somnus had less effect than viable H. somnus. Chromatin margination of neutrophils was greater whenH. somnus was opsonized with adult bovine serum, which facilitates phagocytosis of the bacteria. H. somnus culture filtrates did not cause bovine neutrophil chromatin condensation. These findings suggest that direct contact with H. somnus is required for the maximal effect on bovine neutrophils. Apoptosis was confirmed by flow cytometry, using propidium iodide staining to detect DNA fragmentation. These findings suggest that H. somnus can evade killing by bovine neutrophils, in part, by inducing these cells to undergo apoptosis.

Zheng J, Fu M, Wu M (1998) [Insulin-like growth factor-I upregulates the growth stimulatory effect of epidermal growth factor on HCE16/3 cells]. Zhonghua Zhong Liu Za Zhi 20 :101-104


OBJECTIVES : To examine the role of insulin-like growth factor-I (IGF-I) in growth and gene expression of human papillomavirus (HPV) type 16 DNA-immortalized human cervical epithelial cells (HCE16/3 cells). METHODS : The growth and gene expression of HCE16/3 cells treated with IGF-I and epidermal growth factor (EGF) were examined by flow cytometry, soft-agarose assay and RT-PCR analysis. RESULTS : Physiological concentration of IGF-I had little effect on proliferation of HCE16/3 cells in serum-free and growth factor-free medium but supraphysiological concentration of IGF-I stimulated moderate proliferation of HCE16/3 cells. EGF stimulated proliferation of HCE16/3 cells at physiological concentration. The stimulatory effect was stronger when combined with IGF-I than EGF alone. Furthermore, the combined use of EGF and IGF-I could induce colony formation of HCE16/3 cells in soft agarose. The expression of HPV 16 E7 gene in HCE16/3 cells was not altered by growth factors in RT-PCR analysis. CONCLUSION : IGF-I is a weak regulator of HCE16/3 cells but it enhances the growth stimulatory effect of EGF on HCE16/3 cells.