vendredi 24 avril 2009
par   G. Grégori

Alvarado-Aleman FJ, Gonzalez-Bonilla C, Wong-Arambula C, Gutierrez-Cogco L, Sepulveda-Amor J, Kumate-Rodriguez J (1994) [Identification of Vibrio cholerae O1 by flow cytometry]. Rev Latinoam Microbiol 36 :283-293


A total of 72 peptonated water samples suspected of carrying Vibrio cholerae were assessed by laser flow cytometry (LFC) and compared with positive culture. We used a direct fluorescence technique using polyclonal (PolAb) and monoclonal antibodies (MoAb) conjugated to fluorescein. The PolAb were able to detect 33 positive samples. A clear difference among the 20 positive samples was found with only three V. cholerae O1 false negatives when MoAb were used whereas all 13 V. cholerae Non O1 samples were detected. The correlation index comparing control autofluorescence with peptonated water samples show a R = 0.69, versus 0.96 with pure V. cholerae O1 strains. Our data suggest that the LFC technique is able to recognize V. cholerae O1 from a mixture of microorganisms with high sensitivity and specificity in a few hours.

Asaoka H, Matsuda H (1994) Detection of N-glycolylneuraminic acid-containing glycoproteins from various animal erythrocytes by chicken monoclonal antibody against Hanganutziu-Deicher antigens. J Vet Med Sci 56 :375-377


Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, detecting Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were reactive with bovine, sheep and horse erythrocytes examined by flowcytometry (FCM). The FCM profiles of the three animal erythrocytes treated with trypsin were different from those of non-treated cells and from each other depending on MAbs used. To identify the nature of antigenic molecules, recognized by HU/Ch2-7 and HU/Ch6-1 MAbs, solubilized erythrocytes from the above three animals were analyzed by a Western blotting method. The MAb HU/Ch2-7 identified HD antigen-specific glycoprotein in each of animal erythrocytes. These results indicate that the MAb HU/Ch2-7 is a valuable reagent for the detection of NeuGc-containing gangliosides and glycoproteins.

Challier S, Brown S, Ombrouck C, Desportes-Livage I, De Nay D, Gentilini M (1994) Flow cytometry as a possible method of isolation of spores of the microsporidian Enterocytozoon bieneusi. J Eukaryot Microbiol 41 :27S


Cid VJ, Sanchez M, Nombela C (1994) Characterization of thermosensitive autolytic mutants from diploid Saccharomyces cerevisiae. Microbiology 140 ( Pt 3) :559-568


In order to carry out a systematic search for mutants affected in cell integrity, the diploid strain Saccharomyces cerevisiae D1 was subjected to mutagenesis with ethyl methane sulphonate (EMS), and mutant clones were screened for thermosensitive autolytic phenotypes. The screening was based on examination of cell populations, from individual mutant clones, stained with propidium iodide to establish the proportion of cells lysing under non-permissive conditions by means of flow cytometry. Osmotic remediation of the autolytic phenotype in the presence of 1 M sorbitol was also checked. Out of 13,300 clones surviving mutagenesis, 34 were confirmed to be thermosensitive autolytic and 7 of them showed some osmotic complementation with regard to growth and cell lysis. The osmotic remediation in the other strains was negligible or affected only one of the two parameters. The expression of the mutant phenotype in the strains isolated led to a sporulation defect (40% of the strains) and significant alterations in morphology, such as cells in chains (35%), altered buds (25%) that eventually might elongate, round unbudded and highly vacuolated cells (12%) and large-sized cells (12%). These observations show that alterations in functions related to cell integrity can be correlated with an altered morphology. Genetic analysis of the mutant strains that could sporulate showed that in many instances the mutant phenotype was the result of more than one mutation, the mutations being individually recessive. However, at least one mutant strain, 933, carried a single mendelian mutation that was dominant in the diploid but haploid segregants were non-viable. Dominance of this mutation was also confirmed in tetraploids obtained by means of protoplast fusion.

Cui Y, English D, Neel S, Harvey K, Siddiqui R, Akard L, Jansen J, Hughes CV (1994) Parallel up-regulation of CD11B and CD45 on neutrophilic leukocytes exposed to soluble factors of oral pathogens. Biochem Mol Biol Int 33 :45-54


We tested culture supernatants from a battery of oral bacterial strains for their ability to influence the expression of CD11b and CD45 on the neutrophil plasma membrane. Several bacterial extracts stimulated the up-regulation of both CD11b and CD45 simultaneously. Two supernatants in particular (a clinical isolate of A. actinomycetemcomitans and F. nucleatum ATCC25586) potently stimulated the deployment of CD11b and CD45 from their intracellular storage site to the plasma membrane. Both supernatants inhibited superoxide release stimulated by exposure of neutrophils to formyl methionyl leucyl phenylalanine (FMLP) but had variable effects on superoxide release stimulated by phorbol myristate acetate (PMA). The ability of products of oral bacteria to modulate neutrophil plasma membrane antigen composition may regulate functional reactivity and thus be an important factor in the pathogenesis of periodontal infection and inflammation.

Cullen JM, Linzey DW, Gebhard DH (1994) Nuclear ploidy of normal and neoplastic hepatocytes from woodchuck hepatitis virus-infected and uninfected woodchucks. Hepatology 19 :1072-1078


Flow cytometric analysis of the ploidy of normal and neoplastic hepatocyte nuclei obtained from adult woodchucks, a model of human hepadnavirus-induced hepatocellular carcinoma, was performed. All 36 samples of nuclei from non-neoplastic liver from woodchuck hepatitis virus-infected or uninfected liver were diploid, indicating that age-related nuclear polyploidization does not occur in this species, unlike other rodents. Individual or multiple hepatic neoplasms were obtained from each of 14 woodchuck hepatitis virus-infected woodchucks. Nineteen samples of hepatocellular carcinoma and eight adenomas were examined. Aneuploid nuclei were detected in 10 of the hepatocellular carcinomas and three of the adenoma samples. Similar DNA indexes, ranging from 1.11 to 1.22, were found in 7 of the 10 aneuploid HCCs and all 3 aneuploid adenomas. Nine of the 19 hepatocellular carcinoma samples and 5 of the 8 adenomas were diploid. Four of the diploid hepatocellular carcinomas had increased proportions of tetraploid nuclei. The presence of aneuploid nuclei was not related to histological appearance of the neoplasms or serum gamma-glutamyltranspeptidase levels. Because none of the hepatocellular carcinomas metastasized, the presence of aneuploidy could not be related to biological behavior. We determined the proportion of uninucleate and binucleate hepatocytes in hepatocellular carcinoma and nonneoplastic liver. Approximately 7% of hepatocytes were binucleate in nonneoplastic liver from woodchuck hepatitis virus-infected and uninfected liver. Only 2% of malignant hepatocytes were binucleate. The results of this study indicate that aneuploidy is a common change in hepatic neoplasms from woodchuck hepatitis virus-infected woodchucks.

De Mot R, Schoofs G, Roelandt A, Declerck P, Proost P, Van Damme J, Vanderleyden J (1994) Molecular characterization of the major outer-membrane protein OprF from plant root-colonizing Pseudomonas fluorescens. Microbiology 140 ( Pt 6) :1377-1387


N-terminal sequence analysis of peptides generated by proteolytic treatment of the Pseudomonas fluorescens OE 28.3 major outer-membrane protein OprF, embedded in outer membranes or present in whole cells, indicated a surface-exposed location for the proline-rich region of the protein. This region is absent from the P. aeruginosa and P. syringae OprFs. Evidence was obtained for the presence of additional exposed but less accessible regions in the carboxy half of OprF. Four OprF-specific monoclonal antibodies were all directed to the C-terminal part of the protein but did not recognize a surface-exposed epitope as shown by flow cytometry. Our data support the model previously proposed for P. aeruginosa OprF in which the entire protein is embedded in the outer membrane, unlike the topology proposed for the major outer-membrane protein from Escherichia coli, OmpA, whose carboxy half resides in the periplasmic space. For six other P. fluorescens strains producing OprF proteins with different isoelectric points, the primary structure was determined by sequence analysis of the PCR-amplified oprF genes. The proline-rich domain represented the most conserved region of the different P. fluorescens OprFs. Based on the sequence of its oprF gene, it was shown that the mushroom pathogen P. tolaasii is quite closely related to P. fluorescens. Comparative sequence analysis further showed that the carboxy half of OprF contains a sequence motif that is well conserved in the enterobacterial OmpA proteins but is also present in a number of other outer-membrane proteins, including peptidoglycan-associated lipoproteins.

Diaper JP, Edwards C (1994) Survival of Staphylococcus aureus in lakewater monitored by flow cytometry. Microbiology 140 ( Pt 1) :35-42


The ability of flow cytometry to detect and enumerate viable bacteria during survival in a lakewater microcosm was assessed using Staphylococcus aureus as a model organism. Counts of colony-forming units (c.f.u.) on nutrient agar were not significantly different from those obtained by flow cytometric detection of rhodamine 123 stained bacteria and there was no evidence for a viable but nonculturable state using these methods. However c.f.u. were significantly lower when estimated using mannitol salts agar compared with nutrient agar. S. aureus was also enumerated immunofluorescently after staining with FITC-IgG. There was no significant difference between the population estimated immunofluorescently and by acridine orange direct counting, and unlike estimations of viability, only slight reductions in total cell numbers were observed. Changes in the protein and nucleic acid content of S. aureus during survival were also measured by flow cytometry to investigate any potential heterogeneity arising within the starved population. Flow cytometric determinations were found to correlate significantly with their respective chemical determinations. These results demonstrate the ability of flow cytometry to detect viable bacteria during starvation and to study changes in macromolecular content. They also illustrate the importance of using appropriate methods for the detection of viable bacteria in environmental samples.

Estes DM, Bailey CW, Barnett L, Lafrenz D, Brandt HM, Jensen JB, Allen GK, Carson CA (1994) Fluorescence-activated cell sorting-derived clones of Babesia bigemina show karyotype polymorphism. Parasitol Res 80 :104-107


Use of the fluorescence-activated cell sorter proved to be an accurate and highly efficient means for cloning Babesia parasites. These qualities were examined by separating a mixed population of Babesia-infected bovine erythrocytes composed of two isolates with different karyotypes. Direct evidence of polymorphism was detected during comparison of the resultant clones.

Garside L, Bailey M, Gibson W (1994) DNA content and molecular karyotype of trypanosomes of the subgenus Nannomonas. Acta Trop 57 :21-28


The relative DNA contents of representative stocks of 5 groups within the trypanosome subgenus Nannomonas (Trypanosoma simiae, Godfreyi, T. congolense Savannah, Forest and Kilifi) were measured by flow cytometry. The range of DNA contents formed a continuum. Nevertheless small differences were observed between the groups, with T. simiae/T. congolense Savannah and T. congolense Kilifi/Forest at the lower and higher ends of the range respectively. Analysis of karyotype by pulsed field gel electrophoresis showed all the 5 Nannomonas groups to have minichromosomes and variable numbers of small chromosomes in the 100-700 kb size range. The size and relative number of mini-chromosomes varied from group to group, but no correlation between molecular karyotype and DNA content was observed.

Gilman-Sachs A (1994) Flow cytometry. Anal Chem 66 :700A-707A


Gordon DL, Papazaharoudakis H, Sadlon TA, Arellano A, Okada N (1994) Upregulation of human neutrophil CD59, a regulator of the membrane attack complex of complement, following cell activation. Immunol Cell Biol 72 :222-229


CD59 is a membrane glycoprotein that regulates the membrane attack complex of complement and protects cells from autologous complement damage. Human polymorphonuclear leucocyte (PMN) expression of CD59 was confirmed by flow cytometry following staining with mAb 1F5, and western blotting revealed staining of a 19-23 kDa band. Warming of PMN from 4 to 37 degrees C resulted in spontaneous CD59 upregulation. A dose-dependent increase in expression following PMN stimulation with FMLP was observed and occurred within minutes, indicating that new protein synthesis was not required. Treatment of PMN with calcium ionophore A23187 resulted in similar increases in CD59 expression. This occurred in the presence or absence of extracellular calcium, indicating that upregulation was dependent on release of calcium from intracellular stores. Evidence for a mobilizable intracellular pool of CD59 was obtained by detection of increased binding of 1F5 following PMN permeabilization ; CD59 could also be re-expressed after stripping by phosphatidylinositol specific phospholipase C (PI-PLC) by treatment with FMLP or A23187. There was a correlation between CD59 upregulation and lactoferrin release, suggesting that stores of CD59 may be associated with secondary granules. These studies indicate that PMN expression of CD59 is enhanced by cell activation and suggest the presence of an intracellular pool of CD59 which can be translocated to the cell membrane upon PMN stimulation.

Hager H, Aboagye-Mathiesen G, Petersen PM, Norskov-Lauritsen N, Juhl CB, Villadsen JA, Zdravkovic M, Gildberg A, Dalsgaard AM, Ebbesen P (1994) Human trophoblast interferons enhance major histocompatibility complex class I antigen expression on human term trophoblast cells in culture. Placenta 15 :709-714


The expression and regulation of major histocompatibility complex class I (MHC class I) antigens by virus-induced human trophoblast interferons (tro-IFNs) were examined in term trophoblast cultures. Flow cytometry studies using fluorescence monoclonal antibodies against MHC class I antigens revealed that isolated cytotrophoblasts can express MHC class I antigens. The expression of these antigens increased with stimulation of trophoblast cultures with tro-IFN-alpha and -beta. One hundred IU tro-IFN-alpha and -beta/ml induced no significant higher levels of MHC class I antigens as compared with the control, whereas 1000 IU tro-IFN-alpha and -beta/ml did. The tro-IFN-enhanced expression of MHC class I antigens may be important as it increases the efficiency of local and viral antigen presentation, cytotoxicity by T cell response and local inflammatory processes, thereby preventing virus spread from mother to fetus.

Herles S, Olsen S, Afflitto J, Gaffar A (1994) Chemostat flow cell system : an in vitro model for the evaluation of antiplaque agents. J Dent Res 73 :1748-1755


We developed an experimental in vitro model of dental plaque to assess the potential efficacy of antiplaque agents. The model used a chemostat, which provided a continuous source of 5 species of oral bacteria grown in an artificial "saliva-like" medium. This mixture was pumped through six flow cells, each containing two types of surfaces on which plaque formed and was subsequently measured. Formation of bacterial plaque on hydroxyapatite surfaces was assessed by measurement of the DNA and protein content of the plaque film. The amount of bacterial plaque formed on germanium surfaces was measured by attenuated total reflectance (ATR/FT-IR) spectroscopy. Plaque viability was also assessed by a fluorescent staining technique. The quantity of plaque formed on both types of surfaces gradually increased with the duration of flow (from 24 to 72 h) through the cells during a 72-hour experimental period. The flow cells were then pulsed with experimental treatment solutions for 30 s, twice daily. Parallel to results of human clinical studies, the model was capable of discriminating among water, a placebo mouthrinse, and an active antimicrobial mouthrinse formulation containing 0.03% triclosan. It therefore offers a valuable alternative to animal model testing and allows for more rapid evaluations under well-controlled experimental conditions.

Hou X, Dietrich J, Kuhlmann J, Wegener AM, Geisler C (1994) Structure of the T cell receptor in a Ti alpha V beta 2, alpha V beta 8-positive T cell line. Eur J Immunol 24 :1228-1233


The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Ti alpha beta or Ti gamma delta) and the noncovalently associated CD3 chains (CD3 gamma delta epsilon zeta). The exact number of subunits constituting the TcR is still not known ; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a Ti alpha V beta 2, alpha V beta 8-positive T cell line which expressed the endogenous human TiV beta 8 and the transfected mouse TiV beta 2 both in association with the endogenous Ti alpha and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of Ti alpha V beta 8 by immunoprecipitation with anti V beta 8 monoclonal antibody (mAb) did not reduce the amount of Ti alpha V beta 2 in the lysate, and likewise, depleting the lysate of Ti alpha V beta with anti-V beta 2 mAb did not reduce the amount of Ti alpha V beta 8. Comodulation experiments showed that V beta 8 and V beta 2 did not comodulate with each other. Furthermore, functional tests demonstrated that TcR containing V beta 8 and TcR containing V beta 2 mediated transmembrane activation signals independently of each other. These data demonstrate that mouse V beta 2 and human V beta 8 were not expressed in the same TcR in agreement with a TcR model where each TcR contains only one Ti dimer.

Humphreys MJ, Allman R, Lloyd D (1994) Determination of the viability of Trichomonas vaginalis using flow cytometry. Cytometry 15 :343-348


In clinical laboratories, viability of Trichomonas vaginalis is determined by using light microscopy (differential count of motile to nonmotile organisms). Alternative methods are proposed that utilise flow cytometry. Under an epifluorescence microscope, live organisms fluorescence intensely green with fluorescein diacetate (FDA), whereas dead cells fluoresce orange with propidium iodide (PI). Flow cytometric histograms of green versus red fluorescence reveal distinct populations for live and dead cells. The anionic oxonal probe DiBAC4(3) is a membrane potential sensitive dye that distributes between the inside of the cell and the medium. Live organisms are less fluorescent than dead organisms when stained with the oxonol probe. Valinomycin, dicyclohexylcarbodiimide, and vanadate all give significant changes in the fluorescence intensities of cultures stained with the oxonol probe compared with control cultures, indicating that this probe is detecting changes in plasma membrane potential. Both FDA/PI and oxonol staining protocols allow good discrimination between populations and permit counts that are more statistically significant than those obtained by light microscopy. These methods remove the subjectiveness of microscopic counts and would increase the accuracy of susceptibility assays.

Jobe DA, Callister SM, Lim LC, Lovrich SD, Schell RF (1994) Ability of canine Lyme disease vaccine to protect hamsters against infection with several isolates of Borrelia burgdorferi. J Clin Microbiol 32 :618-622


We used flow cytometry to determine levels of borreliacidal antibodies in hamsters after vaccination with a commercially available canine Lyme disease vaccine. In addition, we evaluated the ability of vaccinated hamsters to resist infection with several isolates of Borrelia burgdorferi. Borreliacidal antibodies could be detected 1 week after a primary vaccination, peaked at weeks 3 to 5, and then rapidly declined. One week after a booster vaccination, borreliacidal activity was detected at a dilution of 1:10,240, and it decreased fourfold by week 10 after the booster vaccination. Vaccinated hamsters were protected against infection with < or = 10(6) B. burgdorferi 297 organisms during the peak borreliacidal response (5 weeks after primary vaccination or 2 weeks after booster vaccination). However, hamsters were not fully protected from development of Lyme arthritis when the titer of borreliacidal antibodies was < 1:5,120. In addition, no significant borreliacidal activity was induced against B. burgdorferi C-1-11, LV4, or BV1, which belong to three other seroprotective groups. These studies demonstrate that vaccination with the canine Lyme disease vaccine induces protective antibodies against B. burgdorferi 297. However, significant levels of borreliacidal antibodies are not produced until 5 weeks after vaccination, and protection is short-lived. In addition, no borreliacidal activity was induced against other isolates of B. burgdorferi. Because of this, the incorporation of multiple isolates or protein subunits may be necessary to increase the effectiveness of future vaccines.

Kamiya I, Okuda K, Hara K (1994) Flow-cytometric identification and detection of Porphyromonas gingivalis by a LPS specific monoclonal antibody. J Periodontol 65 :309-315


The purpose of this study was to identify Porphyromonas gingivalis (P. gingivalis) by flow cytometry (FCM) using a monoclonal antibody (MAb) OMR-Bg1E directed to P. gingivalis-specific lipopolysaccharide (LPS). The P. gingivalis strains ATCC 33277, 381, ESO75, W50, and A7A1 were selected for the study. Fusobacterium nucleatum (F. nucleatum), Prevotella intermedia (P. intermedia), Campylobacter rectus (C. rectus), Streptococcus sanguis (S. sanguis) and Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) served as controls. A suspension of 10(7) bacteria/ml of each bacteria was prepared and then reacted with a P. gingivalis specific MAb OMR-Bg1E and fluorescein isothiocyanate (FITC) labeled second antibody. These samples were analyzed by FCM. Bacterial specific binding aggregate on data was separated out by the forward- and side-angle-scatter characteristics, while non-specific binding (NSB) was eliminated by excluding the region with mouse IgG-positive and second antibody-positive area. FCM detected a mean range of 56.2% to 97.2% P. gingivalis strains. There was a 5.1% non-specific binding using FCM to non-P. gingivalis strains. When the P. gingivalis concentration was adjusted to 10(2), 10(4), and 10(6) bacteria/ml, a detection rate of 35.7%, 48.1%, and 91.4%, was respectively observed. The lower sensitivity of the flow cytometric assay was 10(2) bacteria/ml. When P. gingivalis was added to P. intermedia suspension at 1, 20, 40, 60, and 80%, the MAb-positive fraction yielded by FCM displayed a coefficient of determination of 0.967 with the actual percentage of P. gingivalis and could be regressed to a linear function.(ABSTRACT TRUNCATED AT 250 WORDS)

Kawasaki Y (1994) [Development of detection system of extraterrestrial microorganisms]. Biol Sci Space 8 :103-113


A noble method for the exploration of terrestrial and extraterrestrial soil microorganisms, especially targeted for Mars, has been developed. The method is based on the microscopic observation using fluorescence techniques. Microorganisms could be fluorescent by adsorption, enzymatic cleavage of extrinsic fluorescence chromophores such as acridine orange, ANS and SFDA, and also by intrinsic chromophores. The characteristic points of our fluorescence method are shown below. 1. The present method detected all the culturable cells tested (about 200 species from bacteria to eukaryofic cells). 2. Microorganisms in soil were much brighter than background fluorescence of soil. Cell shapes and location were clearly observed. 3. An esterase substatum SFDA, discriminated vital (reproductive) cells from dead. On the other hand, a membrane probe, ANS, detected both vital and dead cells. 3. Pre-treatment of cells with bleaching reagents improved the detection efficiency. Especially, this pretreatment was effecfive in Fungi with black chromophores. 4. Some anaerobic microorganisms such as methanogenic bacteria with intrinsic chromophores can be detected without stain. 5. Application of the technique to terrestrial soil revealed that more than 100 times larger cell density was obtained compared to the value obtained by the classic plate counting technique. Vertical distribution of microorganism of soil microorganisms from Mt. Shigayama showed that, at surface, cell density was small and maximum was shown below 15 cm from surface. 6. Some pre-biotic cell (cell like aggregates composed of amino acids) could be detected by SFDA or ANS. It can be concluded that the fluorescence technique is one of the most promising method for the exploration of extraterrestrial microorganisms.

Kepley C, Craig S, Schwartz L (1994) Purification of human basophils by density and size alone. J Immunol Methods 175 :1-9


Basophils typically account for approximately 1% of the white cells in peripheral blood. We have developed a unique method for purifying basophils from whole blood of normal subjects to at least 95% purity. Basophils are separated from other cell types by density-dependent sedimentation in Percoll and cell sorting, based solely on their size and granularity. The mean overall yield ranged from 5% to 28%. The procedure is typically completed within 4 h. The highly purified basophils obtained are functionally competent and morphologically intact. They release histamine in response to Fc epsilon RI-mediated stimulation, express Fc epsilon RI and BSP-1 ligand as analyzed by flow cytometry, and exhibit the known characteristic ultrastructural features of basophils by electron microscopy. This procedure avoids positive-selection antibodies that might perturb receptors on basophils or negative-selection antibodies that might activate other cell types, and can be used to obtain basophils for studies in vitro.

Kim YB, Zhang J, Davis WC, Lunney JK (1994) CD11/CD18 panel report for swine CD workshop. Vet Immunol Immunopathol 43 :289-291


Five monoclonal antibodies (mAbs), PNK-I (W #037), H20A (W #077), MUC76A (W #078), MUC93A (W #079) and MHM23 (W #136) of CD11/18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies by competitive binding of mAb by flow cytometric analysis based on PNK-I as CD18 epitope defining antibody resulted H20A and MHM23 mAbs bind to the same shared epitope as PNK-I, but MUC76A and MUC93A mAbs were distinct from PNK-I. Thus, PNK-I, H20A and MHM23 were designated as CD18a mAbs.

Laffin J, Lehman JM (1994) Detection of intracellular virus and viral products. Methods Cell Biol 41 :543-557


Lampisuo M, Lassila O (1994) Intra-embryonic haemopoietic cells and early MHC expression. Scand J Immunol 39 :321-326


In the avian embryo the haemopoietic stem cells originate from the intra-embryonic area near dorsal aorta. The surface-marker expression of haemopoietic stem cells and their potential to produce different haemopoietic cells are still largely unknown. The surface antigen expression and particularly the MHC antigen expression on intra-embryonic haemopoietic cells was studied. Expression of B-F antigens, homologous to mammalian MHC class-I antigens, was found already on embryonic day (ED) 5. The first B-L antigens, analogous to mammalian MHC class-II antigens, were detected also from ED5 onwards. The appearance of surface antigens defined by MoAbs T10A6 and 3-298 during embryogenesis also was studied. The antigen defined with T10A6 was detected from ED4 onwards on endothelial cells but not on haemopoietic cells in the para-aortic region. The first 3-298+ haemopoietic cells were found on ED6, whereas endothelial cells were negative. These findings imply that some surface markers are shared with haemopoietic and endothelial cells indicating either a common embryonic origin or the importance of these molecules in embryonic stem-cell homing.

Lebaron P, Joux F (1994) Flow cytometric analysis of the cellular DNA content of Salmonella typhimurium and Alteromonas haloplanktis during starvation and recovery in seawater. Appl Environ Microbiol 60 :4345-4350


Flow cytometry was used to investigate the heterogeneity of the DNA content of Salmonella typhimurium and Alteromonas haloplanktis cells that were starved and allowed to recover in seawater. Hoechst 33342 (bisbenzimide) was used as a DNA-specific dye to discriminate between DNA subpopulations. The DNA contents of both strains were heterogeneous during starvation. S. typhimurium cells contained one or two genomes, and A. haloplanktis cells contained up to six genomes. S. typhimurium genomes were fully replicated at the onset of starvation. Each replication cycle was completed in the early stage of starvation for A. haloplanktis by stopping cells in the partition step of the cell cycle prior to division. Multigenomic marine cells can undergo rapid cell division without DNA synthesis upon recovery, resulting in large fluctuations in the DNA contents of individual cells. In contrast, the heterogeneity of the DNA distribution of S. typhimurium cells was preserved during recovery. The fluctuations in the DNA fluorescence of this strain seem to be due to topological changes in DNA. Flow cytometry may provide a new approach to understanding dynamic and physiological changes in bacteria by detecting cellular heterogeneity in response to different growth conditions.

Lehman JM, Laffin J, Friedrich TD (1994) DNA content distribution of mouse cells following infection with polyoma virus. Cytometry 16 :138-143


Infection of primary to tertiary mouse embryo fibroblasts or mouse kidney cells with polyoma virus leads to stimulation of cellular DNA synthesis. When either confluent or growing mouse cells were infected, the monolayer cells were found to accumulate cells with a DNA content of S and G2/M phases of the cell cycle as assayed by flow cytometry. A similar pattern of DNA content was also observed in cells in the supernatant, which are probably cells replicating virus and dying. When compared with control cells, the infected monolayer and supernatant cells exhibited a population (5-27%) with a > G2 DNA content. The increase in DNA content of these > G2 cells was calculated to be an average of 26.7%, which is probably due to viral DNA. Polyoma contrasts with another papovavirus, SV40, which stimulates cells into DNA synthesis, with the majority of cells attaining a > G2/tetraploid DNA content, suggesting that there are differences in polyploidization between these two viruses.

Lester JP, 3rd, Evans DL, Leary JH, 3rd, Fowler SC, Jaso-Friedmann L (1994) Identification of a target cell antigen recognized by nonspecific cytotoxic cells using an anti-idiotype monoclonal antibody. Dev Comp Immunol 18 :219-229


Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian natural killer (NK) cells. In the present study an anti-idiotypic monoclonal antibody (mAb 7D12) was generated against idiotopes on an mAb (mAb 6D3.2) that recognizes a putative receptor on NCC. The idiotypic specificity of mAb 7D12 was determined in competition assays by incubating biotinylated mAb 7D12 with mAb 6D3.2 hybridoma cells following preincubation with combinations of biotinylated 7D12 with either nonbiotinylated homologous or heterologous mAb. The ligand recognized by mAb 7D12 (determined by flow cytometry) was found on cells from the anterior kidney, spleen, thymus, PBL, liver, and brain. NCC lysis of IM-9 targets was inhibited 76% following preincubation of the target cells with different concentrations of mAb 7D12. The involvement of the ligand recognized by mAb 7D12 in the NCC lytic cycle was determined by showing that this mAb produced 50% inhibition of NCC conjugate formation with NC-37 target cells. Biochemical analysis using SDS-PAGE and Western blotting revealed that mAb 7D12 recognized 54 and 65 M(r) proteins in IM-9 target cell lysates. These studies demonstrated that an idiotope on a NCC specific anti-receptor mAb was an "internal image" of a target cell ligand. The anti-id mAb generated against this image (idiotope) inhibited NCC cytotoxicity and thus was equivalent to an NCC receptor that binds to a target cell ligand involved in NCC recognition.

Lim LC, Liu YF, Schell K, Lovrich SD, Callister SM, Schell RF (1994) Detection of borreliacidal antibody by using acridine orange and flow cytometry. Clin Diagn Lab Immunol 1 :44-50


Borreliacidal antibody has been shown to be important for the serodiagnosis of Lyme disease and determination of immune status. Our results show that borreliacidal antibody can be rapidly and accurately detected by flow cytometry. Acridine orange was added to normal and immune sera containing Borrelia burgdorferi organisms in the presence and absence of complement prior to data acquisition by flow cytometry. The flow cytometric parameters of side scatter and detection of acridine orange fluorescence were used to determine events per minute (number of labeled spirochetes), percent shift in fluorescence (number of dead spirochetes), and mean channel fluorescence (intensity of fluorescence-labeled spirochetes) of acridine orange-labeled spirochetes. Borreliacidal antibody was detected as early as 4 h, with optimal detection 16 to 24 h after incubation of B. burgdorferi organisms with immune serum and complement. Our results also showed that complement was necessary for detection of borreliacidal antibody. Flow cytometry with acridine orange-labeled spirochetes provides a rapid means for detection of borreliacidal antibody.

Lindqvist C, Karp M, Akerman K, Oker-Blom C (1994) Flow cytometric analysis of bioluminescence emitted by recombinant baculovirus-infected insect cells. Cytometry 15 :207-212


Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), luc YE (578 nm), and luc OR (593 nm) were constructed. All genes were inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda insect cells during viral infection. The biological activity of the different luciferases was characterized by using intact recombinant baculovirus infected cells. Addition of the substrate, D-luciferin, immediately prior to the analysis allowed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. The emission peaks of the infected cells were clearly separated by wavelength scanning. Especially, the firefly luciferase (lucFF) had a broad peak and transient luminescence. The highest maximal intensity values in vivo were recorded for luc GR and luc YG. SDS-PAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity.

Liu YF, Lim LC, Schell K, Lovrich SD, Callister SM, Schell RF (1994) Differentiation of borreliacidal activity caused by immune serum or antimicrobial agents by flow cytometry. Clin Diagn Lab Immunol 1 :145-149


We demonstrated that borreliacidal activity caused by immune serum and complement can easily be differentiated by flow cytometry from killing activity caused by antimicrobial agents that are commonly used for the treatment of Lyme disease. Assay suspensions containing normal or immune serum were incubated with Borrelia burgdorferi in the presence or absence of ceftriaxone, doxycycline, penicillin, and phosphomycin for 2, 8, 16, and 24 h. Samples containing killing activity were identified by using flow cytometry and acridine orange. In 30 min, the effects of immune serum and complement were easily distinguished from the killing of spirochetes by antimicrobial agents by adding fluorescein isothiocyanate-conjugated goat anti-hamster immunoglobulin. This simple procedure greatly enhanced the usefulness of the borreliacidal assay by eliminating a major source of false-positive reactions.

Martin E, Maier F, Bhakdi S (1994) Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum. Antimicrob Agents Chemother 38 :1331-1338


This study addressed the effects of fluconazole and 5-fluorocytosine on the candidacidal activity of amphotericin B in the presence of human serum. A Candida albicans isolate that was susceptible to all three agents according to standard testing procedures was employed. Fungicidal activity was estimated by using a flow cytometric procedure that exploited the fact that yeast cells killed by amphotericin B diminish in size and take up propidium iodide. The following findings were made. (i) Fluconazole and 5-fluorocytosine each failed to inhibit pseudohyphal formation and cell aggregation even when applied at 10 and 50 micrograms/ml, respectively, for up to 10 h. Hence, these agents were not fungistatic when tested in the presence of serum. (ii) Simultaneous application of 5-fluorocytosine had neither enhancing nor inhibitory effects on the fungicidal activity of amphotericin B. However, yeasts that were preincubated for 20 h with 5-fluorocytosine became less susceptible to killing by amphotericin B. (iii) Fluconazole exerted a frank antagonistic effect on the fungicidal activity of amphotericin B. Thus, under our in vitro conditions, both fluconazole and 5-fluorocytosine can overtly antagonize the candidacidal action of amphotericin B.

Mason DJ, Allman R, Stark JM, Lloyd D (1994) Rapid estimation of bacterial antibiotic susceptibility with flow cytometry. J Microsc 176 :8-16


Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis-(1,3-dibutyl-barbituric acid) trimethine oxonol and observing changes in low-angle light scatter (excitation light scattered by up to 15 degrees). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2-5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady-state growth in liquid culture.

May JD, Branton SL, Pruett SB, Ainsworth AJ (1994) Differentiation of two strains of Mycoplasma gallisepticum with monoclonal antibodies and flow cytometry. Avian Dis 38 :542-547


Identification of infecting Mycoplasma spp. is difficult and not routine for strain. This paper describes a procedure for the rapid identification of the strain of M. gallisepticum. Monoclonal antibodies were prepared against M. gallisepticum F and M. gallisepticum S6. Aliquots of 24-hour broth cultures of these organisms were incubated briefly with either of the monoclonal antibodies. A second incubation was made with anti-mouse immunoglobulin conjugated to fluorescein isothiocyanate. Fluorescent intensity associated with the organisms was measured with a flow cytometer. The criterion for identification was a comparative increase in fluorescent intensity when the strain and monoclonal antibody were homologous. The procedure correctly differentiated the F and S6 strains of M. gallisepticum in a blind study.

McClelland RG, Pinder AC (1994) Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry. J Appl Bacteriol 77 :440-447


The use of fluorescently-labelled monoclonal antibodies, with detection by multi-parameter flow cytometry, was investigated for the rapid detection of salmonellas in pure cultures. Accurate detection of specific Salmonella serotypes was demonstrated down to levels of below 10(4) cells ml-1 (within 30 min) and 1 cell ml-1 (after 6 h non-selective pre-enrichment). This level of sensitivity was attained even in the presence of high levels of other bacterial species that would otherwise have interfered with the results. With combinations of different antibodies, each with a unique fluorescent label, simultaneous analysis for two species was possible.

McClelland RG, Pinder AC (1994) Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies. Appl Environ Microbiol 60 :4255-4262


Flow cytometry, combined with fluorescently labelled monoclonal antibodies, offers advantages of speed and sensitivity for the detection of specific pathogenic bacteria in foods. We investigated the detection of Salmonella typhimurium in eggs and milk. Using a sample clearing procedure, we determined that the detection limit was on the order of 10(3) cells per ml after a total analysis time of 40 min. After 6 h of nonselective enrichment, the detection limits were 10 cells per ml for milk and 1 cell per ml for eggs, even in the presence of a 10,000-fold excess of Escherichia coli cells.

Moeck GS, Bazzaz BS, Gras MF, Ravi TS, Ratcliffe MJ, Coulton JW (1994) Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol 176 :4250-4259


The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iron uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, T1, phi 80, and UC-1, for colicin M and microcin 25, and for albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 insertion mutations within the fhuA gene. Each insertion spliced a 13-amino-acid antigenic determinant (the C3 epitope of poliovirus) at a different position within FhuA. Immunoblotting of outer membranes with anti-FhuA and anti-C3 antibodies indicated that 15 of 16 FhuA.C3 proteins were present in the outer membrane in amounts similar to that observed for plasmid-encoded wild-type FhuA. One chimeric protein with the C3 epitope inserted after amino acid 440 of FhuA was present in the outer membrane in greatly reduced amounts. Strains overexpressing FhuA.C3 proteins were subjected to flow cytometric analysis using anti-FhuA monoclonal antibodies. Such analysis showed that (i) the chimeric proteins were properly localized and (ii) the wild-type FhuA protein structure had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferrichrome transport and remained sensitive to FhuA-specific phages. Three FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using anti-C3 antibodies. These three chimeric proteins were all biologically active. We conclude that amino acids 321, 405, and 417 are surface accessible in wild-type FhuA.

Monfort P, Baleux B (1994) Effects of environmental factors present in the St. Lawrence Estuary (Quebec, Canada) on experimental survival of Salmonella salamae as determined by flow cytometry. Can J Microbiol 40 :712-719


Survival of Salmonella salamae in the St. Lawrence Estuary was studied experimentally during an oceanographic cruise using in situ exposure diffusion chambers. The abundance distribution (colony-forming units) of culturable S. salamae on media was compared with the distribution of cells enumerated by flow cytometry. Flow cytometry was also used to characterize the size distribution and DNA content of cells exposed to various environmental factors. Solar radiation, starvation, and a gradual increase in salinity led to an abrupt loss of the ability of S. salamae cells to form cultures and to a gradual reduction in the cell size and DNA content. Conversely, starvation combined with a gradual increase in salinity in the absence of sunlight led to a gradual loss of the cells’ ability to form cultures and an abrupt reduction in cell size and DNA content (i.e., a rapid increase in cell damage). Mortality (i.e., a decrease in total cell count) of S. salamae placed in darkness began at a lower salinity (11.4/1000) than did the mortality of cells exposed to sunlight (23.1/1000). Therefore, the S. salamae cells exposed to sunlight seemed to be more resistant to gradual salinity stress than the cells that were not subjected to sunlight.

Morley PJ, Dash L, Jackson HC (1994) Evaluation of anti-Pneumocystis agents using flow cytometry. J Eukaryot Microbiol 41 :105S-106S


Paradis SE, Dubreuil D, Rioux S, Gottschalk M, Jacques M (1994) High-molecular-mass lipopolysaccharides are involved in Actinobacillus pleuropneumoniae adherence to porcine respiratory tract cells. Infect Immun 62 :3311-3319


Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The major adhesin of A. pleuropneumoniae has been identified as the lipopolysaccharides (LPSs) (M. Belanger, D. Dubreuil, J. Harel, C. Girard, and M. Jacques, Infect. Immun. 58:3523-3530, 1990). Using immunoelectron microscopy and flow cytometry, we showed in the present study that LPSs were well exposed at the surface of this encapsulated microorganism. Immunolocalization with porcine lung and tracheal frozen sections showed that extracted LPS bound to the lung mesenchyme and vascular endothelium and to the tracheal epithelium, respectively. Inhibition of adherence of A. pleuropneumoniae with extracted LPS was also performed with lung and tracheal frozen sections. Acid hydrolysis of LPS revealed that the active component of LPS was not lipid A but the polysaccharides. LPSs from A. pleuropneumoniae serotypes 1 and 2 were separated by chromatography on Sephacryl S-300 SF, in the presence of sodium deoxycholate, according to their molecular masses. The adherence-inhibitory activity was found in the high-molecular-mass fractions. These high-molecular-mass fractions contained 2-keto-3-deoxyoctulosonic acid and neutral sugars, and they were recognized by a monoclonal antibody directed against A. pleuropneumoniae O antigen but not recognized by a monoclonal antibody against capsular antigen.

Pay A, Pirck M, Bogre L, Hirt H, Heberle-Bors E (1994) Isolation and characterization of phosphoprotein phosphatase 1 from alfalfa. Mol Gen Genet 244 :176-182


Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known. In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa. Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa. The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved. Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation. In different plant organs, different pp1Ms transcript levels were observed ; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells. However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.

Pecoraro MR, Kawaguchi Y, Okita M, Inoshima Y, Tohya Y, Kai C, Mikami T (1994) Stable expression of the cDNA encoding the feline CD8 alpha gene. J Vet Med Sci 56 :1001-1003


The stable expression of the alpha chain of the feline cytotoxic T cell differentiation antigen (fCD8 alpha) on Crandell feline kidney cells (CRFK) was carried out by using an expression vector which contains the Rous sarcoma virus long terminal repeat and a neo resistant gene. After three rounds of cloning under G418 selection for over two months, the expression of the feline polypeptide was detected by human monoclonal antibody OKT8.

Pinder AC, McClelland RG (1994) Rapid assay for pathogenic Salmonella organisms by immunofluorescence flow cytometry. J Microsc 176 :17-22


Multi-parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full-fat milk). In all cases, the method was accurate to levels of below 10(4) target cells per ml for a total assay time of about 30 min. After 6 h non-selective enrichment in the presence of a 10,000-fold excess of competing micro-organisms (Escherichia coli) the corresponding detection limit was about 20 cells ml-1. These results suggest that flow cytometry has significant potential for the detection of pathogenic micro-organisms in the food industry.

Pore RS (1994) Antibiotic susceptibility testing by flow cytometry. J Antimicrob Chemother 34 :613-627


The first significant development in antibiotic susceptibility testing in recent years may be the flow cytofluorometric susceptibility test (FCST). The advanced analytical capability of the flow cytometer has provided the means to measure microbial diversity in culture. Membrane integrity and other indicators of microbial viability can be evaluated on a cell-by-cell basis. The FCST measures subtle dosage-response effects as well as the conventional minimum inhibitory and minimum bactericidal concentrations, simultaneously, in rapid tests which have the potential to supersede conventional techniques in terms of sensitivity and reproducibility.

Rainard P, Sarradin P, Poutrel B (1994) Phenotypic variability of X-protein expression by mastitis-causing Streptococcus agalactiae of serotype NT/X and opsonic activities of specific antibodies. Microb Pathog 16 :359-372


This study examined the role of antibodies against the X-protein, a surface-localized antigen frequently associated with streptococci causing mastitis in cattle, in the opsonization and phagocytosis of unencapsulated Streptococcus agalactiae. The analysis of various strains of serotype NT/X by flow cytometry, after labeling with a monoclonal antibody to X-protein, revealed that they consisted of a mixture of unstained and stained bacteria. Cloning of mother strains yielded clones of unstained bacteria but not homogeneous clones of stained bacteria. Analysis by ELISA of an unstained clone (4.1) derived from the reference NT/X strain 24/60 indicated that it expressed low amount of X-protein at its surface, about 25 times less than the stained clone 24/60 5.6. Colloidal gold immunolabeling showed the X-protein at the periphery of bacteria (of clone 5.6 and in lower amount of clone 4.1), at a distance from the cell wall. Bovine antibodies (essentially IgG) to X-protein behaved like the monoclonal antibody in the cytometric assay. They activated the classical pathway of complement as shown by the deposition of C1q and C4 on bacteria. Deposition of C4 also occurred on the low-surface-producing clone 4.1 in the presence of antibodies to X-protein, although less efficiently than on the high-surface-producing clone 5.6. When used alone, antibodies promoted the ingestion of bacteria and heat-inactivated immune serum promoted the chemiluminescence activity and the killing by polymorphonuclear cells. In conclusion, antibodies to X-protein induced the deposition of C3 by the classical pathway and were also able to stimulate opsonophagocytic killing of X-bearing S. agalactiae in the absence of deposited C3.

Raza MW, Blackwell CC, Ogilvie MM, Saadi AT, Stewart J, Elton RA, Weir DM (1994) Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells. FEMS Immunol Med Microbiol 10 :25-30


Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.

Register KB, Ackermann MR, Kehrli ME, Jr. (1994) Non-opsonic attachment of Bordetella bronchiseptica mediated by CD11/CD18 and cell surface carbohydrates. Microb Pathog 17 :375-385


Porcine atrophic rhinitis associated with Bordetella bronchiseptica is characterized by a severe inflammatory response in the mucosa of the nasal turbinates. Initial infiltrates of polymorphonuclear leukocytes (PMN) are followed by accumulations of mononuclear cells. In this report, we have investigated the interaction between porcine PMN and B. bronchiseptica. PMN incubated in PBS with a fluorescently labeled hemagglutinating porcine isolate, but not a non-hemagglutinating variant, had high levels of cell-associated fluorescence as determined by flow cytometry. Light microscopy indicated that most cell-associated bacteria were ingested. Transmission electron microscopy confirmed the presence of intracellular bacteria, which were contained within membrane-bound phagosomes. A monoclonal antibody specific for the leukocyte integrin polypeptide CD18 partially inhibited attachment of B. bronchiseptica to normal PMN but not to PMN genetically deficient in CD11/CD18 integrins. Higher levels of inhibition occurred when bacteria and normal PMN were co-incubated in the presence of galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, mannose and methyl alpha-D-mannopyranoside. D-glucose, L-fucose, alpha-lactose and sialic acid had no inhibitory effect. We conclude that B. bronchiseptica is readily ingested by porcine PMN in the absence of complement and antibody and that internalization is mediated by multiple adhesion mechanisms, including CD18- and carbohydrate-dependent ones.

Reilly BD, Skanes VM, Levine RP (1994) Evidence showing that the 1105 and 1106 isotypic residues of the fourth component of human complement, C4A, are not involved in amide bond formation. Mol Immunol 31 :761-769


Human C4A and C4B have different functions that may stem from their ability to bind hydroxyl or free amino groups on complement activating surfaces. Previous studies suggest that C4B binds to hydroxyl or amino groups whereas C4A binds to free amino groups on acceptor molecules. Comparison of the derived amino acid sequences of C4A and C4B has shown that differences exist between them at positions 1101, 1102, 1105 and 1106. These residues appear to be involved in the binding specificity of C4B. Less is known about the corresponding residues of C4A. It has been suggested that the aspartic acid of C4A at position 1106 is involved in amide bond formation by serving as a catalytic residue for the reaction or by promoting an increased interaction with amino nucleophilic groups. To examine the functional role of residues 1101-1106, we studied the effects of the C4A site-specific antipeptide mAb, AII-1 in assays dependent on the covalent binding properties of C4A ; the C4 mediated inhibition of hemolysis and the C4 mediated inhibition of immune precipitation. This study shows that mAb AII-1 has no effect on C4-mediated hemolysis or its ability to inhibit the rate of immune precipitate formation. The lack of interference by AII-1 in these assays could not be explained by low affinity interaction between antibody and C4A showing that mAb AII-1 does not affect the covalent binding activity of C4A. Furthermore, results from epitope mapping studies show that AII-1 binds to Leu1105 and Asp1106 suggesting that these residues are not critical for amide bond formation by C4A.

Rest RF (1994) Measurement of human neutrophil respiratory burst activity during phagocytosis of bacteria. Methods Enzymol 236 :119-136


Sahar E, Nir R, Lamed R (1994) Flow cytometric analysis of entire microbial colonies. Cytometry 15 :213-221


Much could be gained if the scope of flow cytometry could be broadened to the study of entire cell colonies, rather than to populations of single, separate cells. This can be achieved by encapsulating single microbial cells in small spheres in a way that allows each cell to multiply and form a colony within its respective microbead, which is then amenable to analysis by flow cytometry. Methods for performing the encapsulation within beads of appropriate size and homogeneity have been developed (Nir et al., Appl. Environ. Microbiol. 56:2870-2875, 1990). We describe here how a variety of properties of the entrapped colonies, such as mass, growth rate, enzymatic activity, and expression of specific antigens, can be measured, and we discuss how these constructs can be utilized to select microbial mutants.

Shi J, Goodband RD, Chengappa MM, Nelssen JL, Tokach MD, McVey DS, Blecha F (1994) Influence of interleukin-1 on neutrophil function and resistance to Streptococcus suis in neonatal pigs. J Leukoc Biol 56 :88-94


Nonspecific immunity is usually lower in neonates than adults. Consequently, enhancing the neonate’s nonspecific immune capability may be beneficial for the health and growth performance of young animals. We conducted two experiments in which neonatal pigs were injected with recombinant bovine interleukin-1 beta (rBoIL-1 beta) at 9 to 11 days of age. Three consecutive daily injections of rBoIL-1 beta increased neutrophil and monocyte numbers, which remained elevated until the animals were challenged with Streptococcus suis at 19 days of age. Neutrophil bactericidal activity was greater in interleukin-1-treated pigs than in saline-injected controls. At lower ratios of effector to target cells, neutrophil-mediated, antibody-dependent cellular cytotoxicity was increased in neonates treated with IL-1. However, natural killer cell activity and neutrophil production of superoxide anion were not affected by treatment with IL-1. Expression of CD18 was increased transiently on neutrophils from IL-1-treated pigs at 15 days of age. Severity of the streptococcal infection was less in pigs that were treated with IL-1 at 9 to 11 days of age. These data suggest that IL-1 treatment in neonates may augment nonspecific immune function and disease resistance.

Sommerfelt MA, Sorscher EJ (1994) Use of fluorescence-activated cell sorting for rapid isolation of insect cells harboring recombinant baculovirus. Methods Cell Biol 42 Pt B :563-574


Steinmassl M, Hamprecht K (1994) Double fluorescence analysis of human cytomegalovirus (HCMV) infected human fibroblast cultures by flow cytometry : increase of class I MHC expression on uninfected cells and decrease on infected cells. Arch Virol 135 :75-87


Cultured human foreskin fibroblasts (HFF) were infected with different multiplicities of infection (moi 0.001-0.1) of human cytomegalovirus (HCMV) strain AD 169 or a clinical isolate. Percentage of infected cells was determined by analysis of immediate early (IEA), early (EA), and late (LA) virus antigen expression with flow cytometry or by immunoperoxidase staining. Changes in the expression of class I MHC surface molecules were demonstrated by comparing the mean fluorescence intensities of infected HFF cultures with those of mock infected cell cultures by flow cytometry. At day three post infection single fluorescence analysis showed that infected HFF cultures split into low and high density class I MHC bearing cells. The addition of anti-interferon beta reduced the expression of class I MHC, distinctly. The assumption that infected cells down-regulate and uninfected cells up-regulate their expression of class I MHC molecules was demonstrated by double fluorescence analysis both with flow cytometry and fluorescence microscopy. Analysis of class I MHC-antigen expression versus immediate (IEA, mab E13), early (EA, mab 9221), or late (LA, mab BM219) virus antigen expression yielded three cell populations of HCMV infected HFF cultures three days post infection : 1. uninfected cells with an increase of class I MHC, 2. high density class I MHC, IEA and/or EA expressing cells, and 3. low class I MHC, IEA, EA and LA expressing cells.

Telford WG, King LE, Fraker PJ (1994) Rapid quantitation of apoptosis in pure and heterogeneous cell populations using flow cytometry. J Immunol Methods 172 :1-16


Truyen U, Agbandje M, Parrish CR (1994) Characterization of the feline host range and a specific epitope of feline panleukopenia virus. Virology 200 :494-503


The feline parvovirus subgroup is comprised of viruses isolated from various carnivores, including the dog, cat, mink, raccoon, Arctic fox, and raccoon dog. Those viruses are > 98% identical in their DNA sequences and are very similar antigenically. We have shown that although canine parvovirus (CPV) replicates in numerous feline cell lines in vitro it does not infect cats after parenteral inoculation (U. Truyen and C. R. Parrish, (1992) J. Virol. 66, 5399-5408). Here we use recombination mapping to locate some viral determinants required for feline host range, and show that the ability to replicate in cats was determined by the right-hand 45% of the genome, most likely a function of the capsid protein gene. Efficient replication in the cat appeared to require feline panleukopenia virus sequences from both ends of the VP2 molecule, which contained differences of VP2 amino acid residues 80, 564, and 568. The difference at amino acid 80 was also associated with expression of an FPV-specific antigenic epitope. The differences which affected the feline host range were located in a region of the capsid structure where three VP2 molecules interact, and the mutations gave rise to changes in the conformation of loops of the three adjoining VP2 monomers. The mechanism(s) of the in vivo feline host range restriction were not defined, and we were unable to show in vitro inhibition of virus infectivity by feline serum components or erythrocytes.

Van Amersfoort ES, Van Strijp JA (1994) Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes. Cytometry 17 :294-301


A number of reports have been published describing phagocytosis assays for flow cytometric analysis. In some of these, the fluorescence quenching technique has been used to discriminate between adherent and ingested particles. In this report, we have evaluated the efficacy of a quantitative fluorescence quenching technique with crystal violet and trypan blue for application in a phagocytosis assay with polymorphonuclear leukocytes and sensitized sheep red blood cells. We set the requirements to a high quenching efficiency of the fluorescence of extracellularly bound particles and no intracellular quenching. The latter was determined using polymorphonuclear leukocytes stained with the fluorescent nuclear dye hydroethidine. We observed that both trypan blue and crystal violet efficiently quench the fluorescence of PKH26 (a red fluorescent membrane-associated dye) erythrocytes but that only crystal violet quenches intracellular fluorescence. In testing trypan blue and crystal violet from different manufacturers, there was no real difference between different brands of crystal violet, but only the trypan blue from Merck turned out to be an efficient quencher, whereas the other brands of trypan blue showed low quenching efficiency. Trypan blue at a concentration of 25-50 micrograms/ml proved to be a good quencher of the fluorescent erythrocytes and exerted minimal side effects : over 90% quenching of the erythrocytes, no intracellular quenching, moderate increase in autofluorescence of the polymorphonuclear leukocytes, and no cell loss.

van der Waaij LA, Mesander G, Limburg PC, van der Waaij D (1994) Direct flow cytometry of anaerobic bacteria in human feces. Cytometry 16 :270-279


We describe a flow cytometry method for analysis of noncultured anaerobic bacteria present in human fecal suspensions. Nonbacterial fecal compounds, bacterial fragments, and large aggregates could be discriminated from bacteria by staining with propidium iodide (PI) and setting a discriminator on PI fluorescence and by exclusion of events with large forward scatter. Since anaerobic bacteria, which account for over 99.9% of all fecal bacteria, die during sample preparation, a fixation step was not necessary. A second aim of this study was to investigate the technical possibility of measurement of in vivo IgA coating of fecal anaerobic bacteria as well as their bacterial size. Fecal samples of 22 healthy human volunteers were analyzed. The fluorescence distribution of IgA-coated bacteria labeled with fluorescein isothiocyanate (FITC)-anti-Hu-IgA had overlap with noncoated bacteria. However, with match region subtraction, detection of low levels of specific FITC fluorescence on IgA-coated bacteria was achieved. The median bacterial two-dimensional surface area was 1.0 microns2. To validate flow cytometry data, all samples were analyzed with an image analysis system as well. With this new method, a rapid evaluation of fecal flora with high sensitivity for specific FITC fluorescence is possible without culturing.

Vanderplasschen A, Hanon E, Benarafa C, Greimers R, Op De Beeck A, Loncar M, Pastoret PP (1994) Madin Darby bovine kidney cell synchronization by lovastatin : application to bovine herpesvirus-1 gene expression. Vet Res 25 :555-567


The number of investigations involving cell proliferation has increased rapidly in the last years. One of the major difficulties in studying cell-cycle-related events is obtaining highly synchronous cell populations without metabolic imbalance. This study demonstrates that the Madin Darby bovine kidney (MDBK) cells, a commonly used cell line in veterinary research, can be effectively synchronized using lovastatin (Lov), a drug used to treat hypercholesteremia in humans. This was demonstrated by the following results : (i) Lov inhibits cell proliferation in a dose-dependent manner ; (ii) Lov synchronizes MDBK cells mainly in the G1 and secondarily in the G2+M cell-cycle phases ; (iii) the cytostatic effect of Lov can be specifically inhibited by addition of mevalonate (Mev) (Lov inhibits the synthesis of Mev) ; (iv) removal of Lov from G1-arrested cultures, followed by addition of Mev, resulted in the synchronous recovery of DNA synthesis ; and (v) 5-bromo2’-deoxyuridine incorporation experiments revealed that MDBK cells synchronization by Lov can be followed for at least 3 cycles after removal of Lov and addition of Mev. Furthermore, as an application of investigations based on the availability of synchronized MDBK, we showed that bovine herpesvirus-1 gene expression is independent on the cell cycle.

Vesey G, Hutton P, Champion A, Ashbolt N, Williams KL, Warton A, Veal D (1994) Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. Cytometry 16 :1-6


Cryptosporidium and Giardia are common causes of waterborne disease. The currently used methods of detecting these organisms in water rely on filtration capture, immunofluorescence labelling, and epifluorescence microscopy. These methods are inefficient, labour intensive, and require a highly skilled microscopist. We describe an alternative technique using flocculation concentration, followed by flow cytometry with fluorescence activated cell sorting. Environmental samples were analysed, and protozoan-like particles were sorted and collected before confirmation with epifluorescence microscopy. The technique was found to be significantly more sensitive and considerably faster than the conventional methods.

Vesey G, Narai J, Ashbolt N, Williams K, Veal D (1994) Detection of specific microorganisms in environmental samples using flow cytometry. Methods Cell Biol 42 Pt B :489-522


von Freiesleben U, Rasmussen KV, Schaechter M (1994) SeqA limits DnaA activity in replication from oriC in Escherichia coli. Mol Microbiol 14 :763-772


A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation in this gene, named seqA1, has also been isolated. Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2-containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild-type seqA gene on a low-copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39 degrees C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles. The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold). The introduction of minichromosomes or a low-copy-number plasmid carrying five DnaA-boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild-type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.

Wyrick PB, Davis CH, Wayner EA (1994) Chlamydia trachomatis does not bind to alpha beta 1 integrins to colonize a human endometrial epithelial cell line cultured in vitro. Microb Pathog 17 :159-166


Chlamydia trachomatis is the leading cause of bacterially acquired sexually transmitted diseases in the United States and Europe. As an obligate intracellular pathogen, this bacterium must invade epithelial cells in order to survive and grow. Thus, multiple strategies probably exist for initial binding of chlamydiae to their target cells. Since a variety of bacteria have exploited integrins to colonize tissues, and a precedent existed for the involvement of extracellular matrix components in chlamydial attachment, this study first analyzed, by flow cytometry, integrins expressed by the human endometrial epithelial cell line HEC-1B. The genital cells were then exposed to monoclonal antibodies directed against those integrins and assayed for chlamydial attachment and inclusion development. Monoclonal antibodies bound to the alpha and/or beta 1 subunit of classic integrin receptors displayed by HEC-1B cells were not able to prevent colonization and infection of the epithelial cells by a genital isolate of C. trachomatis.

Yang Q, Chen F, Trempe JP (1994) Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. J Virol 68 :4847-4856


The replication (rep) gene of adeno-associated virus (AAV) is involved in AAV DNA replication, gene regulation, and inhibition of cellular transformation induced by various oncogenes. To study the rep gene’s antiproliferative effects, we have developed cell lines which express the replication proteins under the control of an inducible mouse metallothionein transcription promoter. The Rep78 protein produced in these cell lines binds to the AAV terminal repeat sequences in vitro and supports AAV DNA replication and trans activation of the AAV p40 transcription promoter in vivo. These cell lines are capable of assembling infectious viruses containing a mutant rep gene or a vector bearing a heterologous gene. Growth rate and colony formation efficiency assays indicated that rep gene expression substantially altered cellular proliferation. Long-term induction of the cell lines followed by removal of the inducing agent suggested that constitutive expression of the Rep proteins does not necessarily result in cell death and that the cells can recover from the cytostatic effects. Flow cytometry analysis indicated that the presence of the Rep proteins increased the population of cells in the S phase of the cell cycle. Thus the rep gene’s antiproliferative effects may be realized by interference with cellular DNA replication.

Yeaman MR, Sullam PM, Dazin PF, Bayer AS (1994) Platelet microbicidal protein alone and in combination with antibiotics reduces Staphylococcus aureus adherence to platelets in vitro. Infect Immun 62 :3416-3423


Bacterial adherence to platelets on the cardiac valve surface is believed to be critical in the induction of infective endocarditis. Recent studies have confirmed that thrombin-activated platelets secrete platelet microbicidal protein (PMP), which can both kill and exert nonlethal antiadherence effects against endovascular pathogens. In the present study, we quantified the influence of antibiotic and/or PMP exposures on in vitro platelet adherence of two Staphylococcus aureus strains, identical by DNA restriction and cell wall protein profiles, that differed in their susceptibility to PMP-induced killing (PMPs or PMPr, respectively). Adherence assays were performed by flow cytometry in the presence of sublethal PMP concentrations (1 to 2.5 micrograms/ml) alone or in combination with ampicillin (AMP) alone, sulbactam (SUL) alone, or AMP plus SUL (AMP-SUL), at levels achievable in serum. Exposure of the PMPs and PMPr S. aureus strains to antibiotics (for 2 h at 37 degrees C) prior to flow cytometry resulted in no substantive changes in the percent adherence to platelets compared with that for S. aureus cells not exposed to antibiotics, except for modestly increased adherence of both PMPs and PMPr cells exposed to AMP-SUL (18.5 and 15.8% increases, respectively). Addition of PMP to antibiotic-S. aureus mixtures (final 30 min) caused a significant decrease in S. aureus adherence to platelets, for both the PMPs and PMPr S. aureus strains, compared with antibiotic exposure alone (e.g., reduction in platelet adherence from 57.9 +/- 8.2% to 12.2 +/- 3.6% for PMPs cells exposed to AMP-SUL and PMP [P = 0.01]). Moreover, addition of PMP following exposure of the PMPs and PMPr strains to AMP-SUL reversed the enhanced bacterium-platelet adherence observed with such antibiotic exposures alone (P < or = 0.005). These data demonstrate that PMP exerts a potent antiplatelet adherence effect which is independent of its microbicidal capacity, rendering S. aureus cells less adherent to platelets in the presence or absence of antibiotics. Reduction of microbial adherence to platelets by PMP alone or with antibiotics provides further insight into the mechanism(s) that may be involved in host defense and antibiotic prophylaxis of infective endocarditis and other endovascular infections.

Zorgani AA, Stewart J, Blackwell CC, Elton RA, Weir DM (1994) Inhibitory effect of saliva from secretors and non-secretors on binding of meningococci to epithelial cells. FEMS Immunol Med Microbiol 9 :135-142


Carriage of Neisseria meningitidis B:4:P1.15 was higher among non-secretors during a school outbreak of meningitis ; non-secretors had lower levels of anti-meningococcal salivary IgM. Flow cytometry was used to assess effects of secretor and non-secretor saliva on binding of B:4:P1.15 to buccal epithelial cells : (1) to assess inhibition by IgA and IgM ; and (2) to assess contributions of salivary antibodies to inhibitory activities. Greater inhibition was obtained with secretor saliva : pooled (P = 0.049) ; fresh (P = 0.0001). Purified IgA (P = 0.02) and IgM (P = 0.03) were equally inhibitory. After absorption of anti-meningococcal antibodies, there was still significant inhibitory activity in the pools : secretors (P = 0.018) ; non-secretors (P = 0.005). These results indicate that both secretory immunoglobulins and other factors contribute to protection against colonisation by meningococci and might explain the increased carriage of B:4:1.15 in this population.