vendredi 24 avril 2009
par   G. Grégori

Alfa MJ, DeGagne P (1997) Attachment of Haemophilus ducreyi to human foreskin fibroblasts involves LOS and fibronectin. Microb Pathog 22 :39-46


Haemophilus ducreyi is the causative agent of the genital ulcer disease Chancroid. Chancroid has been shown to increase the risk of heterosexual transmission of HIV. Little is known regarding the attachment or localization of this organism to human cells in either the dermal or epidermal layer. In this study the attachment of H. ducreyi to human foreskin fibroblast (HFF) cells was further characterized. Attachment was mediated by more than one mechanism. Proteinase K treatment but not trypsinization of H. ducreyi significantly reduced attachment suggesting protein involvement. In addition, purified lipooligosaccharide (LOS) was able to inhibit attachment in a dose dependent manner. It appeared that the organism binds to fibronectin in the extracellular matrix of HFF cells, since competition studies using fibronectin showed that it was able to significantly reduce attachment in a dose dependent manner whereas collagen did not. We hypothesize that the attachment of H. ducreyi involves both a protein mediator of attachment (likely pili) as well as LOS and that one or both of these bacterial components interacts with fibronectin in the extracellular matrix to mediate attachment to HFF cells.

Alkout AM, Blackwell CC, Weir DM, Poxton IR, Elton RA, Luman W, Palmer K (1997) Isolation of a cell surface component of Helicobacter pylori that binds H type 2, Lewis(a), and Lewis(b) antigens. Gastroenterology 112 :1179-1187


BACKGROUND & AIMS : Individuals of blood group O and nonsecretors of ABO blood group antigens are more susceptible to peptic ulcers. The aim of this study was to determine if blood group antigens associated with group O or secretor status are epithelial cell receptors for Helicobacter pylori. METHODS : Bacterial binding and binding of monoclonal antibodies to H type 2, Lewis(a), and Lewis(b) to Kato III, buccal epithelial, and gastric mucosal cells were shown by flow cytometry. Bacterial outer membrane proteins eluted from H type 2, Lewis(a), or Lewis(b) were shown by polyacrylamide gel electrophoresis. RESULTS : Kato III and human epithelial cells bound each monoclonal antibody ; O cells bound more anti-H type 2 (P < 0.05). Binding indices for H. pylori correlated with those for anti-H type 2 (P < 0.005) and anti-Lewis(b) (P < 0.001) but not anti-Lewis(a). A 61-kilodalton protein was eluted from H type 2, Lewis(a), or Lewis(b). CONCLUSIONS : Our results indicate that H type 2 is an important receptor for the 61-kilodalton bacterial adhesin, partly explaining increased susceptibility of individuals of blood group O to ulcers. Lewis(b) binds H. pylori more efficiently than Lewis(a). If these interactions occur in vivo, lack of Lewis(b) in mucosal fluids of nonsecretors may contribute to colonization by H. pylori.

Bagai S, Lamb RA (1997) A glycine to alanine substitution in the paramyxovirus SV5 fusion peptide increases the initial rate of fusion. Virology 238 :283-290


Simian virus 5 fusion (F) protein mutant F-G3A, which contains a glycine-to-alanine substitution at position 3 in the conserved hydrophobic fusion peptide at the N-terminus of the F1 subunit, has been shown previously to cause increased syncytium formation compared to wild-type (wt) F protein, when expressed using an SV40 recombinant virus vector system (C. M. Horvath and R. A. Lamb (1992) J. Virol. 66, 2443-2455). The wt F and the F-G3A proteins were expressed in eukaryotic cells using the vaccinia virus-bacteriophage T7 RNA polymerase (vac-T7) expression system, and they showed similar cell surface expression levels as determined by flow cytometry. The final extent of fusion when the vac-T7 expression system was used was not found to be greatly different when examined with a reporter gene activation assay. However, the initial rate of fusion was found to be five- to sixfold higher for the F-G3A mutant protein than the wt F protein, when examined using a quantitative assay for lipid mixing based on relief of self-quenching of fluorescence of the lipid probe octadecyl rhodamine (R18). A microscopic fluorescent dye transfer assay also showed a much earlier spread of dye from R18-labeled red blood cells to the cells expressing the mutant F-G3A protein than the wt F protein. Thus, these data indicate that a single gly-to-ala mutation in the fusion peptide domain, although not affecting the final extent of fusion, significantly increased the rate of fusion. Possible mechanisms for the increased rate of fusion are discussed.

Bernander R, Poplawski A (1997) Cell cycle characteristics of thermophilic archaea. J Bacteriol 179 :4963-4969


We have performed a cell cycle analysis of organisms from the Archaea domain. Exponentially growing cells of the thermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius were analyzed by flow cytometry, and several unusual cell cycle characteristics were found. The cells initiated chromosome replication shortly after cell division such that the proportion of cells with a single chromosome equivalent was low in the population. The postreplication period was found to be long ; i.e., there was a considerable time interval from termination of chromosome replication until cell division. A further unusual feature was that cells in stationary phase contained two genome equivalents, showing that they entered the resting stage during the postreplication period. Also, a reduction in cellular light scatter was observed during entry into stationary phase, which appeared to reflect changes not only in cell size but also in morphology and/or composition. Finally, the in vivo organization of the chromosome DNA appeared to be different from that of eubacteria, as revealed by variation in the relative binding efficiency of different DNA stains.

Blake RD, Wang JZ, Beauregard L (1997) Repetitive sequence families in Alces alces americana. J Mol Evol 44 :509-520


High-resolution derivative melting was used to obtain detailed distributions of local (G + C) contents in a number of ruminant DNAs. Profiles over low (G + C) regions [20-36% (G + C)] are congruent for all ruminants. This region represents 45-50% of the nuclear DNA content and primarily contains intergenic and intron sequences. The high (G + C) region, where most coding sequences are found [38-68% (G + C)], is marked by satellite bands denoting the presence of transcriptionally inert, tandemly repetitive sequence families. These bands can be analyzed for the abundance, base composition, and sequence divergence of satellite families with relatively high precision. Band patterns are unique to each species ; even closely related species can be readily distinguished by their base distribution profiles. Variations in nuclear DNA contents in ruminants, determined by flow cytometry, are primarily due to variations in abundances of these repetitive sequence families. Thus, A. alces (moose) is found to have 8.85 +/- 0.2 pg DNA/cell, 25% more than the average in ruminants, while the base distribution curve indicates the presence of an unusually abundant satellite of 52.6% (G + C). The size (1 kb) and sequence of this satellite corresponds to satellite-I of other cervids, and in consequence it is designated Alces-I. The sequence of a cloned repeat of Alces-I has a length of 968 bp, a (G + C) content of 52.6%, and contributes 35%, or almost 3 million copies to the nuclear DNA, exceeding by approximately 300% the average array size of this repeat family in related cervids. In situ hybridization indicates the repeat is distributed throughout centromeric regions of all 62 acrocentric autosomes. Alces-I has much greater-than-expected numbers of GG, GA, and AG and far fewer numbers of TA and CG duplets, characteristics of all tandem repeats. The sequence is judged to be orthologous with satellite-I sequences from Rangifer tarandus (caribou), Capreolus capreolus (roe deer), Muntiacus muntjac (Chinese muntjac) and Muntiacus reevesi (Indian muntjac), as well as Antilocapra americana (pronghorn), and the bovids Bos taurus and Ovis aries. A tentative tree for the five cervids is in excellent agreement with one proposed on the basis of morphological characteristics. Differences from a consensus sequence indicate transversions exceed transitions by almost twofold, suggesting that substitutions occur randomly, or nearly so.

Colombo E, Colzani C, Butti G (1997) Detection of the cytomegalovirus antigen in the neutrophils of peripheral blood by flow cytometry. Eur J Histochem 41 Suppl 2 :199-200


Cormack BP, Bertram G, Egerton M, Gow NA, Falkow S, Brown AJ (1997) Yeast-enhanced green fluorescent protein (yEGFP)a reporter of gene expression in Candida albicans. Microbiology 143 ( Pt 2) :303-311


The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C. albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.

Diez AA, Farewell A, Nannmark U, Nystrom T (1997) A mutation in the ftsK gene of Escherichia coli affects cell-cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA. J Bacteriol 179 :5878-5883


An insertional mutation in ftsK, encoding an Escherichia coli product similar to the sporulation protein SpoIIIE of Bacillus subtilis, results in uspA overexpression in stationary phase and impairs cell division. The ftsK1 ::cat insertion mutant forms chains which are the result of inhibited cell-cell separation, while chromosome synthesis and partitioning appear to be normal as judged by flow cytometry and electron and light microscopy in combination with DNA staining. The cells of the chains are attached to each other by a small envelope structure, and unlike in a spoIIIE mutant of B. subtilis, there is no DNA trapped in the division plane. In addition, plasmids harboring a truncated ftsK allele lacking the last 195 bp of the gene cause chain formation in wild-type cells. While the mutant cells grow at essentially the same rate as the parent in complex and defined minimal media, they are sensitive to stresses. Specifically, the mutant failed to grow at elevated salt concentrations and survived stationary phase poorly. The phenotypes of the ftsK1 ::cat mutant are complemented by the 3’ end (spoIIIE-like half) of the ftsK locus. In contrast, the 5’ end of the ftsK locus reported to complement ftsK44(Ts) phenotypes does not complement the phenotypes of the ftsK1 ::cat mutant.

Dittmer D, Mocarski ES (1997) Human cytomegalovirus infection inhibits G1/S transition. J Virol 71 :1629-1634


Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.

Drossou V, Kanakoudi F, Tzimouli V, Sarafidis K, Taparkou A, Bougiouklis D, Petropoulou T, Kremenopoulos G (1997) Impact of prematurity, stress and sepsis on the neutrophil respiratory burst activity of neonates. Biol Neonate 72 :201-209


The aim of this study was to evaluate the impact of prematurity, sepsis and stress on the neutrophil respiratory burst activity (NRBA) of neonates. For this purpose 122 healthy neonates (89 term and 33 preterm), 33 preterm stressed neonates, 59 septic neonates (12 term and 47 preterm) and 26 healthy adults were studied. The NRBA was assessed after in vitro stimulation by PMA using a whole blood flow cytometric microassay with dihydrorhodamine 123 (DHR 123). It was found that the percentage of responding neutrophils in term neonates was comparable to that found in adults (medians 83.5 and 89.8%, respectively), whereas it was significantly lower in the healthy preterm neonates (median 70.6%, p < 0.05). The NRBA was further depressed in the stressed (median = 63%) and septic neonates, both term and preterm (medians 60.5 and 54.3%, respectively). No correlation with the levels of G-CSF, TNF-alpha and IL-1 beta, which were found to be higher in the stressed and septic neonates, was observed. These findings indicate that prematurity, sepsis and stress significantly depress the respiratory burst activity of neonatal neutrophils.

Du L, Pellen-Mussi P, Chandad F, Mouton C, Bonnaure-Mallet M (1997) Fimbriae and the hemagglutinating adhesin HA-Ag2 mediate adhesion of Porphyromonas gingivalis to epithelial cells. Infect Immun 65 :3875-3881


The mechanisms by which Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is pathogenic for the periodontium remain largely hypothetical. Invasion of host tissues by P. gingivalis is believed to require adhesion of the bacterium to host cells. The aim of this study was to use monoclonal antibodies (MAbs) to characterize the bacterial cell surface component(s) acting as a ligand binding to a receptor on epithelial cells. Surface antigens of P. gingivalis ATCC 33277 were obtained as a glass bead-EDTA extract (GBE), and antiserum against the GBE was produced in rabbits. Epithelial cell membrane proteins (ECMP) were prepared from a homogenate of the SK-MES-1 cell line with Triton X-100. The antigen/ligand profile of GBE was resolved by crossed immunoaffinity electrophoresis by using ECMP in the first-dimension gel. The migration of one immunoprecipitate (IP) was retarded, indicating a ligand-receptor interaction between a surface antigen of P. gingivalis and a complementary binding site on the epithelial cell membrane. The corresponding IP in the GBE/anti-GBE immunoelectrophoresis profile was excised from replicate gels to immunize mice for production of MAbs specific for the bacterial ligand. Five MAbs were obtained and tested for reactivity with GBE in immunoblots and for inhibition of the interaction between GBE and ECMP. Immunoblots revealed polypeptides at 28, 42, 43, and 49 kDa. Inhibition tests were positive for all five MAbs. These results are conclusive evidence that the MAbs recognize functional epitopes involved in the adherence of P. gingivalis to epithelial cells and that the adhesins are likely associated with fimbriae and the hemagglutinating adhesin HA-Ag2.

Egido JM, Vinuelas J (1997) Flow cytometric quantitation of phagocytosis in heparinized complete blood with latex particles and Candida albicans. Rev Soc Bras Med Trop 30 :441-446


We report a rapid method for the flow cytometric quantitation of phagocytosis in heparinized complete peripheral blood (HCPB), using commercially available phycoerythrin-conjugated latex particles of 1 micron diameter. The method is faster and shows greater reproducibility than Bjerknes’ (1984) standard technique using propidium iodide-stained Candida albicans, conventionally applied to the leukocytic layer of peripheral blood but here modified for HCPB. We also report a modification of Bjerknes’ Intracellular Killing Test to allow its application to HCPB.

Evidente A, Andolfi A, D’Apice L, Iannelli D, Scala F (1997) Identification by flow cytometry of seiridin, one of the main phytotoxins produced by three Seiridium species pathogenic to cypress. Nat Toxins 5 :14-19


Seiridin (SE), one of the main phytotoxins produced in vitro by Seiridium species pathogenic to cypress, was oxidized and the corresponding ketone derivative covalently linked to bovine serum albumin (BSA). The conjugate (SE-BSA) was used to prepare an antiserum to SE. The antibodies were absorbed with BSA and their specificity was assayed by ELISA and flow cytometry against SE, iso-seiridin (ISE), a structural isomer of SE, and some derivatives of these two metabolites. The antibodies tested in a competitive indirect ELISA did not show any binding activity to SE, ISE and their derivatives. The cytometry test, instead, was successful. SE-BSA and SE showed the highest binding activity with the antibodies. SE derivatives having a shift on the adjacent carbon, oxidation, or acetylation of the hydroxy group of the heptyl side chain at C-4 or conversion of the gamma-lactone in the corresponding planar furane ring reacted less than SE. The 2’-dansylhydrazoneSE and the 3,4-dihydroSE having a bulky group attached to the heptyl side chain and a saturated lactone ring, respectively, showed a weak reactivity. SE derivatives in which the gamma-lactone ring was destroyed and ISE derivatives presenting the shift of the hydroxy group at C-3’ and another structural modification had no binding activity.

Forslund T, Sundqvist T (1997) Nitric oxide-releasing particles inhibit phagocytosis in human neutrophils. Biochem Biophys Res Commun 233 :492-495


We have constructed a yeast (Saccharomyces cerevisiae) particle capable of releasing NO, by loading heat-killed yeast particles with a hydrophobic NO-generating substance, GEA-5171. This particle decreased phagocytosis in solution, as measured with flow cytometry, to about 80% of control values. Phagocytosis on a surface, as counted under the microscope, was also decreased by about 20%. The nitric oxide furthermore counteracted the production of oxygen metabolites by neutrophils to about 20% of control values. The inhibitory effect was most pronounced for the intracellular production, as could be seen when neutrophils preincubated with NO-releasing particles were stimulated with chemotactic agent (FMLP) or phorbol ester (PMA). In conclusion, NO has inhibitory effects on both phagocytosis and the respiratory burst of neutrophils. Since nitric oxide is a hydrophobic gas and an air pollutant, there is a possibility that it accumulates in particles which then become more resistant to elimination.

Godaly G, Proudfoot AE, Offord RE, Svanborg C, Agace WW (1997) Role of epithelial interleukin-8 (IL-8) and neutrophil IL-8 receptor A in Escherichia coli-induced transuroepithelial neutrophil migration. Infect Immun 65 :3451-3456


Escherichia coli stimulates neutrophil migration across human uroepithelial cell layers. This study investigated the role of the neutrophil chemokine interleukin-8 (IL-8) in this process. E. coli and IL-1alpha stimulated urinary tract epithelial layers to secrete IL-8 and induced transepithelial neutrophil migration. Anti-IL-8 antibody reduced neutrophil migration across epithelial cell layers, indicating a central role for this chemokine in the migration process. Furthermore, addition of recombinant IL-8 to unstimulated cell layers was sufficient to induce migration. The IL-8 dependence of neutrophil migration was maintained after removal of soluble IL-8 by washing of the cell layers. Flow cytometry analysis with fluorescein isothiocyanate-labelled IL-8 confirmed IL-8’s ability to bind to the epithelial cell surface. Indirect immunofluorescence with confocal laser scanning microscopy showed that IL-8 associated with the epithelial cell layers. Prior incubation of neutrophils with antibodies to IL-8 receptor A (IL-8RA) reduced neutrophil migration. Anti-IL-8 RB antibody had no effect on neutrophil migration. These results demonstrate that IL-8 plays a key role in E. coli- or IL-1alpha-induced transuroepithelial migration and suggest that epithelial cell-produced IL-8 interacts with IL-8RA on the neutrophil surface.

Guindulain T, Comas J, Vives-Rego J (1997) Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry. Appl Environ Microbiol 63 :4608-4611


Three nucleic acid dyes (SYTO-13, TOTO-1, and YOYO-1) were tested on cultures of Escherichia coli and marine prokaryote populations. These dyes stain the RNA and DNA in E. coli but only respond to DNA in marine populations, according to the histograms obtained after DNase and RNase treatments.

Gutierrez-Martin CB, Ojcius DM, Hsia R, Hellio R, Bavoil PM, Dautry-Varsat A (1997) Heparin-mediated inhibition of Chlamydia psittaci adherence to HeLa cells. Microb Pathog 22 :47-57


The adherence of human strains of Chlamydia trachomatis has been recently shown to be inhibitable by heparin and heparitinase, leading to the proposal that Chlamydia binding to host cells may be mediated by a glycosaminoglycan (GAG)-dependent mechanism. We here describe the adherence of the guinea-pig pathogen, Chlamydia psittaci GPIC, to HeLa cells, which was measured by cytofluorometry with chlamydiae whose DNA was fluorescently labelled. Adherence could be inhibited by heat or trypsin pretreatment of the bacteria, and binding was much faster at 37 degrees C (reaching a plateau within 1 h) than 4 degrees C. Little binding remained when host cells were pre-fixed with paraformaldehyde, suggesting that host cell receptor mobility may be required for effective adherence. Visualization by confocal microscopy confirmed that the bacteria were at or near the host cell surface during the entire time-course of these experiments. Adherence increased as a function of pH between pH 6 and pH 8.0-8.5. Both adherence and infection of HeLa cells could be inhibited with heparin when the adherence step was performed at 4 degrees C, but only infection was inhibited when the adherence step was performed at 37 degrees C, even though heparitinase could block adherence at either 4 degrees C or 37 degrees C. Even at 4 degrees C, heparin-mediated inhibition was significantly lower at pH 8 than pH 7.4, suggesting that GAG-independent mechanisms may play a role in the higher adherence observed at basic pH. These results therefore demonstrate that a GAG-dependent adherence step may be operative in C. psittaci, and raise the possibility that other adherence mechanisms may also contribute to binding by this chlamydial strain. Furthermore, they suggest that there may not be a strict correlation between C. psittaci adherence and the ability to cause productive infections.

Hairston PP, Ho J, Quant FR (1997) Design of an instrument for real-time detection of bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic fluorescence. J Aerosol Sci 28 :471-482


A prototype instrument has been constructed to measure individual airborne particles based on their aerodynamic size and their intrinsic fluorescence at selected excitation and emission wavelength bands. The instrument combines features of an aerodynamic particle sizing device with capabilities similar to those of a liquid flow cytometer. The goal of the instrument is to provide real-time data indicative of particle characteristics, and it is especially targeted to respond to bioaerosols from 0.5 to 10 micrometers (aerodynamic diameter) with intrinsic fluorescence exited at a wavelength of 325 nm and emitting from 420 to 580 nm. This size range covers individual airborne bacteria and bacteria clusters, and the fluorescence sensitivity is selected for biological molecules commonly found in cellular systems, for example, reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] and riboflavin. Initial tests with nebulised Bacillus subtilis var. niger (BG, ATCC 9372) spores have shown that, for both individual spores and spore clumps, a low level of fluorescence is detected from 17% of the particles. This detection percentage is on the same order as previous experiments that have measured viability of about 12% for mechanically dispersed BG spores (Ho and Fisher (1993) Defense Research Establishment Suffield Memorandum 1421) and suggests a need for further investigation into the possible relationship between the detected fluorescence and viability of bacterial spores.

Han Y, Kanbe T, Cherniak R, Cutler JE (1997) Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies. Infect Immun 65 :4100-4107


We previously reported that the immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1 protects mice against disseminated candidiasis, whereas the IgM MAb B6 does not. Both MAbs are specific for an adhesin fraction isolated from the cell surface of Candida albicans, but their epitope specificities differ. In the present study, we examined the surface locations of both epitopes and obtained structural information regarding the B6.1 epitope. Immunofluorescence confocal microscopic analysis of C. albicans yeast forms showed that epitope B6.1 is displayed rather homogeneously over the entire cell surface, whereas epitope B6 appears to have a patchy distribution. Both antibodies were essentially nonreactive with the surfaces of mycelial forms of the fungus, indicating that neither epitope is expressed on the surfaces of these forms. For isolation of the B6.1 epitope, the adhesin fraction consisting of cell surface phosphomannan was subjected to mildly acidic (10 mM HCl) hydrolysis and was fractionated into acid-labile and acid-stable portions by size exclusion chromatography. Antibody blocking experiments showed that the B6.1 epitope is an acid-labile moiety of the phosphomannan and that the B6 epitope is located in the acid-stable fraction. The B6 epitope appeared to be mannan because it was stable to heat (boiling) and protease treatments but was destroyed by alpha-mannosidase digestion. The B6.1 epitope eluted from the size exclusion column in two fractions. Mass spectroscopic analyses showed that one fraction contained material with the size of a mannotriose and that the other was a mixture of mannotriose- and mannotetraose-size substances. Dose response inhibition tests of the fractions indicated that the B6.1 epitope is associated with the mannotriose. Nuclear magnetic resonance (NMR) spectroscopic analysis of the epitope yielded data consistent with a beta-(1—>2)-linked mannotriose. The fine structure of the B6 epitope is under investigation. Information derived from these investigations will be useful both in understanding protective versus nonprotective antibody responses to C. albicans and in improving anti-Candida vaccine formulations.

Harris DT, Sakiestewa D, Robledo RF, Witten M (1997) Immunotoxicological effects of JP-8 jet fuel exposure. Toxicol Ind Health 13 :43-55


Chronic exposure to jet fuel has been shown to have adverse effects on human liver function, to cause emotional dysfunction, to cause abnormal electroencephalograms, to cause shortened attention spans, and to decrease sensorimotor speed (3-5). Due to the decision by the United States Air Force to implement the widespread use of JP-8 jet fuel in its operations, a thorough understanding of its potential effects upon exposed personnel is both critical and necessary. Exposure to potential environmental toxicants such as JP-8 may have significant effects on host systems beyond those readily visible (e.g., physiology, cardiology, respiratory, etc.) ; e.g., the immune system. Significant changes in immune consequences, even if short-lived, may have serious consequences for the exposed host that may impinge affect susceptibility to infectious agents. Major alterations in immune function that are long-lasting may result in an increased likelihood of development and/or progression of cancer, as well as autoimmune diseases. In the current study mice were exposed for 1h/day for 7 days to varying concentrations of aerosolized JP-8 jet fuel to simulate occupational exposures. Twenty-four hours after the last exposure the mice were analyzed for effects on their immune systems. It was observed that even at exposure concentrations as low as 100 mg/m3 detrimental effects on the immune system occurred. Decreases in viable immune cell numbers and immune organ weights were found. Jet fuel exposure resulted in losses of different immune cell subpopulations depending upon the immune organ being examined. Further, JP-8 exposure resulted in significantly decreased immune function, as analyzed by mitogenesis assays. Suppressed immune function could not be overcome by the addition of exogenous growth factors known to stimulate immune function. Thus, short-term, low concentration exposure of mice to JP-8 jet fuel caused significant toxicological effects on the immune system. It appears that the immune system may be the most sensitive indicator of toxicological damage due to JP-8 exposure, as effects were seen at concentrations of jet fuel that did not evidence change in other biological systems. Such changes may have significant effects on the health of the exposed individual.

Harris DT, Sakiestewa D, Robledo RF, Witten M (1997) Protection from JP-8 jet fuel induced immunotoxicity by administration of aerosolized substance P. Toxicol Ind Health 13 :571-588


Chronic exposure to jet fuel has been shown to cause human liver dysfunction, emotional dysfunction, abnormal electroencephalograms, shortened attention spans, and decreased sensorimotor speed. The United States Air Force has decided to implement the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Exposure to potential environment toxicants such as JP-8 may have significant effects on host physiology. Previous studies in mice have shown that short-term, low concentration JP-8 exposure had significant effects on the immune system ; e.g., decreased viable immune cell numbers, decreased immune organ weights, and loss on immune function that persisted for extended periods of time (i.e., up to 4 weeks post-exposure). Previous studies have shown that JP-8 induced pulmonary dysfunction was associated with a decrease in levels of the neuropeptide substance P (SP) in lung lavage fluids. It was found that administration of aerosolized SP was able to protect exposed animals from such JP-8 induced pulmonary changes. In the current study, aerosolized SP was analyzed for its effects on JP-i induced immunotoxicity in exposed mice. It was observed that SP administration could protect JP-8 exposed animals from losses of viable immune cell numbers, but not losses in immune organ weights. Further, exposure of animals to SP inhibitors generally increased the immunotoxicity of JP-8 exposure. SP appeared to act on all immune cell populations equally as analyzed by flow cytometry, as no one immune cell population appeared to be preferentially protected by SP. Also, SP administration was capable of protecting JP-8 exposed animals from loss of immune function at all concentrations of JP-8 utilized (250-2500 mg/m3). Significantly, SP only needed to be administered for 15 minutes after JP-8 exposure, and was active at both 1 microM and 1 nM concentrations. Thus, SP administration appears to be a relatively simple and efficient means to reverse the immunotoxicity due to hydrocarbon exposure.

Heinzelmann M, Herzig DO, Swain B, Mercer-Jones MA, Bergamini TM, Polk HC, Jr. (1997) Phagocytosis and oxidative-burst response of planktonic Staphylococcus epidermidis RP62A and its non-slime-producing variant in human neutrophils. Clin Diagn Lab Immunol 4 :705-710


The ability of bacterial organisms to produce an extracellular polysaccharide matrix known as slime has been associated with increased virulence and delayed infections in various prosthetic implants. Within a biofilm, this slime may protect the embedded bacteria from host defense mechanisms, especially phagocytosis by polymorphonuclear leukocytes. To determine whether planktonic Staphylococcus epidermidis is protected in a similar way, a novel flow cytometric assay was performed, measuring ingestion and adherence during phagocytosis and the production of superoxide during oxidative burst. Hydrophobicity was determined by hydrophobic interaction chromatography. Slime-producing S. epidermidis RP62A and its phenotypic variant, non-slime-producing RP62A-NA, were compared. The results showed increased phagocytosis of RP62A at 2, 5, 10, and 30 min ; increased adherence of RP62A at 30 s and 30 min ; and increased superoxide production of RP62A after 2 min. Decreased hydrophobicity of RP62A over RP62A-NA was correlated with a hydrophilic slime coat. The data argue that the host aggressively combats slime-producing S. epidermidis. This biological phenomenon is potentially important during bacteremia to prevent further adhesion, accumulation, and the genesis of a bacterial biofilm on implants or tissue surfaces.

Hoff HS, Donis RO (1997) Induction of apoptosis and cleavage of poly(ADP-ribose) polymerase by cytopathic bovine viral diarrhea virus infection. Virus Res 49 :101-113


The Pestivirus bovine viral diarrhea virus (BVDV) causes the fatal diarrheal syndrome, mucosal disease, because of mutations in the viral genome which convert the common noncytopathic (ncp) BVDV into a cytopathic (cp) biotype. We examined the nature of the cytopathic effect of cp-BVDV in cultured bovine cells in order to accurately describe the process and to gain insight into the mechanism of cp-BVDV-induced cell death. The findings demonstrate that cells infected with cp-BVDV in vitro die by apoptosis, but cells infected with ncp-BVDV do not. Analysis of nuclear morphology by staining with fluorescent DNA dye and cpi-fluorescence microscopy showed chromatin condensation and margination in cells infected with cp-BVDV. Transmission electron microscopy (TEM) confirmed the condensation of chromatin, as well as cell shrinkage and generation of apoptotic bodies. The chromosomal DNA of cells infected with cp-BVDV undergoes fragmentation, generating the typical oligonucleosomal fragments commonly noted during apoptosis. The fragmented DNA was released from the nucleus to the cytoplasm, and eventually to the culture supernatant. Infection with cp-BVDV activates cellular proteases of the ICE family leading to cleavage of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme implicated in genome maintenance. This demonstration that cp-BVDV kills cells by triggering apoptosis suggests the possibility that cp-BVDV is associated with a fatal disease by the acquisition of a new apoptosis-inducing activity. We consider BVDV to be an excellent model system for studies of the biological and medical relevance of apoptosis in viral infections.

Hohenwarter O, Waltenberger A, Strutzenberger K, Katinger H (1997) Human endothelial cell lines established by mutated forms of the simian virus 40 large T oncogene. J Biotechnol 54 :131-137


The large T oncoprotein of Simian Virus 40 is widely used to improve the growth characteristics of primary cells in culture. Beside growth stimulation and immortalization, expression of the large T protein in human cells frequently leads to a loss of differentiated characters and changes in the karyotype. We have constructed mutated forms of the large T protein by deletion of various fragments of the DNA binding domain to test, whether this region is responsible for undesired influences on cell differentiation. After transfection into human umbilical vein endothelial cells, the resulting cell lines showed no improvement in expression of the differentiation marker von Willebrand factor compared to cell lines transfected with the wild type oncogene. Changes in the karyotype were still observed. Our results contribute to the mapping of functional domains of the large T protein. The truncated large T proteins retained growth stimulating activity after removal of 111 and 241 amino acids of the DNA binding region.

Huppertz HI, Heesemann J (1997) Invasion and persistence of Salmonella in human fibroblasts positive or negative for endogenous HLA B27. Ann Rheum Dis 56 :671-676


OBJECTIVE : Analysis of the interaction of enteropathogenic bacteria with HLA B27 transfected murine fibroblasts showed a specific influence of HLA B27 on microbial invasiveness. This possible novel mechanism for the action of HLA B27 should be verified by using endogenous HLA B27 positive and negative human fibroblasts as a model for the direct interaction of arthritogenic bacteria and host cells. METHODS : Fibroblasts were obtained from healthy donors positive or negative for HLA B27 ; cultivated as monolayers ; and infected with Salmonella enterica serovar enteritidis. RESULTS : Invasion and survival of bacteria in human cells was not influenced by the presence of HLA B27. Enhancement of HLA class I molecule expression by treatment of cultures with interferon gamma decreased invasion and survival of bacteria in both HLA B27 positive and negative cells. After disappearance of live bacteria lipopolysaccharide antigens persisted within cells. CONCLUSION : Endogenous HLA B27 does not modulate the direct interaction of Salmonella with human cells. Non-professional phagocytes are able to limit bacterial survival in cells, and interferon gamma accelerates killing of bacteria, but arthritogenic antigens persist after disappearance of live bacteria.

Hyodo T, Kumano K, Haga M, Sakai T, Fukuda M, Isami Y, Okada T (1997) Analysis of urinary red blood cells of healthy individuals by an automated urinary flow cytometer. Nephron 75 :451-457


A newly developed automated urinary flow cytometer allows clear discrimination of erythrocytes from other solid components of urine. In this study, the normal range of the urinary erythrocyte count and the source of urinary erythrocytes in healthy individuals were investigated using this analyzer. For the diagnosis of the source of the urinary erythrocytes, Kitasato University Kidney Center criteria for this analyzer were applied. The subjects were 133 regularly employed volunteers (age range 20-48 years, mean 30.5) who noted no urinary tract symptoms and showed normal blood pressure, consisting of 41 females not in their menstrual period (age range 20-39 years, mean 24.8) and 92 males (age range 20-48 years, mean 33.1). Mid-stream voided urine was collected from these subjects using urine sampling cups, immediately transferred to 50-ml sterilized Spitz tubes, and analyzed within 30 min using the automated urinary flow cytometer. Urinary erythrocytes were derived from glomeruli in all samples of healthy subjects. The urinary erythrocyte count showed a logarithmic normal distribution. Values 2 SD of the urinary erythrocyte count in healthy individuals or higher were regarded as abnormal, and hematuria was considered to be positive when 11.0/microliter or more erythrocytes were observed by this analyzer. The finding by this analyzer corresponded to the report of Birch et al.

Iannelli D, D’Apice L, Cottone C, Viscardi M, Scala F, Zoina A, Del Sorbo G, Spigno P, Capparelli R (1997) Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry. J Virol Methods 69 :137-145


The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 microm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 microm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE ; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 microm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 microm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).

Ibrahim P, Whiteley AS, Barer MR (1997) SYTO16 labelling and flow cytometry of Mycobacterium avium. Lett Appl Microbiol 25 :437-441


Mycobacterium avium cells were harvested from agar at different stages of their growth cycle, exposed to the minimum inhibitory concentration of isoniazid (INH) for 24 h and labelled with the fluorescent nucleic acid stain SYTO16. INH exposure led to a > 10-fold increase in the intensity of labelling in the majority of cells, and revealed discrete fluorescence peaks that were consistent with development of filamentous multinucleate cells during the growth cycle. Similar enhancement of labelling was observed in unfixed INH-treated cells viewed by fluorescence microscopy. INH appears to increase the permeability of Myco. avium cells to SYTO16. A combination of growth cycle-defined inocula, labelling with the new generation of fluorescent dyes and flow cytometry provides new opportunities to study the interrelationships between growth cycle events and antimicrobial susceptibility of mycobacteria.

Imbert-Marcille BM, Robillard N, Poirier AS, Coste-Burel M, Cantarovich D, Milpied N, Billaudel S (1997) Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry. J Clin Microbiol 35 :2665-2669


Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.

Jansson JK, Prosser JI (1997) Quantification of the presence and activity of specific microorganisms in nature. Mol Biotechnol 7 :103-120


Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.

Joyce S (1997) Traffic control of completely assembled MHC class I molecules beyond the endoplasmic reticulum. J Mol Biol 266 :993-1001


It is generally assumed that MHC class I molecules arrive at the plasma membrane following biosynthesis, assembly and architectural editing in the endoplasmic reticulum by constitutive forward movement without requirement for specific signals (bulk flow). If this is true then all overexpressed completely assembled class I molecules should arrive at the cell surface. To study the itinerary of class I traffic beyond the endoplasmic reticulum, mammalian cells that overexpress 20 to 50-fold higher amounts of the constituent heavy and light chains were established. Thorough biochemical analyses revealed that such overexpressed molecules assemble with authentic peptides that contain the canonical class I binding anchor motif in almost 1:1 stoichiometry and impart thermal stability to the heterotrimeric complex. Despite complete assembly, however, only a fraction of the overexpressed molecules reaches the cell surface. Almost all of the overexpressed class I molecules are sialylated, thus traffic as far as the trans-Golgi or the trans-Golgi network. Overexpression of class I molecules do not seem to cause a "traffic jam" in the exocytic pathway because the kinetics of traffic of Sindbis virus structural proteins to the plasma membrane are almost identical when comparing the non-engineered and engineered cells. Thus the steady state expression of class I molecules at the cell surface is further controlled either in the Golgi apparatus or at the plasma membrane.

Kanmogne GD, Bailey M, Gibson WC (1997) Wide variation in DNA content among isolates of Trypanosoma brucei ssp. Acta Trop 63 :75-87


The DNA contents of 18 Trypanosoma brucei ssp. stocks were compared using flow cytometry, karyotype analysis and quantitation of repetitive DNA by Southern blotting and hybridisation. The DNA contents of Type 1 T. b. gambiense stocks were lower than those of non-gambiense stocks, but both groups showed a wide range of variation in DNA content. Amongst T. b. gambiense stocks. Mabia at the lower end of the range had 14% less DNA than Dal 972 at the top of the range. Similarly, amongst non-gambiense stocks. 117R at the lower end of the range had 14% less DNA than LM55 at the top of the range. The T. b. gambiense stock Mabia had 29% less DNA than non-gambiense stock LM55. The DNA content of Type II T. b. gambiense stocks had minichromosomes albeit fewer than non-gambiense stocks. This result was verified by hybridisation with probes for satellite DNA and a telomere-specific repeat. Hybridisation with the probe for the beta-tubulin genes also revealed an apparent reduction of gene copy number T. b. gambiense relative to non-gambiense stocks. In conclusion, there is a wide range of variation in genome size in T. brucei ssp., with T. b. gambiense stocks at the lower end of the range. The reduction in genome size correlates with loss of repeated genes and non-coding sequences in T. b. gambiense stocks, and is not continued to chromosomes of a particular size.

Karpf AR, Blake JM, Brown DT (1997) Characterization of the infection of Aedes albopictus cell clones by Sindbis virus. Virus Res 50 :1-13


We have investigated the infection of Aedes albopictus (mosquito) cell clones by Sindbis virus. Variation in the multiplicity of infection (MOI) from ranges of 50-0.00005 pfu/cell was determined to have no effect on the progression of the infection to high acute phase titer, suggesting that intracellular factors alone are responsible for the restriction of virus production seen as the infection enters the persistent phase : While persistently infected (over 1 year post infection) cell clones are morphologically indistinct from uninfected cells, they do display a uniform 30% reduction in growth rate compared with uninfected cells of the same clone. Using flow cytometry-based DNA content analysis, we found that persistent Sindbis virus infection induces distinct cytological effects on these cells, including an increase in apoptosis and polyploidy in one clone and cell cycle phase effects in another. Finally, the observation that the number of cells in persistently infected cell cultures which are productively infected closely approximates the number of cells dying by apoptosis prompted us to investigate the role that cell death may play in the maintenance of the persistent infection. Persistently infected cell cultures which were artificially induced into apoptosis by short 45 degrees C heat treatments do not display increased Sindbis virus production. This result does not support the hypothesis that infection sensitivity induced by random apoptosis in persistently infected cell cultures is responsible for the long-term maintenance of the persistent infection.

Katayama T, Akimitsu N, Mizushima T, Miki T, Sekimizu K (1997) Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product. Mol Microbiol 25 :661-670


The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli, is regulated by adenine nucleotide binding ; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 degrees C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn5 insertion mutagenesis. Three of the suppressors carried Tn5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over-replication of the chromosome was inhibited in the dnaAcos aroK ::Tn5 double mutant, and initiation of chromosome replication in the dnaA+ aroK ::Tn5 mutant was partially inhibited. The aroK(or B) ::Tn5 cells contained DnaA molecules at a level similar to that in the parental aroBK+ strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo. SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.

Knight DA, Waldman WJ, Sedmak DD (1997) Human cytomegalovirus does not induce human leukocyte antigen class II expression on arterial endothelial cells. Transplantation 63 :1366-1369


Human cytomegalovirus (CMV) has been associated with allograft rejection and, in particular, with transplant-associated arteriosclerosis. However, the role CMV plays in the development of transplant-associated arteriosclerosis remains unclear. CMV can infect the endothelium, the interface between allograft tissue and the host immune cells, but the direct induction of endothelial human leukocyte antigen (HLA) class II by CMV remains controversial. Our previous studies with venous endothelial cells (EC) have shown that CMV does not directly induce this antigen on infected EC and, furthermore, renders these cells refractory to interferon (IFN)-gamma induction. However, questions have arisen regarding the relevance of these findings to arterial endothelia. Thus, we have extended these studies to determine whether similar interactions occur in arterial EC. EC derived from human coronary artery, aorta, and umbilical artery were assayed by immunofluorescence flow cytometry and dual immunohistochemical staining following IFN-gamma treatment and/or inoculation with CMV. Data generated by these experiments demonstrate that regardless of vascular origin : (1) CMV does not directly induce endothelial surface or cytoplasmic HLA class II, and (2) although uninfected arterial EC are HLA class II inducible by IFN-gamma, infected cells are completely refractory to this effect. These results suggest that CMV-mediated inhibition of HLA class II expression is a phenomenon shared by human arterial and venous endothelia of both fetal and adult origin.

Kottom TJ, Nolan LK, Robinson M, Brown J, Gustad T, Horne SM, Giddings CW (1997) Further characterization of a complement-sensitive mutant of a virulent avian Escherichia coli isolate. Avian Dis 41 :817-823


An attempt was made to characterize the mechanism of complement resistance operating in a virulent avian Escherichia coli isolate. Using flow cytometry to detect antibody to C3, we found that there was significantly more antibody bound to a complement-sensitive mutant of this wild type than to the parent organism, suggesting that more C3 subunits were bound to the wild type. Neither the wild type nor the mutant degraded C3. Further, the mutant was phagocytosed to a significantly greater degree than the wild type by cultured phagocytes in the presence of C5-deficient serum. These data suggest that the wild type is resistant to complement, at least in part, because of its ability to restrict C3 deposition on its surface. Therefore, the decrease in virulence seen in the mutant may be related to its increased sensitivity to complement-mediated bacteriolysis or its enhanced susceptibility to complement-opsonized phagocytosis or both.

Kubota K (1997) A killer cell protective antigen expressed by MHC-unrestricted killer hybridomas. Cell Immunol 181 :50-58


We have established MHC-unrestricted killer hybridoma cell lines. A monoclonal antibody (mAb K2D) described in this study destroyed some of these killer hybridoma cell lines when added to their cultures. This destruction of cells required cell-to-cell contact, indicating that the killer hybridoma cells were killed by others of the same killer hybridoma(fratricide). Based on the facts that the killer hybridoma used did not express FcgammaRIII, and that F(ab’)2 fragments of mAb K2D also induced the cell destruction, antibody-dependent cellular cytotoxicity (ADCC) was excluded from a possible mechanism underlying this fratricidal killing. The molecular characteristics of the antigen recognized by mAb K2D and the results obtained by sequential immunoprecipitation led us to conclude that mAb K2D recognizes the Ly-49G2 molecule already described as a member of the Ly-49 family of NK cell inhibitory receptors. Thus, these results suggest that the killer hybridoma cells are protected by the inhibitory signals delivered by Ly-49G2 from killing each other. In addition, we found that mAb K2D recognizes an additional Ly-49-like antigen other than Ly-49G2 on the killer hybridoma, which may reflect the complexity of the Ly-49 family proteins.

Lange JL, Thorne PS, Lynch N (1997) Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria. Appl Environ Microbiol 63 :1557-1563


Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4’,6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment.

Liu L, Elwing H, Karlsson A, Nimeri G, Dahlgren C (1997) Surface-related triggering of the neutrophil respiratory burst. Characterization of the response induced by IgG adsorbed to hydrophilic and hydrophobic glass surfaces. Clin Exp Immunol 109 :204-210


Hydrophilic and hydrophobic glass surfaces precoated with human albumin, fibrinogen, or IgG were investigated with respect to their ability to activate the neutrophil NADPH-oxidase. We found that IgG-coated surfaces induced a substantial and prolonged neutrophil production of reactive oxygen species (ROS). When a hydrophilic surface was used to support protein binding, a somewhat lower neutrophil response (around 35%) was obtained, compared with the response induced by IgG on a hydrophobic surface. The production of ROS was completely eliminated when cytochalasin B was added to the measuring system, suggesting the involvement of the cell cytoskeleton in the activation process. The relation between the intra- and extracellular generation of ROS was further assessed, and we found that most of the ROS produced were released from the cells, in agreement with a model in which the activating surfaces induce a ’frustrated’ phagocytic response. Serum totally inhibited ’frustrated’ phagocytosis provided that the IgG molecules were sticking to a hydrophilic surface.

Lopez-Amoros R, Castel S, Comas-Riu J, Vives-Rego J (1997) Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC. Cytometry 29 :298-305


Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM). A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy. Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction.

Luhtala M, Tregaskes CA, Young JR, Vainio O (1997) Polymorphism of chicken CD8-alpha, but not CD8-beta. Immunogenetics 46 :396-401


We report here the structural basis of CD8 polymorphism in the chicken. Three chicken strains (RPRL Line 7, H.B15.H7, and H.B15. H12) have 14 nucleotide differences in the CD8A cDNA sequence causing eight amino acid replacements in the extracellular part of the molecule. Only two amino acid replacements and four silent mutations were observed in the CD8B cDNA sequence in one (H7) of the strains. Substitutions in CD8alpha were solely responsible for the binding of CD8-specific monoclonal antibodies, as detected by cDNA expression in COS cells. The majority of the amino acid substitutions are located in the immunoglobulin V-like domain and three of the changes (residues 30, 34, and 58) are situated in the putative major histocompatibility complex class I binding CDR1 and CDR2 regions of the chicken CD8alpha. CD8A polymorphism has not been reported in other species and this suggests that CD8A and CD8B have evolved under different selective pressures in the chicken.

MacNeill SA, Fantes PA (1997) Genetic and physiological analysis of DNA replication in fission yeast. Methods Enzymol 283 :440-459


Studies on DNA replication in S. pombe have provided powerful insights into the way in which the genome of this model eukaryote is replicated and how the replication process is controlled. These studies have been facilitated by the simplicity and range of methods available in this organism for physiological and genetic analysis of DNA replication mutants. In the future, continued focus on the analysis of such mutants, coupled with increasingly sophisticated biochemical investigation of the processes of DNA replication in both wild-type and mutant cells, will ensure continued rapid progress in this area.

Mason DJ, Lloyd D (1997) Acridine orange as an indicator of bacterial susceptibility to gentamicin. FEMS Microbiol Lett 153 :199-204


We have studied the response of Escherichia coli NCTC10418 to gentamicin with flow cytometry. The susceptibility of individual bacterial cells to the antibiotic was assessed by differential staining using the metachromatic dye, acridine orange. Exponential phase cultures were exposed to the minimum bactericidal concentration of gentamicin and analysed at regular intervals over 90 min. Within 60 min of exposure to the drug, two sub-populations of organisms could be distinguished in cultures by their different acridine orange-associated fluorescence emissions of < 550 nm and > 550 nm. The number of bacteria exhibiting acridine orange-associated fluorescence at > 550 nm corresponded to counts of colony forming units.

Melin L, Jensen-Waern M, Johannisson A, Ederoth M, Katouli M, Wallgren P (1997) Development of selected faecal microfloras and of phagocytic and killing capacity of neutrophils in young pigs. Vet Microbiol 54 :287-300


Nine healthy piglets, weaned at the age of 35 days and emanating from two litters, were studied from 7 to 63 days of age. The development of their faecal flora was analysed by bacteriological quantification of coliforms, E. coli, enterococci and Clostridium perfringens. The numbers of coliforms, E. coli and enterococci decreased from about 10(8) CFU/g faeces on day 7 to about 10(5) at the end of the study. Clostridium perfringens, with an initial value of 10(4) CFU/g faeces, was not found in any faecal sample from day 21 onwards. At each sampling occasion the similarity between the floras of different pigs were investigated by biochemical fingerprinting and calculated as correlation coefficients between metabolic fingerprints. This was performed for the coliform and the enterococcal floras. Initially, the coliform floras had a low homogeneity (rmean = 0.6), indicating large initial differences between the piglets. From day 14 post-partum until weaning the homogeneity was stable at a high level (rmean = 0.9). On day 3 post-weaning a marked decrease of the homogeneity was seen (rmean = 0.5), which later returned to the level before weaning. The enterococcal floras had a high homogeneity (rmean = 0.8-0.9) through the study and was only slightly affected by weaning. The function and development of the phagocytic and killing capacity of neutrophil granulocytes was monitored by flow cytometry and chemiluminescence. No changes in these functions were seen over time or between litters.

Moayeri M, Welch RA (1997) Prelytic and lytic conformations of erythrocyte-associated Escherichia coli hemolysin. Infect Immun 65 :2233-2239


Flow cytometry was developed as a method to assess the conformation of erythrocyte-bound Escherichia coli hemolysin polypeptide (HlyA). Topology of membrane-associated hemolysin (HlyA(E)) was investigated by testing surface accessibility of HlyA regions in lytic and nonlytic bound states, using a panel of 12 anti-HlyA monoclonal antibodies (MAbs). Hemolysin associates nonlytically with erythrocytes at 0 to 2 degrees C. To test the hypothesis that the nonlytic HlyA(E) conformation at 0 to 2 degrees C differs from the lytic conformation at 23 degrees C, MAb epitope reactivity profiles at the two temperatures were compared by flow cytometry. Four MAbs have distinctly increased reactivity at 0 to 2 degrees C compared to 23 degrees C. HlyA requires HlyC-dependent acylation at lysine residues 563 and 689 for lytic function. Toxin with cysteine substitution mutations at each lysine (HlyA(K563C) and HlyA(K689C)) as well as the nonacylated form of hemolysin made in a HlyC-deficient strain were examined by flow cytometry at 0 to 2 and 23 degrees C. The three mutants bind erythrocytes at wild-type toxin levels, but there are conformational changes reflected by altered MAb epitope accessibility for six of the MAbs. To test further the surface accessibility of regions in the vicinity of MAb-reactive epitopes, HlyA(E) was proteolytically treated prior to testing for MAb reactivity. Differences in protease susceptibility at 0 to 2 degrees and 23 degrees C for the reactivities of three of the MAbs further support the model of two distinct conformations of cell-associated toxin.

Nelson PJ, Gelman IH (1997) Cell-cycle regulated expression and serine phosphorylation of the myristylated protein kinase C substrate, SSeCKS : correlation with culture confluency, cell cycle phase and serum response. Mol Cell Biochem 175 :233-241


We recently identified a novel myristylated protein kinase C (PKC) substrate, named SSeCKS (pronounced essex), whose transcription is suppressed > 15 fold in src- or ras-transformed rodent fibroblasts, but not in raf-transformed cells [1, 2]. SSeCKS associates with and controls the elaboration of a cortical cytoskeletal matrix in response to phorbol esters [2], and overexpression of SSeCKS causes growth arrest of untransformed NIH3T3 cells [3]. Our preliminary data suggested that SSeCKS functions as a negative mitogenic regulator by controlling cytoskeletal architecture and that serine phosphorylation of SSeCKS by kinases such as PKC alters its interaction with cytoskeletal matrices and its ability to control mitogenesis. Here, we determine the effects of culture confluency, growth arrest and serum response on the steady-state abundance of SSeCKS RNA and protein and on the relative level of phosphoserine-free SSeCKS. SSeCKS transcription is initially induced by serum factors and by contact-inhibited growth rather than by cell-cycle arrest induced by serum starvation, hydroxyurea or nocodazole, and following serum-induced G1/S progression, SSeCKS transcription is suppressed. SSeCKS protein is hyperphosphorylated on serine residues during G1/S progression but not during the G2/M phase. Finally, we show that the induction of SSeCKS protein expression by contact inhibition is independent of SSeCKS’ serum responsiveness. These data suggest that SSeCKS expression and function can be controlled at either the transcriptional or post-translational level in response to serum factors and culture confluency. The data strengthen the notion that SSeCKS plays an important, yet transient, role in cell cycle progression from G0 to G1 that differs from its role in controlling contact-inhibited growth.

Ogawa T, Ogo H, Kinoshita A (1997) Antagonistic effect of synthetic peptides corresponding to the binding regions within fimbrial subunit protein from Porphyromonas gingivalis to human gingival fibroblasts. Vaccine 15 :230-236


Specific binding region within fimbrial subunit protein (fimbrilin) from Porphyromonas gingivalis strain 381 was studied in cultured human gingival fibroblasts. Fluorescent micrographs visualised FITC-labelled fimbriae of P. gingivalis specifically bound to normal human fibroblast cell line (Gin-1) along the cell surface. Flow cytometric analysis also revealed the binding of FITC-labelled fimbriae to Gin-1 cells. Synthetic peptides composed of residues 1-20 (AFGVGDDESKVAKLTVMVYN) of the fimbrilin from P. gingivalis, FP381 (1-20), FP381 (69-80 ; ALTTELTAENQE) and FP381 (171-181 ; DA-NYLTGSLTT) definitely inhibited P. gingivalis fimbria-binding to Gin-1 cells by enzyme-linked immunosorbent assay (ELISA). Furthermore, based on the Scatchard plot analysis of the binding of 125I-labelled P. gingivalis fimbriae to Gin-1 cells, the apparent dissociation constant (Kd) was calculated as 15.9 pM, and the number of binding sites (Rt) was estimated as 150 sites/cell. Binding studies of 125I-labelled FP381(171-181) also revealed the presence of a non-interacting, single class of affinity binding sites : the apparent Kd and Rt were 29.2 nM and 18440 sites/cell on Gin-1 cells, respectively. These results demonstrate that specific binding regions on P. gingivalis fimbriae to human gingival fibroblasts are present, and certain corresponding peptides clearly inhibited the binding of P. gingivalis fimbriae to human gingival fibroblasts.

Oleksiewicz MB, Alexandersen S (1997) S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus. J Virol 71 :1386-1396


We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.

Pavic I, Hartmann A, Zimmermann A, Michel D, Hampl W, Schleyer I, Mertens T (1997) Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet. Antimicrob Agents Chemother 41 :2686-2692


We established a quantitative flow cytometric method for determination of herpes simplex virus type 1 (HSV-1) susceptibility to acyclovir (ACV), ganciclovir, and foscarnet in vitro. Susceptibility was defined in terms of the drug concentration which reduced the number of cells expressing HSV-1 glycoprotein C (gpC) with a fluorescence intensity of > or =10(2) by 50% (IC50). Flow cytometry allowed us to use a high (1.0) as well as a low (0.005) multiplicity of infection, and determination of the IC50 was possible after one or more viral replicative cycles. IC50s were dependent on virus input and on time postinfection. In mixture experiments, 1 to 2% resistant viruses added to a sensitive strain could be detected. The results obtained by flow cytometry showed a good qualitative correlation with those achieved by cytopathic effect inhibitory assay. However, flow cytometry might detect more quantitative differences in drug susceptibility, especially among resistant strains, as confirmed also by determination of intracellular drug phosphorylation. The mean IC50s for ACV-sensitive strains were 0.45 to 1.47 microM, and those for ACV-resistant strains were between 140 and 3,134 microM. Flow cytometric analysis was fast and accurate, automatizable, and highly reproducible. Flow cytometry may be a more powerful tool than standard cytopathic effect-based assays and could have advantages for the detection of low levels of drug resistance or mixtures of sensitive and resistant virus strains.

Perin F, Laurence D, Savary I, Bernard S, Le Pape A (1997) Radioactive technetium-99m labelling of Salmonella abortusovis for the assessment of bacterial dissemination in sheep by in vivo imaging. Vet Microbiol 57 :171-180


We report the development and validation of a 99mTc-labelling technique of bacteria, applied to Salmonella abortusovis. The radioactive labelling is obtained using a pre-tinning step of the cells followed by direct incubation of S. abortusovis suspension with 99mTc-pertechnetate. Several procedures with different amounts of stannous tin (SnF2 or SnCl2) were evaluated. The selected method, respectful of bacterial viability, provided a 30% labelling yield. Viability of 99mTc-labelled bacteria was assessed by flow cytometry using rhodamine 123 and was demonstrated to be unchanged, turbidimetric measurements showing only a slight increase in the growth rate for radiolabelled cells. Incubation of 99mTc-labelled S. abortusovis with pronase, saponine and urea demonstrated labelling stability and suggested an intra-cellular localization for 99mTc. A preliminary study was also conducted in sheep to evaluate the value of the imaging of radiolabelled S. abortusovis. Spatial and temporal patterns of their in vivo dissemination in the lymphatic system after a sub-cutaneous injection were compared with control lymphoscintigraphic agents. These imaging data supported the assumption that the radioactivity detected in vivo was proportional to the number of 99mTc-labelled bacteria.

Quessy S, Busque P, Higgins R, Jacques M, Dubreuil JD (1997) Description of an albumin binding activity for Streptococcus suis serotype 2. FEMS Microbiol Lett 147 :245-250


This study was undertaken to investigate the binding activity of Streptococcus suis serotype 2 to albumin. Using flow cytometry we observed a binding activity of S. suis to albumin for virulent as well as for avirulent isolates. Western immunoblots analysis revealed that a 39-kDa S. suis protein was responsible, at least in part, for this binding activity. This protein showed high N-terminal homology (95.6% for the first 23 residues) with a group A Streptococcus glyceraldehyde-3-phosphate dehydrogenase. Furthermore, the addition of albumin to the culture broth resulted in an increase in the virulence of S. suis strains in mice. These results suggest that an interaction with albumin could play a role in the pathogenesis of S. suis serotype 2 infections.

Rall GF, Manchester M, Daniels LR, Callahan EM, Belman AR, Oldstone MB (1997) A transgenic mouse model for measles virus infection of the brain. Proc Natl Acad Sci U S A 94 :4659-4663


In addition to the rash, fever, and upper respiratory tract congestion that are the hallmarks of acute measles virus (MV) infection, invasion of the central nervous system (CNS) can occur, establishing a persistent infection primarily in neurons. The recent identification of the human membrane glycoprotein, CD46, as the MV receptor allowed for the establishment of transgenic mice in which the CD46 gene was transcriptionally regulated by a neuron-specific promoter. Expression of the measles receptor rendered primary CD46-positive neurons permissive to infection with MV-Edmonston. Notably, viral transmission within these cultures occurred in the absence of extracellular virus, presumably via neuronal processes. No infection was seen in nontransgenic mice inoculated intracerebrally with MV-Edmonston. In contrast, scattered neurons were infected following inoculation of transgenic adults, and an impressive widespread neuronal infection was established in transgenic neonates. The neonatal infection resulted in severe CNS disease by 3-4 weeks after infection. Illness was characterized initially by awkward gait and a lack of mobility, and in later stages seizures leading to death. These results show that expression of the MV receptor on specific murine cells (neurons) in vivo is absolutely essential to confer both susceptibility to infection and neurologic disease by this human virus. The disparity in clinical findings between neonatal and adult transgenic mice indicates that differences exist between the developing and mature CNS with respect to MV infection and pathogenesis.

Rumeu MT, Suarez MA, Morales S, Rotger R (1997) Enterotoxin and cytotoxin production by Salmonella enteritidis strains isolated from gastroenteritis outbreaks. J Appl Microbiol 82 :19-31


Seventy-six Salmonella enteritidis, three Salmonella virchow and one Salmonella bradenrup strains were screened for enterotoxigenicity by using the Chinese hamster ovary (CHO), Y1 adrenal, Vero and HeLa cell tests. All the strains gave positive reactions for enterotoxin production, except one, and the relative sensitivity to the toxin exhibited by the different cell lines was evaluated. An enterotoxic activity has been identified in sonicated extracts of Salm. enteritidis. This enterotoxin was purified on Agarose A-5m (Bio-Rad) and Superose 12 HR 10/30 column. The enterotoxic activity was eluted from the Superose column in the first peak. Like Vibrio cholerae toxin CT and Escherichia coli enterotoxin LT, it was blocked by GM1 ganglioside, but at a higher concentration. In addition, a cytotoxic factor has been partially identified. The procedure for isolating the cytotoxin included ammonium sulphate precipitation, size-exclusion chromatography and anion exchange chromatography. This cytotoxin factor caused inhibition of protein synthesis in cultured cells, as determined by flow cytometry and [3H]-leucine incorporation. Flow cytometry analysis also showed an activation of CHO cells when exposed to this cytotoxic factor resulting in a state of active growth. Cytotoxic activity was not blocked by gangliosides.

Satoh E, Osawa M, Tomiyasu K, Hirai H, Shimazaki C, Oda Y, Nakagawa M, Kondo M, Kinoshita S, Mazda O, Imanishi J (1997) Efficient gene transduction by Epstein-Barr-virus-based vectors coupled with cationic liposome and HVJ-liposome. Biochem Biophys Res Commun 238 :795-799


We show here a novel non-viral strategy to transduce human cells by using an EBV-based vector system. The EBV-based vectors, the plasmid vectors carrying EBV oriP (origin for plasmid replication) and EBNA (EBV nuclear antigen) 1 gene from EBV genome, were combined with 2 gene delivery systems, i.e., cationic liposome and HVJ-liposome. By both methods, EBV-based vectors could be more efficiently transfected into HeLa cells than non-EBV, conventional plasmid vectors. When human primary fibroblasts were transfected, EBV-based vectors coupled with cationic liposome but HVJ-liposome resulted in successful gene transduction, while human bone marrow cells were transduced with both HVJ-liposome- and cationic liposome-EBV vectors. These results suggest the potential applications of the EBV-based vector system for gene therapy.

Sgorbati S, Barbesti S, Neri MG, Davegna C, Citterio S (1997) Rapid flow cytometric immunodetection of bacteria to monitor aquatic environments. Eur J Histochem 41 Suppl 2 :187-188


Shahgasempour S, Woodroffe SB, Sullivan-Tailyour G, Garnett HM (1997) Alteration in the expression of endothelial cell integrin receptors alpha 5 beta 1 and alpha 2 beta 1 and alpha 6 beta 1 after in vitro infection with a clinical isolate of human cytomegalovirus. Arch Virol 142 :125-138


Human cytomegalovirus infection of human umbilical vein endothelial cells reduces the ability of these cells to bind to fibronectin, collagen type IV and laminin. This suppression requires active virus, since UV-inactivated virus did not alter the binding ability of these cells to adhere to fibronectin, collagen type IV, and laminin. In an attempt to elucidate the molecular mechanism of this altered interaction, the surface expression of alpha 5 beta 1, alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins on cytomegalovirus-infected endothelial cells was examined using attachment inhibition assay and flow cytometric analysis. The results presented here show that infection with human cytomegalovirus selectively alters the expression of integrin on human endothelial cells, with the ability to induce downregulation of alpha 5 beta 1 and alpha 2 beta 1 (p = 0.001) and p = 0.03, respectively), while significantly upregulating alpha 6 beta 1 (p = 0.03), and marginally upregulating alpha 3 beta 1 (p = 0.05).

Shanahan T (1997) Application of flow cytometry in transplantation medicine. Immunol Invest 26 :91-101


Immunological rejection remains a major barrier to successful organ transplantation. Consequently, immunosuppressive intervention to prevent or control the rejection process renders the transplant recipient susceptible to infectious diseases. Flow cytometry has become a useful tool for monitoring immunological responses in transplant recipients. There are three areas of clinical transplantation immunology that may benefit from this technology. First, characterizing and classifying alloreactive antibodies by flow cytometry identifies high-risk donor and recipient combinations with greater precision. Second, the ability to detect subtle changes in the cellular components of the immune system cytometrically may facilitate the differential diagnosis of rejection, infection, and iatrogenic toxicity. Finally, the ease with which flow cytometry determines the adequacy or inadequacy of immunosuppressive therapy through T cell receptor analyses serves to maximize the beneficial effects of engraftment.

Slepushkin VA, Simoes S, Dazin P, Newman MS, Guo LS, Pedroso de Lima MC, Duzgunes N (1997) Sterically stabilized pH-sensitive liposomes. Intracellular delivery of aqueous contents and prolonged circulation in vivo. J Biol Chem 272 :2382-2388


Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.

Sorensen BB, Jakobsen M (1997) The combined effects of temperature, pH and NaCl on growth of Debaryomyces hansenii analyzed by flow cytometry and predictive microbiology. Int J Food Microbiol 34 :209-220


Flow cytometry was applied to determine growth of Debaryomyces hansenii in a laboratory medium. Viable yeasts were enumerated after staining with the fluorogenic ester fluorescein diacetate (FDA). Initial studies showed that the flow cytometric determinations correlated well with viable yeast populations determined as colony forming units (CFU) whereas the relationship between CFU and optical density was only linear over a narrow range of cell concentrations, 10(5.5)-10(7.5) cells/ml. The flow cytometric measurements could reliably detect D. hansenii at concentrations as low as 10(2) cells/ml whereas the lower detection limit using optical density measurements was 10(5)-10(6) cells/ml. Growth was determined by flow cytometry at different combinations of temperatures (10-30 degrees C), pH (4.7-6.0) and NaCl concentrations (1-12% w/v). Growth curves were generated by fitting a modified Gompertz equation to the growth data using non-linear regression analysis. Lag phase duration and maximum specific growth rates were derived and quadratic polynomial models were developed describing the effects of environmental conditions on the growth parameters. Model validation based upon repetition of experiments and use of another laboratory medium showed good agreement between observed and predicted maximum specific growth rates whereas predicted lag phases were shorter than the observed lag phases.

Srivastava IK, Rottenberg H, Vaidya AB (1997) Atovaquone, a broad spectrum antiparasitic drug, collapses mitochondrial membrane potential in a malarial parasite. J Biol Chem 272 :3961-3966


At present, approaches to studying mitochondrial functions in malarial parasites are quite limited because of the technical difficulties in isolating functional mitochondria in sufficient quantity and purity. We have developed a flow cytometric assay as an alternate means to study mitochondrial functions in intact erythrocytes infected with Plasmodium yoelii, a rodent malaria parasite. By using a very low concentration (2 nM) of a lipophilic cationic fluorescent probe, 3,3’dihexyloxacarbocyanine iodide, we were able to measure mitochondrial membrane potential(DeltaPsim) in live intact parasitized erythrocytes through flow cytometry. The accumulation of the probe into parasite mitochondria was dependent on the presence of a membrane potential since inclusion of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, dissipated the membrane potential and abolished the probe accumulation. We tested the effect of standard mitochondrial inhibitors such as myxothiazole, antimycin, cyanide and rotenone. All of them except rotenone collapsed the DeltaPsim and inhibited respiration. The assay was validated by comparing the EC50 of these compounds for inhibiting DeltaPsim and respiration. This assay was used to investigate the effect of various antimalarial drugs such as chloroquine, tetracycline and a broad spectrum antiparasitic drug atovaquone. We observed that only atovaquone collapsed DeltaPsim and inhibited parasite respiration within minutes after drug treatment. Furthermore, atovaquone had no effect on mammalian DeltaPsim. This suggests that atovaquone, shown to inhibit mitochondrial electron transport, also depolarizes malarial mitochondria with consequent cellular damage and death.

Taguchi H, Yamaguchi H, Osaki TY, Yamamoto T, Ogata S, Kamiya S (1997) Flow cytometric analysis for adhesion of Vibrio cholerae to human intestinal epithelial cell. Eur J Epidemiol 13 :719-724


The adhesion of Vibrio cholerae O1 strains to human intestinal epithelial cell, Intestine 407, was analyzed by flow cytometer. According to positive percentages of Intestine 407 cells adhered by V. cholerae, two groups of V. cholerae strains were classified as follows : more adhesive (more than 50%), less adhesive (less than 50%) strains. In addition, the fluorescence intensity after attachment of V. cholerae was directly correlated to the number of the microorganisms. It was concluded that flow cytometry is a useful and objective method for analyzing adhesion of V. cholerae to cultured cells.

Thomas JC, Desrosiers M, St-Pierre Y, Lirette P, Bisaillon JG, Beaudet R, Villemur R (1997) Quantitative flow cytometric detection of specific microorganisms in soil samples using rRNA targeted fluorescent probes and ethidium bromide. Cytometry 27 :224-232


Specific detection and accurate enumeration of microorganisms in the environment have been hampered by the lack of suitable techniques. A three-parameter flow cytometric method (FCM) was developed to detect quantitatively Sphingomonas sp. strain 107 inoculated into soil samples. By combining light scattering profiles (i.e., morphological properties), ethidium bromide (EtBr) influx (i.e., wall permeability), and fluorescence in situ hybridization against the 16S rRNA (i.e., detection specificity), we could accurately discriminate the bacterium of interest from the indigenous microflora and soil debris. EtBr was used, first, to determine the optimal cell wall permeabilization treatment to allow oligonucleotide probes to enter the bacterial cells and, second, to achieve clear discrimination of fixed cells from debris in soil samples. This method allowed effective qualitative and quantitative analysis by fluorescence in situ hybridization. The results showed that the detection threshold by FCM was 3 x 10(4) cells/g of dry soil. Cell counts deduced from FCM analysis were similar to those obtained by the colony forming unit assay when soils contained fewer than 3 x 106 cells/g dry soil. This method should be useful for either quantitative monitoring of microorganisms inoculated in contaminated soil samples during bioremediation or detecting known bacterial strains in environmental samples.

Tortorello ML, Stewart DS, Raybourne RB (1997) Quantitative analysis and isolation of Escherichia coli O157:H7 in a food matrix using flow cytometry and cell sorting. FEMS Immunol Med Microbiol 19 :267-274


Flow cytometry is a potentially valuable analytical method in microbiology providing the ability to analyze rapidly large numbers of individual microorganisms by several parameters. With a flow cytometer with enhanced light scatter sensitivity and a conventionally configured sorting cytometer, a series of comparative studies to determine the ability of the two flow systems and the antibody-direct epifluorescent filter technique (Ab-DEFT) to detect and enumerate Escherichia coli O157:H7 were made. Initial experiments used culture-derived mixtures of non-pathogenic E. coli and serial dilutions of E. coli O157:H7. Subsequent studies involved analysis of enrichment cultures from ground beef inoculated with E. coli O157:H7. Comparison of flow cytometry with microscopy and plate counts produced similar results at higher concentrations in both culture mixtures and beef enrichments. At the lowest concentrations Ab-DEFT was more sensitive, however, the time required for analysis was much less with flow cytometry. With a cytometer with enhanced light scatter sensitivity designed for bacterial analysis, O157:H7 could be distinguished from E. coli strain HB101 on the basis of light scatter. This instrument also provided direct count data for selected populations. In experiments using cell sorting to isolate target organisms, the purity of fluorescent-labeled E. coli O157:H7 sorted from beef enrichment cultures and plated was not affected by the level of background organisms, as is often the case in conventional plating procedures.

Ueckert JE, Nebe von-Caron G, Bos AP, ter Steeg PF (1997) Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury. Lett Appl Microbiol 25 :295-299


Flow cytometry in combination with fluorescent molecular markers 5- (and 6-) carboxyfluorescein succinimidylester (CFSE) and propidium iodide (PI) have been applied to determine lag times, numbers of cell divisions and injury after mild heat (50 degrees C, 5 min) and nisin treatments (0.1 and 1.0 microgram ml-1) of Lactobacillus plantarum. Initial labelling with covalently bound dye CFSE (20 and 100 micrograms ml-1) allowed determination of lag times and cell proliferation for up to eight generations. Double-labelling with CFSE and PI (5 micrograms ml-1) provided additional information about damage levels and distributions within populations. Subpopulations surviving treatment could be identified easily and selectively sorted.

van der Zee H, Huis in’t Veld JH (1997) Rapid and alternative screening methods for microbiological analysis. J AOAC Int 80 :934-940


Automated analytical instruments for enumerating indicator organisms and diagnostic test kits for pathogens can be used in food microbiology to screen samples and to replace conventional cultural and confirmation steps. Such methods are now available for rapid detection or estimation of groups of (indicator) organisms, pathogenic micro-organisms, bacterial toxins and mycotoxins, and molds. These alternative methods can be classified by the principles on which they are based : modified conventional methods, instrumental measurement of bacterial metabolism, bioluminescence, immunological techniques, DNA techniques, and combinations of these techniques. To meet user expectations, test kits must be accurate, sensitive, specific, rapid (24 h or less), easy to use, and labor-saving. They must also offer the possibility of computerization, a low detection limit, and low investment and running costs. The paper compares the ability of alternative methods to meet these criteria. Variations were found, depending on the techniques used and the target organism of the analysis. Economic reasons can determine whether alternative methods can be used routinely. Adoption of these screening systems also can be hampered by lack of internationally coordinated and accepted validation protocols.

Vesey G, Deere D, Gauci MR, Griffiths KR, Williams KL, Veal DA (1997) Evaluation of fluorochromes and excitation sources for immunofluorescence in water samples. Cytometry 29 :147-154


Fluorescent labelling methods for detecting microorganisms in water have limited sensitivity partly due to the natural autofluorescence from environmental particles. The aim of this study was to examine the autofluorescence of water samples to determine the optimal excitation source and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporidium oocysts. Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluorescent properties. Two major peaks were identified emitting at 390 to 510 nm and at 640 to 700 nm. Flow cytometry was used to define the optical properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cytometry and the number of particles with fluorescence and light scatter properties similar to the fluorescently labelled oocysts recorded. Fluorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having optical properties similar to labelled oocysts, detected in 10 litre concentrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrhodamine B thioisocyanate (TRITC) excited at 542 nm were the most suitable labels for oocysts in drinking water with less than 40 particles having optical properties similar to labelled oocysts, detected in 100 litre concentrates.

Vesey G, Griffiths KR, Gauci MR, Deere D, Williams KL, Veal DA (1997) Simple and rapid measurement of Cryptosporidium excystation using flow cytometry. Int J Parasitol 27 :1353-1359


In vitro excystation is commonly used to determine the viability of samples of purified Cryptosporidium parvum oocysts. Following exposure to conditions that stimulate excystation, samples are examined microscopically to determine the number of excysted oocysts. The microscopy procedure is tedious and time consuming, and difficult to apply to most oocyst samples without a purification step. A simple flow cytometric method was developed for determining the numbers of oocysts that had excysted following the in vitro excystation procedure. Differences in light-scatter properties were used to differentiate intact, partially empty and empty oocysts. By staining samples with a monoclonal antibody specific to the oocyst wall it was possible to apply the technique to unpurified oocysts from faeces. Correlation of the flow cytometric and microscopic method was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. The flow cytometry method is faster and more sensitive than the microscopy procedure, and enables analysis of large numbers of samples and of many thousands of oocysts in each sample.

Walberg M, Gaustad P, Steen HB (1997) Rapid discrimination of bacterial species with different ampicillin susceptibility levels by means of flow cytometry. Cytometry 29 :267-272


An in vitro model for flow cytometric detection of heterogeneous drug response in exponentially growing Escherichia coli and Klebsiella pneumoniae was studied to evaluate the potential of this technology for rapid antibiotic susceptibility testing in polymicrobial samples. The cells, which exhibited 80-fold difference in in ampicillin susceptibility, were cultivated in the presence of ampicillin at a concentration equaling 1 MIC of the low-susceptibility strain (E. coli). Prior to flow cytometric analysis, the cells were fixed in 70% ethanol and stained with a DNA-specific dye. After 1 h of ampicillin incubation, the light scattering and fluorescence intensities of the susceptible cells increased 4.3-fold and 5-fold, respectively, but remained about constant for the resistant cells. The corresponding cell number increase for the resistant and the susceptible cells was 9.4 and 1.7, respectively. The two strains could be distinguished in the histograms on the basis of their light scattering and fluorescence intensities and by cell number. With an incubation of up to 3 h, the light scattering and fluorescence intensities of the susceptible cells grew stronger, at the expense of cell number. By combination of histograms, the discrimination of different cell populations could be improved. The results demonstrate the ability of flow cytometry to discriminate between species in heterogeneous cultures and suggest the potential of the technique for rapid assessment of bacterial susceptibility. However, the present results are preliminary, and the application to biological liquids and clinical samples has to be demonstrated further.

Wallner G, Fuchs B, Spring S, Beisker W, Amann R (1997) Flow sorting of microorganisms for molecular analysis. Appl Environ Microbiol 63 :4223-4231


Not only classical cultivation-based methods but also the new molecular approaches may result in incomplete and selective information on the natural diversity of microbial communities. Flow sorting of microorganisms from environmental samples allows the deliberate selection of cell populations of interest from highly diverse systems for molecular analysis. Several cellular parameters that can be measured by flow cytometry are useful as sort criteria. Here, we report sorting of bacteria from activated sludge, lake water, and lake sediment according to differences in light scattering, DNA content, and/or affiliation to certain phylogenetic groups as assessed by fluorescein-labeled, rRNA-targeted oligonucleotide probes. Microscopy of the sorted cells showed that populations of originally low abundance could be strongly enriched by flow sorting (up to 280-fold), depending on the original abundance of the cells of interest and the type of sample sorted. The purity of the cells of interest could be further increased by repeated sorting, but this increase was limited by cell aggregation in the case of activated-sludge samples. It was possible to amplify almost full-length 16S ribosomal DNA (rDNA) fragments from sorted microbial cells by PCR, even after fixation with paraformaldehyde and in situ hybridization. Dot blot hybridization and sequencing demonstrated that most of the amplified rDNA originated from those cells that had been selected for by flow sorting. Comparative analysis of 16S rDNA sequences revealed previously unknown species of magnetotactic or activated-sludge bacteria.

Xue W, Zhang S, Minocha HC (1997) Characterization of a putative receptor protein for bovine viral diarrhea virus. Vet Microbiol 57 :105-118


In a previous communication, we reported a 50-kDa cell surface protein from Madin-Darby bovine kidney (MDBK) cells as a putative receptor for bovine viral diarrhea virus (BVDV). The present study delineates further characterization of the receptor protein. Protease treatment of cultured MDBK cells adversely affected the receptor, thus abolishing the binding of anti-D89 (BVDV anti-idiotypes) to the cells. However, pretreatment of the cells with either phospholipases or glycosidases did not significantly change the anti-D89 binding to the cells. Additionally, pretreatment of cell monolayers with proteases decreased BVDV attachment and replication in the cells. These results suggested that the receptor for BVDV is a protein in nature, and glycosylation and phosphorylation may not play a direct role in BVDV attachment to cells. The BVDV receptor gradually regenerated on the cell surface after the protease-treated cells were cultured in normal growth medium. Regeneration of the BVDV receptor to a normal level took about 4 h as indicated by flow cytometric analysis and this process was inhibited in the presence of cycloheximide, a protein synthesis inhibitor. The 50-kDa receptor protein purified by electro-elution inhibited BVDV infection in a plaque reduction assay. It also inhibited anti-D89 binding to cells as analyzed by flow cytometry. These data demonstrated the nature of the 50-kDa protein as a specific receptor for BVDV.

Yamaguchi H, Osaki T, Taguchi H, Hanawa T, Yamamoto T, Fukuda M, Kawakami H, Hirano H, Kamiya S (1997) Growth inhibition of Helicobacter pylori by monoclonal antibody to heat-shock protein 60. Microbiol Immunol 41 :909-916


The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth. The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.

Yule TD, Roth MB, Dreier K, Johnson AF, Palmer-Densmore M, Simmons K, Fanton R (1997) Canine parvovirus vaccine elicits protection from the inflammatory and clinical consequences of the disease. Vaccine 15 :720-729


Inflammatory changes following infection are central to the clinical manifestation of disease. However, information regarding such changes in animal disease is limited. In canine parvovirus infected puppies we measured the levels of acute phase proteins and changes in leukocyte phenotypes and cell trafficking by flow cytometry. These parameters correlated with conventional assessment of clinical disease in a vaccine efficacy study. Seropositive (CPV-2) 6-week-old puppies given three doses of a CPV-2 containing vaccine developed significant antibody titers and remained healthy after experimental infection with CPV-2b. Unvaccinated controls developed clinical signs and shed virus. Importantly, acute phase proteins became elevated, and lymphopenia, neutropenia and modulation of neutrophil-CD4 were detected in controls but not in vaccinates.

Zielinska M, Fenrych W (1997) The application of a flow cytometric assay for evaluation of phagocytosis of neutrophils. Acta Biochim Pol 44 :121-125


Phagocytosis and the release of oxidative products generated by the respiratory burst have been studied in vitro under the influence of non-steroidal anti-inflammatory drugs : naproxen and ibuprofen, using phagocytes of peripheral blood from healthy human donors. Phagocytosis was monitored by flow cytometry in order to investigate the uptake of propidium iodide-labelled bacteria (Staphylococcus aureus) by polymorphonuclear leucocytes. In addition, the phagocytic capacity and percentage of killed bacteria was measured in isolated neutrophils using the Pantazis & Kniker method. It was found that naproxen and ibuprofen affect the phagocytic function and hydrogen peroxide production in the examined granulocytes. These methods might be useful in investigations on neutrophil functions.