vendredi 24 avril 2009
par   G. Grégori

Achilles J, Muller S, Bley T, Babel W (2004) Affinity of single S. cerevisiae cells to 2-NBDglucose under changing substrate concentrations. Cytometry A 61 :88-98


BACKGROUND : Saccharomyces cerevisiae is a widely employed microorganism in biotechnological processes. Since proliferation and product formation depend on the capacity of the cell to access and metabolize a carbon source, a technique was developed to enable for analyzing the S. cerevisiae H155 cells’ affinity to extracellular glucose concentrations. METHODS : The fluorescent glucose analogue 2-NBDglucose was employed as a functional parameter to analyze the cells’ affinity to glucose. Structural parameters (proliferation, neutral lipid content, granularity, and cell size) were also investigated. Cells were grown both in batches and in chemostat regimes. RESULTS : The 2-NBDglucose uptake in individual cells proceeds in a time- and concentration-dependent manner and is affected by respiratory and respirofermentative modes of growth. The process is inhibited by D-glucose, D-fructose, D-mannose, and sucrose, but not L-glucose, D-galactose or lactose ; maltose is a weak inhibitor. The affinity of the individual cells to 2-NBDglucose was found to be high at low extracellular glucose concentrations, and weak at high concentrations. An additional, underlying pattern in the cells’ affinity to glucose was detected, illustrated by the recurrent appearance of two subpopulations showing distinctly differing quantities of this substrate. CONCLUSIONS : A multiparameter flow cytometry approach is presented that enables, for the first time, for analysis of the affinity of individual S. cerevisiae cells to glucose. Besides the adjustment of the yeast cell metabolism to extracellular glucose concentrations by altering their affinity to glucose, at least one further mechanism is clearly involved. Two subpopulations of cells were resolved, with different affinities not correlated with other cellular parameters measured.

Ananta E, Knorr D (2004) Evidence on the role of protein biosynthesis in the induction of heat tolerance of Lactobacillus rhamnosus GG by pressure pre-treatment. Int J Food Microbiol 96 :307-313


It was the aim of this work to evaluate, whether and to which extent heat resistance of Lactobacillus rhamnosus GG is affected by mild pressure treatments prior to exposure to lethal temperatures, such as during spray-drying. It was observed that cells pressure pre-treated at 100 MPa at 37 degrees C for 10 min showed higher survival than untreated cells when exposed to heat challenge at 60 degrees C. To gain more insights on the cellular mode of action of pressure induced heat tolerance, flow cytometric analysis was applied in combination with functional dye LIVE/DEAD BacLight bacterial viability kit. Dot plot analysis showed that a lower degree of membrane damage was observed at pressure pre-treated cells upon heat treatment at 60 degrees C for 3 min. Evaluation of heat inactivation kinetics of cells pressure treated in the presence of chloramphenicol, a protein synthesis inhibitor, pointed out the potential contribution of pressure-induced protein biosynthesis in the enhancement of bacterial heat tolerance.

Atreya CD, Kulkarni S, Mohan KV (2004) Rubella virus P90 associates with the cytokinesis regulatory protein Citron-K kinase and the viral infection and constitutive expression of P90 protein both induce cell cycle arrest following S phase in cell culture. Arch Virol 149 :779-789


In utero infection of developing fetus by Rubella virus (RV) causes cell division inhibition of critical precursor cells in organogenesis, CNS-associated birth defects and induction of apoptosis in cell culture. The underlying mechanisms of RV-induced congenital abnormalities are not known. Here, we identified a novel interaction between RV replicase P90 protein and a cytokinesis-regulatory protein, the Citron-K kinase (CK), in a yeast two-hybrid cDNA library screen. Aberrations in cytokinesis and subsequent apoptosis do occur in specific cell types when the CK gene is knocked out or, its regulatory function is perturbed. Our analysis found that full-length P90 binds CK and in RV-infected cells P90 colocalizes with CK in the cytoplasm. Furthermore, during RV infection as well as cellular expression of P90 alone, we identified a discrete subpopulation of cells containing 4N DNA content, indicating that these cells are arrested in the cell cycle following S phase, suggesting that cellular expression of viral P90 during RV infection perturbs cytokinesis. Previous reports by others established that RV infection leads to apoptosis in cell culture. These observations together taken to the fetal organogenesis level, favor the idea that RV P90, by binding to cellular CK, invokes cell cycle aberrations resulting in the cell- and organ-specific growth inhibition and programmed cell death during RV infection in utero, which commonly is referred to as RV-induced teratogenesis.

Babu U, Dalloul RA, Okamura M, Lillehoj HS, Xie H, Raybourne RB, Gaines D, Heckert RA (2004) Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge. Vet Immunol Immunopathol 101 :251-257


Our previous work showed that the cell-mediated immunity (CMI) was enhanced by live Salmonella vaccine (LV). The objective of this study was to evaluate the impact of live and killed Salmonella vaccines on Salmonella enteritidis (SE) clearance and to determine if the clearance was mediated by cell-mediated and/or humoral immunity. Chickens were first immunized at 2 weeks of age followed by a booster dose at 4 weeks, challenged with live SE 2 weeks later (6-week-old) and tested for CMI, antibody response and SE clearance 1-week post SE-challenge (7-week-old). Spleen cell proliferation induced by SE-flagella and Concanavalin A (Con A) were significantly higher and SE shedding was significantly lower in the LV group. The splenic CD3 population was significantly lower and B cells were higher in the control group compared to all the SE-challenged groups (with and without vaccination). Serum antibody to SE-flagella and envelope were significantly higher in the KV group compared to all the other groups. These results suggest that LV protects against SE infection, probably by enhancing the CMI.

Backman A, Maraha N, Jansson JK (2004) Impact of temperature on the physiological status of a potential bioremediation inoculant, Arthrobacter chlorophenolicus A6. Appl Environ Microbiol 70 :2952-2958


Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28 degrees C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system ("viable cells") whereas propidium iodide (PI) was used to stain cells with damaged membranes ("dead cells"). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28 degrees C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28 degrees C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5 degrees C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.

Bacon LD, Fulton JE, Kulkarni GB (2004) Methods for evaluating and developing commercial chicken strains free of endogenous subgroup E avian leukosis virus. Avian Pathol 33 :233-243


The genome of nearly all chickens contains various DNA proviral insertions of retroviruses of subgroup E avian leukosis virus (ALVE). However, the elimination or control of ALVE gene expression is desirable to improve productivity, to improve resistance to avian leukosis virus (ALV)-induced tumours, and to develop safer live virus vaccines in chick embryos and cultured chick cells. Restriction fragment length polymorphism and polymerase chain reaction methods are used to define the presence of ALVE genes ; and the expression of ALVE in chicken plasma or on cells, and the susceptibility of cells to ALVE is determined by flow cytometry using a specific (R2) antibody. ADOL line 0 chickens have been selected to be free of ALVE genes, while being resistant (i.e. lack receptors to ALVE), but susceptible to exogenous ALV (i.e. ALVA, ALVB, ALVC and ALVJ). To develop improved line 0-type chickens, ADOL line 0 was outcrossed to a commercial line that had one ALVE gene and evidence for ALVE resistance. Rous sarcoma virus (RSV) challenge was used to confirm resistance of F1 chickens to ALVE, and susceptibility of F2 breeders to ALVA and ALVB using test chicks produced by matings to line 7(2). Selected F2 breeders were resistant to ALVE, but susceptible to exogenous ALVA, ALVB, ALVC and ALVJ, based on challenge tests of progeny chick cells using an enzyme-linked immunosorbent assay. The new line, 0(1), has evidence for improved egg size, productivity, fertility and hatchability. Similar procedures may be used for development of productive ALVE free chicken lines with preferred ALV susceptibility traits.

Baek YU, Kim YR, Yim HS, Kang SO (2004) Disruption of gamma-glutamylcysteine synthetase results in absolute glutathione auxotrophy and apoptosis in Candida albicans. FEBS Lett 556 :47-52


Glutathione is the most abundant non-protein thiol and a major source of reducing equivalents in eukaryotes. We examined the role of glutathione in Candida albicans by the disruption of gamma-glutamylcysteine synthetase (GCS1), an essential enzyme in glutathione biosynthesis. The gcs1/gcs1 null mutants exhibited glutathione auxotrophy, which could be rescued by supplementing with reduced and oxidized glutathione and gamma-glutamylcysteine. When the mutants were depleted of glutathione, they showed typical markers of apoptosis. These results suggest that glutathione itself is an essential metabolite and C. albicans lacking GCS1 undergoes apoptosis.

Bahl MI, Sorensen SJ, Hestbjerg Hansen L (2004) Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry. FEMS Microbiol Lett 232 :45-49


A method was developed to study plasmid stability in Escherichia coli cells, which utilised the high speed analysis properties of flow cytometry. To discriminate between plasmid-harbouring cells and plasmid-free cells a plasmid-encoded Lac repressor protein was used to regulate the expression of a chromosomally inserted green fluorescent protein gene in the host cells. Flow cytometric analysis enabled detection and quantification of plasmid-free cells due to their green fluorescent phenotype. The reported system offers real-time analysis in combination with a very low detection level of plasmid loss in bacterial populations. This could be useful in future investigations of plasmid stability and population selection in bacterial communities.

Banks DJ, Porcella SF, Barbian KD, Beres SB, Philips LE, Voyich JM, DeLeo FR, Martin JM, Somerville GA, Musser JM (2004) Progress toward characterization of the group A Streptococcus metagenome : complete genome sequence of a macrolide-resistant serotype M6 strain. J Infect Dis 190 :727-738


We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

Barc MC, Bourlioux F, Rigottier-Gois L, Charrin-Sarnel C, Janoir C, Boureau H, Dore J, Collignon A (2004) Effect of amoxicillin-clavulanic acid on human fecal flora in a gnotobiotic mouse model assessed with fluorescence hybridization using group-specific 16S rRNA probes in combination with flow cytometry. Antimicrob Agents Chemother 48 :1365-1368


Predominant groups of bacteria from a human fecal flora-associated mouse model challenged with amoxicillin-clavulanic acid were quantified with fluorescence in situ hybridization combined with flow cytometry using specific 16S rRNA targeted oligonucleotide probes. This approach provides a useful tool with high throughput to evaluate fecal microflora under antibiotic treatment.

Batista da Silva AP, Lee W, Bajenova E, McCulloch CA, Ellen RP (2004) The major outer sheath protein of Treponema denticola inhibits the binding step of collagen phagocytosis in fibroblasts. Cell Microbiol 6 :485-498


Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads ; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.

Belibasakis GN, Mattsson A, Wang Y, Chen C, Johansson A (2004) Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans : involvement of the cytolethal distending toxin. Apmis 112 :674-685


The cytolethal distending toxin (Cdt) is produced by several Gram-negative bacterial species and causes growth arrest and morphological alterations in mammalian cells. Actinobacillus actinomycetemcomitans, which is involved in the pathogenesis of localized aggressive periodontitis, also produces a Cdt that affects periodontal connective tissue cells. The aim of this study was to investigate in which phase of the cell cycle these cells are arrested and enlarged when challenged with A. actinomycetemcomitans, and to evaluate the involvement of its Cdt. Human gingival fibroblasts and periodontal ligament cells were challenged with A. actinomycetemcomitans extract, or with purified Cdt, and cell cycle analysis was performed by propidium iodide staining and flow cytometry. Cells exposed to an A. actinomycetemcomitans wild-type strain, or to purified Cdt, were arrested in both G1 and G2/M phases, and appeared enlarged compared to the corresponding controls. The cellular enlargement occurred in both G1 and G2/M arrested cells. In contrast, cells exposed to an A. actinomycetemcomitans cdt-knockout mutant strain showed cell cycle phase distribution and size similar to the controls. In conclusion, A. actinomycetemcomitans causes a combined G1 and G2/M growth arrest and enlargement in periodontal connective tissue cells, which is attributed to its Cdt.

Berro AI, Perry GA, Agrawal DK (2004) Increased expression and activation of CD30 induce apoptosis in human blood eosinophils. J Immunol 173 :2174-2183


Eosinophils are one of the major effector cells in asthma, and controlling the number and survival of eosinophils might attenuate the severity of asthma. This result could be achieved by inducing eosinophil apoptosis. Apoptosis allows the removal of cells without inducing an inflammatory response. Our knowledge of the factors involved in regulating eosinophil apoptosis remains limited. CD30 molecule has been associated with T cell-negative selection and in TCR-mediated apoptosis. In this study we examined the expression and role of CD30 in apoptosis of human blood eosinophils. Percentage of apoptotic eosinophils was determined by annexin V-propidium iodide labeling, and CD30 expression was examined by flow cytometry. Spontaneous apoptosis was induced by serum deprivation, and survival was conferred by incubating cells with 10% FBS and IL-5. CD30 surface expression was up-regulated in eosinophils incubated for 24 h as compared with freshly isolated eosinophils, and both CD30 expression and eosinophil apoptosis increased in a time-dependent manner. We also measured CD30 mRNA expression by quantitative real-time RT-PCR and determined that CD30 transcripts increased in eosinophils undergoing apoptosis only under serum deprivation conditions. The agonistic CD30 Abs, Ber-H8 and HeFi-1, significantly enhanced eosinophil apoptosis. FBS and IL-5 failed to inhibit or suppress the CD30 agonistic-induced apoptosis. These data support the role of CD30 activation in eosinophil apoptosis. This research will help in furthering our understanding of eosinophil apoptosis and therefore might contribute to the development of better therapeutic modalities in the treatment and/or cure of allergic inflammation in bronchial asthma.

Beyer M, Bartz H, Horner K, Doths S, Koerner-Rettberg C, Schwarze J (2004) Sustained increases in numbers of pulmonary dendritic cells after respiratory syncytial virus infection. J Allergy Clin Immunol 113 :127-133


BACKGROUND : Respiratory syncytial virus (RSV) bronchiolitis in infants can lead to wheezing and early allergic sensitization. In mice, RSV infection enhances allergic airway inflammation and airway hyperresponsiveness. Dendritic cells are critical in inducing T-cell responses to both viruses and allergens and could be pivotal in regulating interactions between these. OBJECTIVE : This study addresses the effects of RSV infection on phenotype and function of pulmonary dendritic cells. METHODS : BALB/c mice were infected with RSV, and expression of CD11c, MHC II, and CD86 on lung and spleen cells was monitored by flow cytometry for 21 days after infection. CD11c(+) cells were isolated to assess their phagocytic capacity and their ability to induce proliferation in allogenic T cells. RESULTS : Numbers of pulmonary CD11c(+) MHC II(hi) cells increased 13-fold starting from day 6 after RSV infection. This was associated with increased CD86 expression, reduced phagocytosis, and increased allogenic T-cell stimulatory capacity in CD11c(+) cells. These changes in the lung outlasted acute infection and were not observed in spleens. CONCLUSION : RSV infection results in sustained increases in numbers of mature dendritic cells in the lung. These might well contribute to the development of intense airway inflammation and airway hyperresponsiveness after RSV infection and to enhancement of subsequent responses to allergen exposure.

Bhatia V, Sinha M, Luxon B, Garg N (2004) Utility of the Trypanosoma cruzi sequence database for identification of potential vaccine candidates by in silico and in vitro screening. Infect Immun 72 :6245-6254


Glycosylphosphatidylinositol (GPI)-anchored proteins are abundantly expressed in the infective and intracellular stages of Trypanosoma cruzi and are recognized as antigenic targets by both the humoral and cellular arms of the immune system. Previously, we demonstrated the efficacy of genes encoding GPI-anchored proteins in eliciting partially protective immunity to T. cruzi infection and disease, suggesting their utility as vaccine candidates. For the identification of additional vaccine targets, in this study we screened the T. cruzi expressed sequence tag (EST) and genomic sequence survey (GSS) databases. By applying a variety of web-based genome-mining tools to the analysis of approximately 2,500 sequences, we identified 348 (37.6%) EST and 260 (17.4%) GSS sequences encoding novel parasite-specific proteins. Of these, 19 sequences exhibited the characteristics of secreted and/or membrane-associated GPI proteins. Eight of the selected sequences were amplified to obtain genes TcG1, TcG2, TcG3, TcG4, TcG5, TcG6, TcG7, and TcG8 (TcG1-TcG8) which are expressed in different developmental stages of the parasite and conserved in the genome of a variety of T. cruzi strains. Flow cytometry confirmed the expression of the antigens encoded by the cloned genes as surface proteins in trypomastigote and/or amastigote stages of T. cruzi. When delivered as a DNA vaccine, genes TcG1-TcG6 elicited a parasite-specific antibody response in mice. Except for TcG5, antisera to genes TcG1-TcG6 exhibited trypanolytic activity against the trypomastigote forms of T. cruzi, a property known to correlate with the immune control of T. cruzi. Taken together, our results validate the applicability of bioinformatics in genome mining, resulting in the identification of T. cruzi membrane-associated proteins that are potential vaccine candidates.

Bhuyan PK, Kariko K, Capodici J, Lubinski J, Hook LM, Friedman HM, Weissman D (2004) Short interfering RNA-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes. J Virol 78 :10276-10281


RNA interference (RNAi) is an antiviral mechanism that is activated when double-stranded RNA is cleaved into fragments, called short interfering RNA (siRNA), that prime an inducible gene silencing enzyme complex. We applied RNAi against a herpes simplex virus type 1 (HSV-1) gene, glycoprotein E, which mediates cell-to-cell spread and immune evasion. In an in vitro model of infection, human keratinocytes were transfected with siRNA specific for glycoprotein E and then infected with wild-type HSV-1. RNAi-mediated gene silencing reproduced the small plaque phenotype of a gE-deletion mutant virus. The specificity of gene targeting was demonstrated by flow cytometry and Northern blot analyses. Exogenous siRNA can suppress HSV-1 glycoprotein E expression and function during active infection in vitro through RNAi. This work establishes RNAi as a genetic tool for the study of HSV and provides a foundation for development of RNAi as a novel antiviral therapy.

Bobryshev YV, Cao W, Phoon MC, Tran D, Chow VT, Lord RS, Lu J (2004) Detection of Chlamydophila pneumoniae in dendritic cells in atherosclerotic lesions. Atherosclerosis 173 :185-195


Dendritic cells (DCs) populate atherosclerotic lesions and might be involved in the regulation of immune reactions in atherosclerosis. The present work was undertaken to examine a possible association of DCs with Chlamydophila pneumoniae in human atherosclerotic plaques obtained by endarterectomy. C. pneumoniae was identified in 17 of 60 (28%) atherosclerotic plaques by a combination of immunohistochemistry and polymerase chain reaction (PCR). Double immunohistochemistry identified the presence of C. pneumoniae within S100(+) DCs that were localised predominantly in the deep layer of the intima under the necrotic core. Quantitative analysis showed that there were no differences in the numbers of DCs between C. pneumoniae(+) and C. pneumoniae(-) groups of atherosclerotic specimens. There were also no differences in the expression of Lag-antigen and HLA-DR by DCs between the groups of specimens. Markers of DC activation CD80 and CD86 were absent from both groups of specimens. Flow cytometry analysis of the effects of C. pneumoniae infection on immature monocyte-derived DCs in vitro showed no changes in the expression of CD1a, MHC class II, CD80 and CD86. The results of this study demonstrate that C. pneumoniae might infect DCs within the atherosclerotic intima but whether the presence of C. pneumoniae in DCs affects the intensity of immune reactions in atherosclerosis needs further clarification.

Bohm E, Voglauer R, Steinfellner W, Kunert R, Borth N, Katinger H (2004) Screening for improved cell performance : selection of subclones with altered production kinetics or improved stability by cell sorting. Biotechnol Bioeng 88 :699-706


One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins. Usually, the most important selection criterion is the cell specific production rate. Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process. However, the time and work required to select for high production rates as well as the lack of methods to specifically select for other cellular properties, usually prevents researchers from including such criteria into their screening program.With the Single Cell Secretion Assay it is possible to measure the specific production rates of individual cells by catching secreted product in an artificial matrix applied to the cell surface. Flow cytometric cell sorting then allows selection of rare cells with high production rates, which occur at frequencies as low as 10(-6). By combining this method with culture conditions that bring out a desired cellular property, we were able to isolate subclones with similar production rates, but improved performance from a recombinant Chinese hamster ovary cell line producing a human monoclonal antibody. The two desired cellular properties screened for were a non-growth associated production kinetic and improved stability in the absence of selective pressure.

Bosch A, Pinto RM, Comas J, Abad FX (2004) Detection of infectious rotaviruses by flow cytometry. Methods Mol Biol 268 :61-68


Human rotaviruses are considered the main cause of viral gastroenteritis in infants and young children throughout the world. Their transmission is through the fecal-oral route, mostly after ingestion of contaminated water and food. Since an extremely high number of virus particles are present in the feces during the acute gastroenteritis, methods based on electron microscopy, passive particle agglutination tests, or enzyme-linked immunosorbent assays are readily employed for clinical diagnosis. However, the sensitivity of these procedures is not high enough to detect the low number of viral particles sometimes present in the environment. In the case of environmental samples, amplification of viral nucleic acids by polymerase chain reaction assays coupled to reverse transcription (RT-PCR) has been increasingly applied to detect rotaviruses in water and shellfish samples. However, procedures based on molecular approaches have to face the drawback that they do not differentiate between infectious and noninfectious particles, which is of major relevance from the public health point of view.Virus propagation in cell culture prior to detection by immunological or molecular procedures accomplishes the dual purpose of increasing the amount of target material and incorporating an infectivity assay as well.Wild-type rotaviruses present difficulties in their in vitro replication, although some of them may be adapted to grow in several cell lines such as the monkey kidney cell line MA104 or the human intestinal cell line CaCo-2. More than a decade ago, an assay for the specific detection of infectious rotaviruses in environmental samples, involving an indirect immunofluorescence test (IIF) and optical microscopy (OM) counting of infected foci in infected MA-104 cell monolayers, was described. On the other hand, CaCo-2 cells have been successfully employed in our laboratory for infectivity assays of several fastidious enteric virus strains present in water samples.

Brehm-Stecher BF, Johnson EA (2004) Single-cell microbiology : tools, technologies, and applications. Microbiol Mol Biol Rev 68 :538-559


The field of microbiology has traditionally been concerned with and focused on studies at the population level. Information on how cells respond to their environment, interact with each other, or undergo complex processes such as cellular differentiation or gene expression has been obtained mostly by inference from population-level data. Individual microorganisms, even those in supposedly "clonal" populations, may differ widely from each other in terms of their genetic composition, physiology, biochemistry, or behavior. This genetic and phenotypic heterogeneity has important practical consequences for a number of human interests, including antibiotic or biocide resistance, the productivity and stability of industrial fermentations, the efficacy of food preservatives, and the potential of pathogens to cause disease. New appreciation of the importance of cellular heterogeneity, coupled with recent advances in technology, has driven the development of new tools and techniques for the study of individual microbial cells. Because observations made at the single-cell level are not subject to the "averaging" effects characteristic of bulk-phase, population-level methods, they offer the unique capacity to observe discrete microbiological phenomena unavailable using traditional approaches. As a result, scientists have been able to characterize microorganisms, their activities, and their interactions at unprecedented levels of detail.

Brussaard CP (2004) Optimization of procedures for counting viruses by flow cytometry. Appl Environ Microbiol 70 :1506-1513


The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.

Bryndorf T, Kirchhoff M, Larsen J, Andreasson B, Bjerregaard B, Westh H, Rose H, Lundsteen C (2004) The most common chromosome aberration detected by high-resolution comparative genomic hybridization in vulvar intraepithelial neoplasia is not seen in vulvar squamous cell carcinoma. Cytogenet Genome Res 106 :43-48


We analyzed genetic changes in condylomas (four cases), vulvar intraepithelial neoplasia I-III (VIN I-III, eleven cases), and primary vulvar squamous cell carcinomas (VSCC, ten cases) by high-resolution comparative genomic hybridization (HR-CGH) and flowcytometry. All samples were also human papilloma virus (HPV)-genotyped. Gain of chromosome 1, the aberration most often seen in VIN III (67%), was not seen in HPV-positive or -negative VSCCs (0%). Both VIN III and VSCC frequently showed gain of 3q (56 and 70%, respectively). The VIN III samples often demonstrated gain of 20q (56%) and 20p (44%), and the VSCC samples gain of 8q (60%), loss of 3p (50%), and 8p (40%). None of the four most frequent changes in the VSCC samples occurred exclusively in the HPV-positive or -negative samples. As expected, we did not find any cytogenetic changes in condylomas and nearly any changes in VIN I-II.

Budge PJ, Li Y, Beeler JA, Graham BS (2004) RhoA-derived peptide dimers share mechanistic properties with other polyanionic inhibitors of respiratory syncytial virus (RSV), including disruption of viral attachment and dependence on RSV G. J Virol 78 :5015-5022


Large polyanionic molecules, such as sulfated polysaccharides (including soluble heparin and dextran sulfate), synthetic polyanionic polymers, and negatively charged proteins, have been shown to broadly inhibit several enveloped viruses. We recently reported the antiviral activity of a peptide derived from amino acids 77 to 95 of a potential binding partner of respiratory syncytial virus F protein (RSV F), the GTPase RhoA. A subsequent study with a truncated peptide (amino acids 80 to 94) revealed that optimal antiviral activity required dimerization via intermolecular disulfide bonds. We report here that the net negative charge of this peptide is also a determining factor for its antiviral activity and that it, like other polyanions, inhibits virus attachment. In a flow cytometry-based binding assay, peptide 80-94, heparin, and dextran sulfate inhibited the attachment of virus to cells at 4 degrees C at the same effective concentrations at which they prevent viral infectivity. Interestingly, time-of-addition experiments revealed that peptide 80-94 and soluble heparin were also able to inhibit the infectivity of a virus that had been prebound to cells at 4 degrees C, as had previously been shown for dextran sulfate, suggesting a potential role for postattachment effects of polyanions on RSV entry. Neutralization experiments with recombinant viruses showed that the antiviral activities of peptide 80-94 and dextran sulfate were diminished in the absence of the RSV attachment glycoprotein (G). Taken together, these data indicate that the antiviral activity of RhoA-derived peptides is functionally similar to that of other polyanions, is dependent on RSV G, and does not specifically relate to a protein-protein interaction between F and RhoA.

Busetto S, Trevisan E, Patriarca P, Menegazzi R (2004) A single-step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane-bound and ingested Candida albicans in phagocytosing neutrophils. Cytometry A 58 :201-206


BACKGROUND : Distinguishing ingested particles from those attached to the cell surface is an essential requirement when performing quantitative studies of phagocytosis. In the present report, we describe a simple, sensitive and reliable flow cytofluorometric method that achieves this goal in a Candida albicans-human neutrophils (PMN) system. METHODS : The assay is based on the observation that the vital dye trypan blue (TB), while quenching the green fluorescence of fluorescein-labeled C. albicans, causes them to fluoresce red. PMN were incubated with fluorescein-labeled yeast particles for the required time. Aliquots of the incubation mixtures were then promptly diluted with an equal volume of a TB solution at pH 4.0, and subsequently analyzed by flow cytometry for green and red fluorescence. RESULTS : Since TB does not penetrate into the cells, ingested yeasts retain their green fluorescence, while membrane-bound particles display a red fluorescence. CONCLUSIONS : Our fluorescence flow cytometric method enables to simultaneously distinguish, within the leukocyte population, cell subsets with attached and ingested yeast particles. Its major features are : (1) accuracy, sensitivity and reproducibility ; (2) no further sample manipulations after completion of phagocytosis ; (3) possibility of counting free, attached and internalized yeast particles ; and (4) use of a nontoxic reagent (TB).

Caballero S, Abad FX, Loisy F, Le Guyader FS, Cohen J, Pinto RM, Bosch A (2004) Rotavirus virus-like particles as surrogates in environmental persistence and inactivation studies. Appl Environ Microbiol 70 :3904-3909


Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20 degrees C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20 degrees C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

Callister SM, Jobe DA, Schell RF (2004) Detection of borreliacidal antibodies by flow cytometry. Curr Protoc Cytom Chapter 11 :Unit 11 15


Lyme disease is a multisystem disorder that usually begins with a skin lesion called erythema migrans and with constitutional symptoms. If the disease is left untreated or treated inappropriately, dissemination of the organism can lead to more severe sequelae, including nervous system disorders or arthritis. Vaccinations with B. burgdorferi or several individual B. burgdorferi proteins induce borreliacidal antibodies that provide protection against infection by inducing a complement cascade that kills the spirochetes without the necessity of scavenging by phagocytic cells. Detection of borreliacidal antibodies is therefore useful for serodiagnosing Lyme disease and monitoring immune status after vaccination. This unit provides a technique for detecting anti-B. burgdorferi antibodies, as well as for preparing and determining the quality of Barbour-Stoenner-Kelly (BSK medium) and complement. In addition, methods are provided for preparation of a B. burgdorferi stock and Mueller-Hinton agar containing Bacillus subtilis spores.

Cao ZY, Zhao J, Liao QP, Yang YS, Zhou L, Zeng Y (2004) [Immortalization of human embryonic cervical epithelial cells induced by E6, E7 genes of human papillomavirus 16]. Zhonghua Fu Chan Ke Za Zhi 39 :486-488


OBJECTIVE : To establish an immortalized cell line derived from the embryonic cervical epithelium by infection with the recombinant adeno-associated virus (rAAV) containing human papillomavirus (HPV)16 E6, E7, and to study the biological features of cervical cancer cell line. METHODS : Human embryonic cervical tissues were cultured in keratinocyte free serum (K-FS) medium and infected with rAAV containing HPV16 E6, E7. Morphological features and growth rate were examined by light, electronic and fluorescence microscopies. The fragments of E6, E7 were detected by polymerase chain reaction (PCR) and laser confocal microscopy. The biological characteristics of human cervical epithelium were observed by soft agar culture, scid mice inoculation and chromosome analysis. Cell proliferative dynamics was plotted by flow cytometry. RESULTS : After a long-term culture, the phenotype kept the characteristics of primary epithelial cells. They showed monolayer, anchorage-dependent and attachment-inhibited growth without forming colonies in soft agar culture. They were non-oncogenic when inoculated into scid mice. The tonofilament expression in the cervical cancer cells was inspected by electronic microscopy, demonstrating that the cells were squamous epithelium in origin. The cell line contained HPV16 E6, E7 genes by PCR and laser confocal detection. Chromosome analysis disclosed that the karyotype was diploid or polyploid. The 11th chromosome was assumed to be the integration site by rAAV containing HPV16 E6, E7. CONCLUSIONS : Establishment of the immortalized cervical epithelial cell line by infection with rAAV containing HPV16 E6, E7, supports that HPV16 E6, E7 may be the primary etiology of cervical cancer. It will facilitate further research on the etiology and pathogenesis of cervical cancer.

Carneiro CR, Postol E, Nomizo R, Reis LF, Brentani RR (2004) Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus. Microbes Infect 6 :604-608


We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.

Chandler DP, Jarrell AE (2004) Automated purification and suspension array detection of 16S rRNA from soil and sediment extracts by using tunable surface microparticles. Appl Environ Microbiol 70 :2621-2631


Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA ( approximately 10(4) cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA ( approximately 10(5) cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.

Chia JS, Lin YL, Lien HT, Chen JY (2004) Platelet aggregation induced by serotype polysaccharides from Streptococcus mutans. Infect Immun 72 :2605-2617


Platelet aggregation plays an important role in the pathogenesis of infective endocarditis induced by viridans streptococci or staphylococci. Aggregation induced in vitro involves direct binding of bacteria to platelets through multiple surface components. Using platelet aggregometry, we demonstrated in this study that two Streptococcus mutans laboratory strains, GS-5 and Xc, and two clinical isolates could aggregate platelets in an irreversible manner in rabbit platelet-rich plasma preparations. The aggregation was partially inhibited by prostaglandin I(2) (PGI(2)) in a dose-dependent manner. Whole bacteria and heated bacterial cell wall extracts were able to induce aggregation. Cell wall polysaccharides extracted from the wild-type Xc strain, containing serotype-specific polysaccharides which are composed of rhamnose-glucose polymers (RGPs), could induce platelet aggregation in the presence of plasma. Aggregation induced by the serotype-specific RGP-deficient mutant Xc24R was reduced by 50% compared to the wild-type strain Xc. In addition, cell wall polysaccharides extracted from Xc24R failed to induce platelet aggregation. The Xc strain, but not the Xc24R mutant, could induce platelet aggregation when preincubated with plasma. Both Xc and Xc24R failed to induce platelets to aggregate in plasma depleted of immunoglobulin G (IgG), but aggregation was restored by replenishment of anti-serotype c IgG. Analysis by flow cytometry showed that S. mutans RGPs could bind directly to rabbit and human platelets. Furthermore, cell wall polysaccharides extracted from the Xc, but not the Xc24R, strain could induce pseudopod formation of both rabbit and human platelets in the absence of plasma. Distinct from the aggregation of rabbit platelets, bacterium-triggered aggregation of human platelets required a prolonged lag phase and could be blocked completely by PGI(2). RGPs also trigger aggregation of human platelets in a donor-dependent manner, either as a transient and reversible or a complete and irreversible response. These results indicated that serotype-specific RGPs, a soluble product of S. mutans, could directly bind to and activate platelets from both rabbit and human. In the presence of plasma containing IgG specific to RGPs, RGPs could trigger aggregation of both human and rabbit platelets, but the degree of aggregation in human platelets depends on the donors.

Comitini F, Di Pietro N, Zacchi L, Mannazzu I, Ciani M (2004) Kluyveromyces phaffii killer toxin active against wine spoilage yeasts : purification and characterization. Microbiology 150 :2535-2541


The killer toxin secreted by Kluyveromyces phaffii (KpKt) is active against spoilage yeast under winemaking conditions and thus has potential applications in the biocontrol of undesired micro-organisms in the wine industry. Biochemical characterization and N-terminal sequencing of the purified toxin show that KpKt is a glycosylated protein with a molecular mass of 33 kDa. Moreover, it shows 93% and 80% identity to a beta-1,3-glucanase of Saccharomyces cerevisiae and a beta-1,3-glucan transferase of Candida albicans, respectively, and it is active on laminarin and glucan, thus showing a beta-glucanase activity. Competitive inhibition of killer activity by cell-wall polysaccharides suggests that glucan (beta-1,3 and beta-1,6 branched glucans) represents the first receptor site of the toxin on the envelope of the sensitive target. Flow cytometry analysis of the sensitive target after treatment with KpKt and K1 toxin of S. cerevisiae, known to cause loss of cell viability via formation of pores in the cell membrane, suggests a different mode of action for KpKt.

Condron CM, Toomey DM, Casey RG, Creagh T, Bouchier-Hayes DJ (2004) Taurine protects against PMN dysfunction and death in urine. Urol Res 32 :338-345


The polymorphonuclear neutrophil (PMN) is the primary pro-inflammatory cell in the host response to bacterial infection and, as the first line of defence, is the principal cell responsible for the recognition, phagocytosis and killing of bacteria. PMN function is known to be defective in the urine. High osmolarity is physiologic in the urine and this hypertonic environment has been shown to compromise neutrophil function. In this study, PMN function was found to be suppressed in urine. This correlated with significant cell death, both by apoptosis and necrosis. The amino acid taurine down regulated PMN cell death and preserved function in the urine, suggesting taurine as a therapeutic option for urinary tract infection.

Crapnell KB, Almeida-Porada G, Khaiboullina S, St Jeor SC, Zanjani ED (2004) Human haematopoietic stem cells that mediate long-term in vivo engraftment are not susceptible to infection by human cytomegalovirus. Br J Haematol 124 :676-684


A human/sheep xenograft model was used to evaluate whether long-term engrafting haematopoietic stem cells (HSC) are susceptible to human cytomegalovirus (HCMV) infection. CD34+ Lin- HSC were isolated by fluorescence-activated cell sorting (FACS) from the bone marrow (BM) of HCMV-positive and HCMV-negative normal donors. Cells from the latter group were infected in vitro with HCMV. HCMV DNA was detected in both cell populations by nested-polymerase chain reaction (PCR) and fluorescence in situ hybridization. Cells were transplanted into separate groups of fetal sheep at concentrations of 1.3-5.0 x 105 cells per fetus. Multilineage human haematopoietic cell engraftment, including CD34+ cells, was detected in the BM and peripheral blood of recipients up to 16 months post-transplant as assessed by FACS analysis and PCR for HLA-DQalpha. Levels of engraftment varied (1.2-24.3%) but no sheep exhibited HCMV-positive cells. To ensure that our inability to detect HCMV-positive cells was not due to immune-elimination of HCMV-infected cells, 3.8-10 x 105 HCMV-positive uncharacterized BM stromal cells were transplanted into fetal sheep. At 5 weeks post-transplant several organs were HLA-DQalpha- and HCMV-positive, confirming that HCMV was detectable. These results provide evidence that the long-term engrafting HSC is not a primary target of HCMV and suggest that HCMV infection of human haematopoietic cells is exercised at the level of committed progenitors.

Dave S, Carmicle S, Hammerschmidt S, Pangburn MK, McDaniel LS (2004) Dual roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and factor H. J Immunol 173 :471-477


Streptococcus pneumoniae, also known as the pneumococcus, contains several surface proteins that along with the polysaccharide capsule function in antiphagocytic activities and evasion of the host immune system. These pneumococcal proteins interact with the host immune system in various ways and possess a wide range of biological activities that suggests that they may be involved at different stages of pneumococcal infection. PspC, also known as CbpA and SpsA, is one of several pneumococcal surface proteins that binds host proteins, including factor H (FH) and secretory IgA (sIgA) via the secretory component. Previous work by our laboratory has demonstrated that PspC on the surface of live pneumococcal cells binds FH. This paper provides evidence that FH activity is maintained in the presence of PspC and that the PspC binding site is located in the short consensus repeat 6-10 region of FH. We also report for the first time that although both FH and sIgA binding has been localized to the alpha-helical domain of PspC, the binding of FH to PspC is not inhibited by sIgA. ELISA, surface plasmon resonance, and flow cytometry indicate that the two host proteins do not compete for binding with PspC and likely do not share the same binding sites. We confirmed by Western analysis that the binding sites are separate using recombinant PspC proteins. These PspC variants bind FH yet fail to bind sIgA. Thus, we conclude that FH and sIgA can bind concurrently to the alpha-helical region of PspC.

Dave S, Pangburn MK, Pruitt C, McDaniel LS (2004) Interaction of human factor H with PspC of Streptococcus pneumoniae. Indian J Med Res 119 Suppl :66-73


BACKGROUND & OBJECTIVES : Streptococcus pneumoniae has acquired virulence factors such as the polysaccharide capsule and various surface proteins, which prevent opsonization mediated by the complement system. PspC is one of the multi-functional pneumococcal surface proteins capable of eliciting an antibody response in mice. Our study further explores the role of pneumococcal surface proteins in resistance to complement mediated opsonophagocytosis by providing evidence that PspC binds human Factor H (FH), a regulatory protein of the alternative complement pathway. The present study was carried out to map the binding regions on PspC and FH, and to assess the functional activity of FH upon binding to PspC. METHODS : FH binding to D39 and other pneumococcal strains was observed by flow cytometry. A series of FH truncated and deletion mutants and PspC mutants were used to localize binding regions within these molecules. The functional activity of FH upon binding to PspC was measured by a haemolysis assay. RESULTS : FH binding to D39 and not to TRE108 (PspC-) cells was demonstrated by flow cytometry. Pneumococcal isolates of 14 different strains varied in their ability to bind FH. The binding region of FH within PspC to the first 225 amino acids of the alpha-helical domain was localized. The corresponding binding site for PspC is located within the SCR 6-10 region of FH. Haemolysis of rabbit red blood cells was inhibited by FH even in the presence of PspC. INTERPRETATION & CONCLUSION : FH binding is specific to PspC on the pneumococcal cell surface. The binding region on PspC mapped to the non-conserved N-terminal region of the alpha-helical domain. The binding site on FH to PspC is different from the active site that functions in degradation of C3b. A haemolysis assay provided evidence that the functional activity of FH was maintained upon binding to PspC. Thus, binding of FH to PspC might be an important mechanism by which S. pneumoniae resist complement activation and opsonophagocytosis.

Ding Y, Chung CS, Newton S, Chen Y, Carlton S, Albina JE, Ayala A (2004) Polymicrobial sepsis induces divergent effects on splenic and peritoneal dendritic cell function in mice. Shock 22 :137-144


Dendritic cells (DCs) are professional antigen-presenting cells that act as sentinels in the cell-mediated response against invading pathogens associated with septic challenge. The purpose of the present study was to determine whether there is a loss of dendritic cells and/or changes in function of these cells in septic mice. Here we report that the number of DCs, in both spleen and peritoneum, decreased over 24 h postsepsis [cecal ligation and puncture (CLP)] when compared with sham. The most dramatic change was seen in the peritoneal cavity. This decrease appeared to be caused mainly by the depletion of immature DCs rather than mature DCs. This change was LPS independent and minimally affected by FasL ; however, overexpression of human Bcl-2 gene provides protection of the septic peritoneal DCs. Moreover, although the level of IL-12 release decreased significantly in splenic DCs obtained from CLP mice, IL-12 secretion was markedly elevated by peritoneal DCs as well as in both plasma and peritoneal fluid at 24 h post-CLP. In peritoneal cells, the expression of CD40, CD80, and CD86 was unchanged, but their respective ligands CD40L, CD28, and CD152 all increased in mice 24 h after CLP, although no such change was observed in splenocytes. Regardless of the presence or absence of antigen, peritoneal DCs from CLP mice showed higher capacity to stimulate T-cell proliferation than those cells from the sham control. However, splenic DCs from CLP mice only showed augmented capacity to induce antigen-dependent stimulation of T-cell proliferation. Together, these data indicate that sepsis produces divergent functional changes in splenic and peritoneal DC populations.

Du C, Zhang Q, Li C, Miao D, Gui J (2004) Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus. Virus Res 101 :119-126


A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells.

Elliott DE, Setiawan T, Metwali A, Blum A, Urban JF, Jr., Weinstock JV (2004) Heligmosomoides polygyrus inhibits established colitis in IL-10-deficient mice. Eur J Immunol 34 :2690-2698


Inflammatory bowel disease (IBD) is prevalent in industrialized countries, but rare in less-developed countries. Helminths, common in less-developed countries, may induce immunoregulatory circuits protective against IBD. IL-10(-/-) mice given piroxicam develop severe and persistent colitis. Lamina propria mononuclear cells from colitic IL-10(-/-) mice released IFN-gamma and IL-12. The ongoing piroxicam-induced colitis could be partially blocked with anti-IL-12 monoclonal antibody suggesting that the inflammation was at least partly IL-12 dependent. Colonization of piroxicam-treated colitic IL-10(-/-) mice with Heligmosomoides polygyrus (an intestinal helminth) suppressed established inflammation and inhibited mucosal IL-12 and IFN-gamma production. H. polygyrus augmented mucosal IL-13, but not IL-4 or IL-5 production. Transfer of mesenteric lymph node (MLN) T cells from IL-10(-/-) animals harboring H. polygyrus into colitic IL-10(-/-) recipients inhibited colitis. MLN T cells from worm-free mice did not. Foxp3 (scurfin) drives regulatory T cell function. H. polygyrus enhanced Foxp3 mRNA expression in MLN T cells that had regulatory activity. This suggests that H. polygyrus inhibits ongoing IL-10(-/-) colitis in part through blocking mucosal Th1 cytokine production. Resolution of inflammation is associated with increased IL-13 production and can be adoptively transferred by MLN T cells.

Emerson SU, Nguyen H, Graff J, Stephany DA, Brockington A, Purcell RH (2004) In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. J Virol 78 :4838-4846


Hepatitis E virus (HEV) RNA replication occurred in seven of nine primate cell cultures transfected with in vitro transcripts of an infectious cDNA clone. Cell-to-cell spread did not occur in cell cultures, but rhesus monkeys inoculated with lysates of HEV-transfected PLC/PRF/5 and Huh-7 cells became infected with HEV. A replicon with the ORF2 and ORF3 genes deleted and replaced with the green fluorescent protein gene also replicated in the same primate cells that supported the replication of the full-length genome. Fluorescence-activated cell sorter analysis confirmed that the 7mG cap structure was critical for efficient infectivity, although replication could be initiated at a very low level in its absence. HEV virions were also able to infect a limited number of cells of certain lines.

Engelmann P, Molnar L, Palinkas L, Cooper EL, Nemeth P (2004) Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge. Cell Tissue Res 316 :391-401


Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences : (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.

Ferrari BC, Oregaard G, Sorensen SJ (2004) Recovery of GFP-labeled bacteria for culturing and molecular analysis after cell sorting using a benchtop flow cytometer. Microb Ecol 48 :239-245


Exciting opportunities exist for the application of simple fluorescence-activated cell sorting (FACS) to microbiology. The technology is widely available, but critical reports on the efficiency of cell sorting using benchtop instruments are lacking. It is vital that single cell sorting be of the highest purity possible. If purity is compromised detrital material or unwanted cells will be captured along with target cells of interest. Here, the isolation of fluorescent bacteria using a benchtop FACSCalibur-sort flow cytometer is described. The efficiency and purity of isolated cells was determined using fluorescence microscopy, culturing, and molecular analysis. To achieve high purity it was essential that the total event rate did not exceed 300 cells per second. This instrument was capable of recovering >55% sorted Escherichia coli cells, coupled with a purity exceeding 99%. However, the purity of recovered cells was substantially reduced (<25%) when the event rate increased. Cell sorting onto polycarbonate membranes did not reduce the ability of E. coli to form colonies, and sorting of 1000 E. coli cells was sufficient for 16S rDNA amplification. Additionally, as few as 100 isolated Erwinia sp. carrying the gfp gene were amplified using seminested PCR targeting the single copy gfp gene. With such low numbers of bacteria being required for molecular identification, FACS can be achieved without the requirement for high-speed droplet cell sorters.

Flynn JC, Rao PV, Gora M, Alsharabi G, Wei W, Giraldo AA, David CS, Banga JP, Kong YM (2004) Graves’ hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA. Clin Exp Immunol 135 :35-40


Familial and twin studies in Caucasians have established that the MHC class II allele HLA-DRB1*0301 (DR3) is a strong susceptibility gene in Graves’ hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II-knockout NOD mice (H2Ag7-). DR3+g7- mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL-4 or GM-CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid-stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid-stimulating blocking Abs and/or TSH-binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimoto’s thyroiditis model induced with human thyroglobulin.

Freeman A, Cohen-Hadar N, Abramov S, Modai-Hod R, Dror Y, Georgiou G (2004) Screening of large protein libraries by the cell immobilized on adsorbed bead approach. Biotechnol Bioeng 86 :196-200


Screening of mutant libraries for in vitro enzyme evolution is carried out primarily by physical separation of the cells, followed by growth of individual clones and screening of biocatalytic activity on the basis of color or fluorescence signal development. Currently, most frequently employed methods are labor-intensive or require robotic equipment, resulting in screening limited to a relatively small fraction of the potential inherent in a given library. In this study we present a design, development, and feasibility demonstration of a new screening approach, providing convenient handling of large libraries consisting of 106 to 107 clones and screening based on a simultaneous enzymatic assay with commercially available substrates. This new screening method is based on the "cell immobilized on adsorbed bead" approach : the cell population to be screened is mixed with an excess of medium pre-equilibrated polyacrylamide beads, chemically derivatized to affect quantitative cell immobilization by adsorption. The resulting bead population, comprising of single cell on a bead or blank beads, is then immobilized on a solid glass support. After removal of the freely flowing liquid, the cells immobilized on the adsorbed beads are allowed to grow into microcolonies, utilizing the medium retained within the supporting hydrogel matrix. These colonies are subsequently equilibrated with chromogenic or fluorogenic substrate and screening is affected under a stereomicroscope, resulting in readily retrieved of the most active colonies. This technique may be particularly useful when the screened mutants are expressed and displayed on the cell surface, providing an active and homogeneous "naturally immobilized" enzyme population with minimal substrate diffusion limitations.

Fulford MR, Walker JT, Martin MV, Marsh PD (2004) Total viable counts, ATP, and endotoxin levels as potential markers of microbial contamination of dental unit water systems. Br Dent J 196 :157-159 ; discussion 153


OBJECTIVES : To determine if either ATP or endotoxin concentrations in water supplied by dental unit water systems (DUWS) correlated with total viable counts (TVC), and therefore could be used as a rapid, chairside measure of levels of microbial contamination. DESIGN : A prospective trial. METHOD : Fifty-seven water samples were taken from the ’triple spray’, air rotor and source water supplies from 25 dental units in eight practices. The samples were assayed for endotoxin concentration, total ATP and TVC. A pilot study was performed to assess the relationship between TVC and total cell counts, as determined by flow cytometry. RESULTS : ATP concentrations ranged from 22 to 958 relative light units (RLU) and free endotoxin ranged from 25 to 600 EU ml(-1). TVC varied from not detected to 2.16 x 10(4) CFU ml(-1). The ATP method proved to be a simple and rapid method that could be used at the chairside. However, there was no correlation between ATP or endotoxin concentrations and TVC in DUWS. TVC generally underestimated the total cell count by 50 to 500 fold. CONCLUSION : Half of the water samples from DUWS exceeded recommended levels of TVC. However, ATP and endotoxin concentrations in DUWS water samples did not correlate with these TVC data and therefore could not be recommended as an alternative assay to TVC for measuring bacterial contamination or for monitoring water treatment efficacy.

Fuller ME, Mailloux BJ, Streger SH, Hall JA, Zhang P, Kovacik WP, Vainberg S, Johnson WP, Onstott TC, DeFlaun MF (2004) Application of a vital fluorescent staining method for simultaneous, near-real-time concentration monitoring of two bacterial strains in an Atlantic coastal plain aquifer in Oyster, Virginia. Appl Environ Microbiol 70 :1680-1687


Two differentially labeled bacterial strains were monitored in near-real time during two field-scale bacterial transport experiments in a shallow aquifer in July 2000 and July 2001. Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107 were grown and labeled with the vital fluorescent stain TAMRA/SE (5 [and -6]-carboxytetramethylrhodamine, succinimidyl ester) or CFDA/SE (5 [and -6]-carboxyfluorescein diacetate, succinimidyl ester). Fluorescently labeled cells and a conservative bromide tracer were introduced into a suboxic superficial aquifer, followed by groundwater collection from down-gradient multilevel samplers. Cells were enumerated in the field by microplate spectrofluorometry, with confirmatory analyses for selected samples done in the laboratory by epifluorescence microscopy, flow cytometry, and ferrographic capture. There was general agreement in the results from all of the vital-stain-based enumeration methods, with differences ranging from <10% up to 40% for the analysis of identical samples between different tracking methods. Field analysis by microplate spectrofluorometry was robust and efficient, allowing thousands of samples to be analyzed in quadruplicate for both of the injected strains. The near-real-time data acquisition allowed adjustments to the predetermined sampling schedule to be made. The microplate spectrofluorometry data sets for the July 2000 and July 2001 experiments allowed the transport of the injected cells to be related to the site hydrogeology and injection conditions and enabled the assessment of differences in the transport of the two strains. This near-real-time method should prove effective for a number of microbial ecology applications.

Garcia-Hermoso D, Dromer F, Janbon G (2004) Cryptococcus neoformans capsule structure evolution in vitro and during murine infection. Infect Immun 72 :3359-3365


Cryptococcus neoformans capsule structure modifications after prolonged in vitro growth or in vivo passaging have been reported previously. However, nothing is known about the dynamics of these modifications or about their environmental specificities. In this study, capsule structure modifications after mouse passaging and prolonged in vitro culturing were analyzed by flow cytometry using the glucuronoxylomannan-specific monoclonal antibody E1. The capsule structures of strains recovered after 0, 1, 8, and 35 days were compared by using the level of E1-specific epitope expression and its cell-to-cell heterogeneity within a given cell population. In vitro, according to these parameters, the diversity of the strains was higher on day 35 than it was initially, suggesting the absence of selection during in vitro culturing. In contrast, the diversity of the strains recovered from the brain tended to decrease over time, suggesting that selection of more adapted strains had occurred. The strains recovered on day 35 from the spleen and the lungs had different phenotypes than the strains isolated from the brain of the same mouse on the same day, thus strongly suggesting that there is organ specificity for C. neoformans strain selection. Fingerprinting of the strains recovered in vitro and in vivo over time confirmed that genotypes evolved very differently in vitro and in vivo, depending on the environment. Overall, our results suggest that organ-specific selection can occur during cryptococcosis.

Gaur S, Trayner E, Aish L, Weinstein R (2004) Bronchus-associated lymphoid tissue lymphoma arising in a patient with bronchiectasis and chronic Mycobacterium avium infection. Am J Hematol 77 :22-25


We describe a 67-year-old woman with bronchiectasis and Mycobacterium avium complex infection who underwent wedge resection of her pulmonary infiltrates because they were progressing despite antibiotic therapy. In addition to the expected granulomatous changes, she was found to have a B-cell lymphoma of bronchus associated lymphoid tissue (BALT). Despite normal bone marrow morphology, marrow involvement was demonstrated by flow cytometry. Her lymphoma remains suppressed with antimycobacterial therapy 6 months after resection of bulk disease.

Goodrum F, Jordan CT, Terhune SS, High K, Shenk T (2004) Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations. Blood 104 :687-695


The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.

Halmos B, Anastopoulos HT, Schnipper LE, Ballesteros E (2004) Extreme lymphoplasmacytosis and hepatic failure associated with sulfasalazine hypersensitivity reaction and a concurrent EBV infection—case report and review of the literature. Ann Hematol 83 :242-246


We present an unusual case of a patient with extreme lymphoplasmacytosis and hepatic failure in association with a reaction to sulfasalazine and a concurrent Epstein-Barr virus (EBV) infection. Sulfa drugs can cause a wide range of allergic and hypersensitivity reactions and occasionally can lead to a fulminant illness. In the case under discussion the patient had hepatotoxicity, skin rash, fever, and peripheral blood atypical lymphocytosis. Initial impressions suggested the possibility of a malignant lymphoproliferative disorder. Flow cytometry of peripheral blood and a bone marrow biopsy provided clear evidence for a reactive, polyclonal process as opposed to a malignant disorder. Cessation of the offending drug and administration of steroids led to dramatic improvement. This case illustrates that drug hypersensitivity reactions can be manifested by an extreme lymphocytoid leukemoid reaction.

Heinemann L, Dillon S, Crawford A, Backstrom BT, Hibma MH (2004) Flow cytometric quantitation of the protective efficacy of dendritic cell based vaccines in a human papillomavirus type 16 murine challenge model. J Virol Methods 117 :9-18


A murine model for the assessment of protective immunity to human papillomavirus (HPV) type 16, a virus that does not naturally infect mice, is described. In this system, protection was tested following intranasal challenge of mice with a recombinant vaccinia virus expressing both the selected HPV antigen and a beta-galactosidase (beta-gal) reporter. The extent of viral infectivity was determined by measuring beta-gal positive lung cells using flow cytometry. The efficacy of this model to measure protective immunity was evaluated by priming mice with the beta-gal vaccinia virus then challenging the mice with the same virus. Vaccinia primed mice had negligible numbers of beta-gal positive cells in the lung 5 days following viral challenge indicating protection, whereas around 50% of cells were infected in immunologically naive, challenged mice. The protective efficacy of two dendritic cell vaccines for HPV16 was measured in this model. Both vaccines provided some protection to subsequent viral challenge, compared with their controls. Although this protection model was applied to HPV in this study, it may also have broad application to other viruses that do not infect mice naturally.

Hiasa Y, Takahashi H, Shimizu M, Nuriya H, Tsukiyama-Kohara K, Tanaka T, Horiike N, Onji M, Kohara M (2004) Major histocompatibility complex class-I presentation impaired in transgenic mice expressing hepatitis C virus structural proteins during dendritic cell maturation. J Med Virol 74 :253-261


Hepatitis C virus (HCV) infection is often persistent, but its mechanism and pathogenesis remain unclear. One mechanism through which HCV escapes systemic immunosurveillance might be via impaired dendritic cells (DCs), which are the most potent type of antigen-presenting cells. We examined whether HCV causes immunosuppression in DCs during maturation. We isolated immature DCs from the bone marrow of two founder lineages of transgenic mice harboring HCV cDNA expressing HCV structural proteins (nucleotides 294-3435), and studied how DC function is modified by HCV expression. Our data showed that the capacity of DCs expressing HCV structural proteins to stimulate T-cells was significantly impaired. Moreover, the surface expression of major histocompatibility complex (MHC) class-I molecules was significantly impaired on infected DC, especially with respect to H-2D. The transportation of H-2D to the cell surface during DC maturation was inhibited by HCV expression. However, the total amount of H-2D molecules produced by DC expressing HCV was not impaired. These results indicated that the immune response of DC infected with HCV is diminished and might be associated with the mechanism of persistent HCV infection.

Holm C, Mathiasen T, Jespersen L (2004) A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk. J Appl Microbiol 97 :935-941


AIMS : The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. METHODS AND RESULTS : A total of 75 Danish bulk tank milk samples exceeding the grading level of 3.0 x 10(4) CFU ml(-1) were examined by both flow cytometry and traditional microbiological analyses. The correlation coefficient (r) between the two methods was 0.71. For the differential analyses of the dominant bacterial populations four different parameters were used to give a species-characteristic pattern. The four parameters were as follows : staining with Oregon Green conjugated wheat germ agglutinin that binds to the cell wall of bacteria, staining with hexidium iodide that binds to all bacterial DNA, the flow cytometric forward scatter and the flow cytometric side scatter. Three regions in the flow cytometric plot were defined : region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine samples was examined. Comparing these results with results obtained by traditional microbiological analyses for identification showed that for 81% of the samples the two techniques agreed on the main cause of an elevated bacterial count. CONCLUSIONS : The ability of the presented flow cytometric technique to enumerate and differentiate bacteria in bulk tank milk according to the main cause of elevated counts was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY : This study described the first step in development of a technique suitable for routine analyses of bulk tank milk samples. A technique indicating the main cause of an elevated count will enable the farmer to eliminate the contamination source within a short time limit.

Hornbaek T, Nielsen AK, Dynesen J, Jakobsen M (2004) The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain. FEMS Microbiol Lett 236 :145-151


Shorter lag phases were obtained in cultivations of Bacillus licheniformis using early-compared to late-stationary growth phase inocula and using liquid versus solid propagation medium. Flow cytometry and fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), confirmed that liquid early-stationary growth phase inoculum had a higher vitality and was more homogeneous than solid late-stationary growth phase inoculum. DNA-microarray analyses indicated that liquid early-stationary growth phase inoculum was in a more active state in terms of cell multiplication whereas solid late-stationary growth phase inoculum was induced to some spore formation potentially causing delayed growth initiation.

Hou LH, Du Y, Zhang XP, An XP, Chen W (2004) [Expression of prostate stem cell antigen (PSCA) and selection of its specific binding peptide]. Sheng Wu Gong Cheng Xue Bao 20 :694-698


Prostate stem cell antigen (PSCA), a homologue of the Ly-6/Thy-1 family of cell surface antigen, is expressed by a majority of human prostate cancers and is a promising target for prostate cancer immunotherapy. To obtain the specific peptide binding with PSCA for targeted immunotherapy, PSCA gene was obtained by RT-PCR from human prostate cancer cell line DU145 and the transcated PSCA (tPSCA) gene was cloned into vector pQE30 for soluble expression in E. coli. The identity of recombinant tPSCA was confirmed through ELISA and western blot by use of anti-PSCA monoclonal antibody. Then the 12-peptide phage display library was screened with the purified tPSCA protein for its specific binding peptide through 3 rounds panning. For identifying the peptide’s specificity, the peptide was coupled with EGFP (enhanced green fluorecent protein) by recombinant DNA technology and the recombinant coupled protein was termed 11-EGFP. The binding specificity with tPSCA of 11-EGFP was further confirmed by ELISA and competitive inhibition experiment. Flow cytometry demonstrated its binding specificity with cell line DU145. In conclusion, a 12-amino-acid peptide which could bind with PSCA specifically was found and it may be a potential tool for targeted immunotherapy of prostate carcinoma.

Ivanov V, Tay JH, Tay ST, Jiang HL (2004) Removal of micro-particles by microbial granules used for aerobic wastewater treatment. Water Sci Technol 50 :147-154


Microbial granules with a diameter from 0.4 mm to 3.0 mm have been produced by fast sedimentation and retention of microbial aggregates in sequencing batch airlift reactors used for model wastewater treatment. The wastewater was with or without addition of calcium salt. The granules were able not only to degrade organic matter but to remove nano- and micro-particles from wastewater due to microchannels and pores in the matrix of the granules. To detect the removal of 0.1 microm, 0.6 pm, 4.2 microm fluorescent microspheres, and cells of Escherichia coli, stained by permeable nucleic acid stain SYTO9, the granules were incubated with these particles. The rate of particle removal and their accumulation in the granules was measured by a Fluoview300 confocal laser scanning microscope (CLSM) (Olympus, Japan) ; a FACSCalibur flow cytometer (Becton Dickinson, CA, USA), and a fluorescence spectrometer LS-50B (Perkin-Elmer, UK). The release or removal of biological and non-biological particles was analyzed by a flow cytometer after DNA staining. Total number of the particles bigger than 0.1 microm in the reactors was approximately 4 x 10(7) per ml, and 23% of these particles were bacterial cells. The 0.1 microm and 4.2. microm microbeads were accumulated within 250 microm in the upper layer of the microbial granule but externally added cells of Escherichia coli penetrated to the depth of approximately 800 microm in the granules without calcium addition. Microbial granules contained also attached ciliates but accumulation of the particles in protozoan cells was smaller than in the granule matrix. Kinetics of particle sorption was revealed by flow cytometry and fluorescence spectrometry. Almost half of the stained cells of E. coli can be removed by the granules for one hour. The ability of the microbial granules to remove the particles can enhance their function in aerobic treatment of wastewater.

Ivanov VN, Wang JY, Stabnikova OV, Tay ST, Tay JH (2004) Microbiological monitoring in the biodegradation of sewage sludge and food waste. J Appl Microbiol 96 :641-647


AIM : To study the microbiology of intensive, in-vessel biodegradation of a mixture of sewage sludge and vegetable food waste. METHODS AND RESULTS : The biodegradation was performed in a closed reactor with the addition of a starter culture of Bacillus thermoamylovorans SW25 under conditions of controlled aeration, stirring, pH and temperature (60 degrees C). The content of viable bacterial cells, determined by flow cytometry, increased from 5 x 108 g-1 of dry matter to 61 x 108 g-1 for 6 days of the process and then dropped to the initial value at the end of the process. The reductions of organic matter, 16S rRNA of methanogens and coenzyme F420 fluorescence during 10 days of the treatment were 67, 54 and 87% of the initial values, respectively. The biodegradability of the organic matter decreased during the 10 days of the treatment from 3.8 to 1.3 mg CO2 g-1 of organic matter per day. The treatment of sewage sludge and food waste at 60 degrees C did not remove enterobacteria, which are the agents of intestinal infections, from the material. The percentage of viable enterobacterial cells, determined by fluorescent in situ hybridization (FISH) with Enterobacteriaceae-specific oligonucleotide probe and flow cytometry, varied from 1 to 14% of the viable bacterial cells. CONCLUSIONS : The mixture of sewage sludge and food waste can be degraded by the aerobic thermophilic bacteria ; the starter culture of Bacillus thermoamylovorans SW25 can be used to perform this process ; and enterobacteria can survive under treatment of sewage sludge and food waste at 60 degrees C for 13 days. SIGNIFICANCE AND IMPACT OF THE STUDY : The results show that FISH with an oligonucleotide probe can be used to study not only the growth but also the degradation of biomass. Obtained results could be used to design the bioconversion of sewage sludge and food waste into organic fertilizer.

Joachimsthal EL, Ivanov V, Tay ST, Tay JH (2004) Bacteriological examination of ballast water in Singapore Harbour by flow cytometry with FISH. Mar Pollut Bull 49 :334-343


In this study the concentrations of total bacteria, enterobacteria, Vibrio spp., and E. coli have been compared for ballast water samples taken from ships in Singapore Harbour. The cell concentrations were enumerated using FISH and flow cytometry. The data were highly variable, reflecting the many influences upon ballast water as it is utilized in the shipping industry. The concentration of bacterial species was determined as a proportion of the total concentration of cells for the ballast water sampled. For the ballast water sampled these concentrations were 0.67-39.55% for eubacteria, 0-2.46% for enterobacteria, 0.18-35.82% for Vibrio spp., and 0-2.46% for E. coli. Using FISH and flow cytometry, an informative determination of the bacterial hazards of ship ballast water can be made.

Jomantaite I, Dikopoulos N, Kroger A, Leithauser F, Hauser H, Schirmbeck R, Reimann J (2004) Hepatic dendritic cell subsets in the mouse. Eur J Immunol 34 :355-365


The CD11c(+) cell population in the non-parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c(+) DC population from immunocompetent or immunodeficient [recombinase-activating gene-1 (RAG1)(-/-)] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220(+) CD11c(int) subset of ’plasmacytoid’ DC, and a B220(-) CD11c(+) DC subset. The latter DC population could be subdivided into a major, immature (CD40(lo) CD80(lo) CD86(lo) MHC class II(lo)) CD11c(int) subset, and a minor, mature (CD40(hi) CD80(hi) CD86(hi) MHC class II(hi)) CD11c(hi) subset. Stimulated B220(+) but not B220(-) DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220(-) DC three- to fourfold within 18 h post-injection, and up-regulated their surface expression of activation marker, while it contracted the B220(+) DC population. Early in virus infection, the hepatic B220(+) DC subset expanded, and both, the B220(+) as well as B220(-) DC populations in the liver matured. In vitro, B220(-) but not B220(+) DC primed CD4(+) or CD8(+)T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220(+) and B220(-) subsets in CD11c(+) DC populations freshly isolated from the mouse liver.

Kacmar J, Zamamiri A, Carlson R, Abu-Absi NR, Srienc F (2004) Single-cell variability in growing Saccharomyces cerevisiae cell populations measured with automated flow cytometry. J Biotechnol 109 :239-254


Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences. However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured. In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system. To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared. The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp. The other two plasmids are 2 microm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter. The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics. The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible "fingerprint" of the culture dynamics. The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability. Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability.

Kadono T, Kawano T, Hosoya H, Kosaka T (2004) Flow cytometric studies of the host-regulated cell cycle in algae symbiotic with green paramecium. Protoplasma 223 :133-141


Paramecium bursaria (green paramecium) possesses endosymbiotically growing chlorella-like green algae. An aposymbiotic cell line of P. bursaria (MBw-1) was prepared from the green MB-1 strain with the herbicide paraquat. The SA-2 clone of symbiotic algae was employed to reinfect MBw-1 cells and thus a regreened cell line (MBr-1) was obtained. The regreened paramecia were used to study the impact of the host’s growth status on the life cycle of the symbiotic algae. Firstly, the relationship between the timing of algal propagation and the host cell division was investigated by counting the algal cells in single host cells during and after the host cell division and also in the stationary phase. Secondly, the changes in the endogenous chlorophyll level, DNA content, and cell size in the symbiotic algae were monitored by flow cytometry and fluorescence microscopy. The number of algae was shown to be doubled prior to or during the host cell division and the algal population in the two daughter cells is maintained at constant level until the host cell cycle reenters the cytokinesis, suggesting that algal propagation and cell cycle are dependent on the host’s cell cycle. During the host’s stationary growth, unicellular algal vegetatives with low chlorophyll content were dominant. In contrast, complexes of algal cells called sporangia (containing 1-4 autospores) were present in the logarithmically growing hosts, indicating that algal cell division leading to the formation of sporangia with multiple autospores is active in the dividing paramecia.

Kampen AH, Tollersrud T, Lund A (2004) Flow cytometric measurement of neutrophil respiratory burst in whole bovine blood using live Staphylococcus aureus. J Immunol Methods 289 :47-55


A rapid and simple method for measurement of respiratory burst in neutrophil granulocytes in whole bovine blood is described. The respiratory burst was stimulated by live Staphylococcus aureus, and the production of reactive oxygen species quantified by the conversion of intracellular dihydrorhodamine 123 to the green fluorescent rhodamine 123, measured by flow cytometry. Assay conditions, including bacterial and dihydrorhodamine 123 concentrations and incubation time, were determined. Repeatability and precision of the method were assessed by testing parallel samples from clinically healthy dairy cows. In vitro and in vivo inhibition of respiratory burst was investigated, and labelling with a granulocyte marker antibody was performed. Stimulation with live S. aureus induced green fluorescence in the neutrophil granulocytes in a whole blood preparation. The fluorescence intensity increased with increasing bacterial concentration and increasing incubation time. Agreement analysis showed that the method gave repeatable results, and the intra-assay variability of the method was relatively low. The method is considered a useful technique for measurement of neutrophil respiratory burst in whole bovine blood.

Kespichayawattana W, Intachote P, Utaisincharoen P, Sirisinha S (2004) Virulent Burkholderia pseudomallei is more efficient than avirulent Burkholderia thailandensis in invasion of and adherence to cultured human epithelial cells. Microb Pathog 36 :287-292


Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular gram-negative bacillus that is closely related to its avirulent counterpart, Burkholderia thailandensis. However, pathogenic mechanisms and virulence factors of B. pseudomallei remain elusive. In the present study, we compared the invasiveness, adherence, and replication of B. pseudomallei and B. thailandensis in human respiratory epithelial cells A549. Invasion was determined after 4 h of coculturing using antibiotic protection assay. Adherence was demonstrated by coculturing the cells with fluorescein-labeled bacteria for 1 h and the number of positive cells was analyzed by flow cytometry. The results obtained with this in vitro study demonstrated that compared with its avirulent counterpart, B. pseudomallei is significantly more efficient (P<0.01) in invasion, adherence and inducing cellular damage, as represented by plaque formation.

Kim JS, Choi SE, Yun IH, Kim JY, Ahn C, Kim SJ, Ha J, Hwang ES, Cha CY, Miyagawa S, Park CG (2004) Human cytomegalovirus UL18 alleviated human NK-mediated swine endothelial cell lysis. Biochem Biophys Res Commun 315 :144-150


Human cytomegalovirus UL18, a MHC class I homologue, is known to serve as a natural killer cell (NK) decoy and to ligate NK inhibitory receptors to prevent lysis of an infected target cell. To explore whether the cell surface expression of UL18 represents a potential immune suppressive approach to evade NK-mediated cytotoxicity in the prevention of xenograft rejection, we examined the effect of the UL18 expression in vitro upon human NK-mediated cytotoxicity against swine endothelial cells (SECs). UL18 expression on SECs by a retroviral vector (PLNCX2) significantly suppressed NK-mediated SEC lysis by approximately 25-100%. The protective effect of UL18 could be mediated through ILT-2 inhibitory receptor on NKs. Additionally, the interaction between UL18 and NKs resulted in the significant reduction of IFN-gamma production. This study demonstrates that UL18 can serve as an effective tool for the evasion of NK-mediated cytotoxicity and for the inhibition of IFN-gamma production during xenograft rejection.

Kim M, Mackenzie JM, Westaway EG (2004) Comparisons of physical separation methods of Kunjin virus-induced membranes. J Virol Methods 120 :179-187


The two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting.

Klasen M, Wabl M (2004) Silent point mutation in DsRed resulting in enhanced relative fluorescence intensity. Biotechniques 36 :236-238


Kong XL, Wang QW, Chen ML, Cai Y, He ZX, Yang M (2004) [Human cytomegalovirus aggravates apoptosis of human megakaryocytes via direct infection in vitro]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 12 :70-73


The megakaryocyte and platelet lineage may be one of the major sites of human cytomegalovirus (HCMV) infection. However, whether HCMV aggravates apoptosis in normal megakaryocytes was not well investigated. Megakaryocytic cell line CHRF-288-11 and HCMV AD 169 strain were co-cultured in this study. PCR was used to detect the direct infection of the cells by HCMV IEA expression. The apoptotic cells were analyzed by morphologic observation, DNA ladder formation, annexin V/PI and PI assay with flow cytometry. The results showed that HCMV significantly inhibited the growth of CHRF cells in three different concentrations of viral infection groups (10(-3), 10(-2), 10(-1)). The viability levels in each infection groups were 77%, 73% and 68% respectively after incubation for 7 days, compared with 98% in the control group. Using annexin V/PI with flow cytometry, it was shown that the percentages of apoptotic cells viral infection in groups (10(-3), 10(-2), 10(-1)) were (21.3 +/- 2.49)%, (25.8 +/- 3.65)% and (31.4 +/- 3.91)% at 7 days after infection, while the control was (3.68 +/- 1.47)%. The apoptotic cells were further confirmed by morphologic observation and DNA ladder formation. Furthermore, PCR detection also showed the direct infection by identification of HCMV IEA expression in CHRF cells. This study suggested that HCMV could directly infect megakaryocytes and aggravated apoptosis in HCMV-infected megakaryocytes.

Koo JT, Choe J, Moseley SL (2004) HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli. Mol Microbiol 52 :1813-1826


Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.

Krut O, Sommer H, Kronke M (2004) Antibiotic-induced persistence of cytotoxic Staphylococcus aureus in non-phagocytic cells. J Antimicrob Chemother 53 :167-173


OBJECTIVES : After infection of non-phagocytic cells, some Staphylococcus aureus strains are able to survive and kill their host cells. The purpose of this study was to determine the action of various antibiotics on the survival of host cells and/or intracellular S. aureus. METHODS : Murine keratinocyte (PAM212) and fibroblast (mKSA) cell lines were infected with cytotoxic S. aureus and cultured in the presence of various antibiotics at graded concentrations. The viability of host cells was measured 24 h after infection. To determine the bacterial viability within host cells, cellular lysates were prepared and colony forming units were quantified using a spiral plater. Host cells infected with fluorescein isothiocyanate (FITC)-labelled S. aureus were analysed by flow cytometry and microscopy to determine the subcellular localization S. aureus. RESULTS : Oxacillin, vancomycin, gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole did not rescue host cells from cell death induced by intracellular S. aureus. In contrast, linezolid, rifampicin, azithromycin, clindamycin, erythromycin and quinupristin/dalfopristin suppressed the cytotoxic action of S. aureus. After withdrawal of antibiotics, intracellular S. aureus regained cytotoxic activity and killed their host cells. Only rifampicin was able to eliminate intracellular S. aureus completely within 72 h. In contrast, clindamycin, azithromycin and linezolid induced a state of intracellular persistence of viable S. aureus. CONCLUSIONS : Antibiotics commonly used for the management of S. aureus infections appear to create a niche for invasive intracellular S. aureus, which may play an important role for persistence and recurrence of infection. Because of its unique ability to eliminate intracellular S. aureus, rifampicin appears to be valuable for the treatment of invasive S. aureus infections.

Lai DZ, Weng SJ, Qi LQ, Yu CM, Fu L, Yu T, Chen W (2004) [Construction of two robust CHO cell lines resistant to apoptosis and adapted to protein-free medium by over-expression of Igf-1/bcl-2 or bcl-2/cyclin E genes]. Sheng Wu Gong Cheng Xue Bao 20 :66-72


Serum used widely in mammalian cell culture is also a potential source of bacterial, mycoplasmal and viral contaminations. In addition, the complex biological components in serum make harder the subsequent product recovery process. High cost, high batch variation and potential source limitation are among the other shortcomings. So serum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO-dhfr- cells were genetically engineered to make them adapted to IMEM, a protein-free medium, and resistant to apoptosis. The genes in choice are insulin-like factor (Igf-1), Bcl-2 and cyclin E. Bcl-2 is a mitochondrial membrane-integrated protein. It can block the release of cytochrome c by maintaining the integrity of mitochondrial membrane, and thus inhibit apoptosis. Igf-1 is similar both in structure and function to insulin, a growth factor added to serum-free medium to promote cell growth and is the only protein component in many currently used serum-free media. cyclin E is a cell cycle protein expressed continuously in G1 phase. When cyclin E accumulates to certain amount, cell cycle was driven to S phase. So cyclin E is a proliferation-promoting protein. By co-express Igf-1/Bcl-2 or Bcl-2/ cyclin E in CHO-dhfr- cells with a dicistronic expression vector, we constructed two cell lines : CHO-IB and CHO-BC. The high expression of each protein was confirmed by Western blot and flow cytometry. Apoptosis was analyzed by flow cytometry and DNA ladder detection, and the two cell lines were both found much more resistant to apoptosis induced by withdrawal of serum or addition of actinomycin D than the CHO-dhfr- parent cell. Cell proliferation assay by MTT method showed that the two cell lines proliferated much faster than CHO-dhfr- in IMDM medium without serum. Continuously culture assay proved that the two cell lines grow very well in IMEM protein-free medium supplemented with fibronectin and vitronectin to ease adherence. When compared to CHO-dhfr-, the two cell lines exhibited much more viable cell numbers and faster growth rate.

Lavrentyev PJ, McCarthy MJ, Klarer DM, Jochem F, Gardner WS (2004) Estuarine microbial food web patterns in a Lake Erie coastal wetland. Microb Ecol 48 :567-577


Composition and distribution of planktonic protists were examined relative to microbial food web dynamics (growth, grazing, and nitrogen cycling rates) at the Old Woman Creek (OWC) National Estuarine Research Reserve during an episodic storm event in July 2003. More than 150 protistan taxa were identified based on morphology. Species richness and microbial biomass measured via microscopy and flow cytometry increased along a stream-lake (Lake Erie) transect and peaked at the confluence. Water column ammonium (NH4+) uptake (0.06 to 1.82 microM N h(-1)) and regeneration (0.04 to 0.55 microM N h(-1)) rates, measured using 15NH4+ isotope dilution, followed the same pattern. Large light/dark NH4+ uptake differences were observed in the hypereutrophic OWC interior, but not at the phosphorus-limited Lake Erie site, reflecting the microbial community structural shift from net autotrophic to net heterotrophic. Despite this shift, microbial grazers (mostly choreotrich ciliates, taxon-specific growth rates up to 2.9 d(-1)) controlled nanophytoplankton and bacteria at all sites by consuming 76 to 110% and 56 to 97% of their daily production, respectively, in dilution experiments. Overall, distribution patterns and dynamics of microbial communities in OWC resemble those in marine estuaries, where plankton productivity increases along the river-sea gradient and reaches its maximum at the confluence.

Li B, New JY, Tay YK, Goh E, Yap EH, Chan SH, Hu HZ (2004) Delaying acute graft-versus-host disease in mouse bone marrow transplantation by treating donor cells with antibodies directed at l-selectin and alpha4-integrin prior to infusion. Scand J Immunol 59 :464-468


Acute graft-versus-host disease (GVHD) is still a major hurdle for successful bone marrow transplantation (BMT). Although many immunosuppressive drugs are available, none of them alone or in combination are able to completely abolish acute GVHD. The lifelong immunosuppression profoundly reduces the quality of life of BMT recipients. Therefore, new therapeutic approaches are needed. We previously reported that, in an acute GVHD model using SCID mice as recipient, incubating donor spleen cells with antibodies directed at CD49d and CD62L could significantly delay the occurrence of acute GVHD. To test the potential usefulness of this treatment in BMT, we examined this therapeutic protocol in a mouse BMT model. The present mouse BMT study confirmed our previous results that incubation of donor cells with antibodies directed at CD49d and CD62L prior to infusion into the recipient can effectively delay acute GVHD, allowing the recipients to recover from the side effects of total body irradiation. This one-time treatment is easy and simple and may be modified for clinical usage.

Li H, Chunsong H, Guobin C, Qiuping Z, Qun L, Xiaolian Z, Baojun H, Linjie Z, Junyan L, Mingshen J, Jinquan T (2004) Highly up-regulated CXCR3 expression on eosinophils in mice infected with Schistosoma japonicum. Immunology 111 :107-117


CXCR3, predominately expressed on memory/activated T cells, is a receptor for both interferon-gamma inducible protein-10/CXC ligand 10 (CXCL10) and monokine induced by interferon-gamma/CXCL9. We reported here that CXCR3 was highly up-regulated on infiltrating eosinophils in Schistosoma japonicum egg-induced granuloma in the mouse liver. It was also highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum. The phenomena were demonstrated at protein and mRNA levels using immunohisto- and immunocytochemistry evaluation of biopsy, flow cytometry and real-time quantitative reverse transcriptase-polymerase chain reaction technique, and verified by Northern blotting and chemotaxis assay in vitro. We also found that CCR3 expression on the infiltrating and peritoneal exudate cells was significantly decreased, CXCR4 expression was unchanged during the 42-day period of infection. We screened mRNA expression levels of the all known chemokine receptors in purified peritoneal exudate eosinophils and liver granuloma dominated by eosinophils. CXCR3 was highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum, meanwhile CCR3 was significantly and functionally down-regulated in these cells. The findings could lead to a better understanding of the chemokine receptor expression pattern of eosinophils at inflamed tissue sites caused by parasites. These could be also crucial for establishing a therapeutic strategy for eosinophilic inflammation via intervention in chemokine actions.

Lin HJ, Xue J, Bai Y, Wang JD, Zhang YL, Zhou DY (2004) Pathogenicty and immune prophylaxis of cag pathogenicity island gene knockout homogenic mutants. World J Gastroenterol 10 :3289-3291


AIM : To clarify the role of cag pathogenicity island (cagPAI) of Helicobacter pylori (H pylori ) in the pathogenicity and immune prophylaxis of H pylori infection. METHODS : Three pairs of H pylori including 3 strains of cagPAI positive wildtype bacteria and their cagPAI knockout homogenic mutants were utilized. H pylori binding to the gastric epithelial cells was analyzed by flow cytometry assays. Apoptosis of gastric epithelial cells induced by H pylori was determined by ELISA assay. Prophylaxis effect of the wildtype and mutant strains was compared by immunization with the sonicate of the bacteria into mice model. RESULTS : No difference was found in the apoptasis between cagPAI positive and knockout H pylori strains in respective of the ability in the binding to gastric epithelial cells as well as the induction of apoptosis. Both types of the bacteria were able to protect the mice from the infection of H pylori after immunization, with no difference between them regarding to the protection rate as well as the stimulation of the proliferation of splenocytes of the mice. CONCLUSION : The role of cagPAI in the pathogenicity and prophylaxis of H pylori infection remains to be cleared.

Lin JC, Chang FY, Fung CP, Xu JZ, Cheng HP, Wang JJ, Huang LY, Siu LK (2004) High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess. Microbes Infect 6 :1191-1198


To better understand the role of capsular polysaccharide (CPS) K1 or K2 in Klebsiella pneumoniae liver abscess as well as the development of metastasis to eye, neutrophil phagocytosis of 70 CPS isolates including K1 (n = 23)/K2 (n = 10), non-K1/K2 (n = 37) was evaluated by flow cytometry, fluorescence imaging, and electron microscopy. K1/K2 isolates were significantly more resistant to phagocytosis (P < 0.0001) than non-K1/K2 isolates and displayed increased resistance to intracellular killing. Although mucoid phenotype (M-type) K1/K2 isolates were significantly more resistant to phagocytosis (P = 0.0029) than M-type non-K1/K2, no significant difference in the phagocytosis rate was observed between K1/K2 isolates with M-type and non-M-type (P = 0.0924). Mucoidy is an associated factor that was predominant in K1/K2 isolates, but which itself is not an independent influence on phagocytic resistance. The K1/K2 CPS proved significantly more resistant to phagocytosis than non-K1/K2 CPS in liver abscess isolates (P < 0.0001) and non-abscess isolates (P = 0.0001), suggesting that K1/K2 isolates were generally more virulent in both liver abscess and in non-liver abscess conditions. These findings indicate that resistance of CPS K1 or K2 K. pneumoniae to phagocytosis and intracellular killing presumably contributes to their high prevalence in liver abscess and uniquely in endophthalmitis.

Lipoglavsek L, Avgustin G (2004) Obstacles to flow cytometric analysis of rumen microbial samples. Folia Microbiol (Praha) 49 :183-186


Several methods were tested that would improve the fluorescence signal from hybridized rumen bacterial cells. Disruption of cell envelopes by lysozyme, EDTA, proteinase K and/or SDS caused only a minor increase in fluorescence signal. Use of helper unlabeled oligonucleotide probes was successful only with the Puni[H672] probe which, however, when used with specific PBBl4-labeled probe, gave fluorescence signal drop. No substantial rise in fluorescence signal was also observed with cells subjected to growth-without-cell-division treatment. Further improvements are needed to make the fluorescent in situ hybridization (FISH)-flow cytometry combination applicable to rumen bacteria.

Liu J, Olsson G, Mattiasson B (2004) Short-term BOD (BODst) as a parameter for on-line monitoring of biological treatment process. Part I. A novel design of BOD biosensor for easy renewal of bio-receptor. Biosens Bioelectron 20 :562-570


A novel design of a biochemical oxygen demand (BOD) biosensor has been developed for on-line monitoring of easily biodegradable organic compounds in aqueous samples. The biological recognition element of the sensor could be easily renewed by injecting new bacterial paste without disassembling the sensor system. The sensor measurements were carried out in the initial-rate mode using a flow injection (FI) system, resulting in 60 s for one sample analysis followed by a recovery time less than 10 min. The sensor performance achieved showed a wide detection linearity over the range of 5-700 mg BOD5.l(-1) and a generally good agreement between the BOD values estimated by the biosensor and the conventional 5-day test. Furthermore, the precision test was in the control range (i.e. repeatability < or = /+/-7.5%/, reproducibility < or = /+/-7.3%/). The sensor could be used over 1 week in continuous test, however, the best performance was found within the first 24 h where standard deviation of the sensor response was +/-2.4%. The design of the sensor allows easy and fast renewal of the cells used as sensing elements. Replacement of biological recognition element and calibration of the sensor responses can be performed in a rather simple procedure on a daily regular basis. By using a mixed culture as the bio-receptor, one gets a sensor that reacts to a wide range of substrates. The new sensor construction will thus allow fast and convenient replacement of the bio-receptor and on-line assay of a broad range of substrates. This makes the sensor being an interesting and promising candidate for on-line monitoring of biological treatment process.

Liu J, Olsson G, Mattiasson B (2004) Short-term BOD (BODst) as a parameter for on-line monitoring of biological treatment process ; Part II : instrumentation of integrated flow injection analysis (FIA) system for BODst estimation. Biosens Bioelectron 20 :571-578


An instrument with integrated flow injection analysis (FIA) system has been developed for on-line monitoring a process for conversion of biomass under field condition. The instrument consists of a newly designed biosensor for easy renewal of the bio-receptor without disassembling the sensor, a FIA controller for controlling the analysis operations, and a computer-based data acquisition system for data recording and processing. The instrument performed a sequence operations automatically including preparation of sample in the desired concentration, sample loading, sample injection, signal recording, data processing, and self-cleaning of the system. This makes the instrument being an interesting and promising device for on-line process monitoring.

Lloyd D, Harris JC, Biagini GA, Hughes MR, Maroulis S, Bernard C, Wadley RB, Edwards MR (2004) The plasma membrane of microaerophilic protists : oxidative and nitrosative stress. Microbiology 150 :1183-1190


The trans-plasma-membrane electrochemical potential of microaerophilic protists was monitored by the use of voltage-sensitive charged lipophilic fluorophores ; of the many available probes, the anionic oxonol dye bis(1,3-dibarbituric acid)-trimethine oxonol [DiBAC(4)(3)] is an example of one which has been successfully employed using fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. Several microaerophilic protists have been investigated with this dye ; these were Giardia intestinalis, Trichomonas vaginalis, Tritrichomonas foetus, Hexamita inflata and Mastigamoeba punctachora. Under conditions where they exhibit normal vitality, these organisms exclude DiBAC(4)(3) by virtue of their maintenance of a plasma-membrane potential (negative inside). Uptake of the fluorophore is indicative of disturbance to this membrane (i.e. by inhibition of pump/leak balance, blockage of channels or generation of ionic leaks), and is indicative of metabolic perturbation or environmental stress. Here, it is shown that oxidative or nitrosative stress depolarizes the plasma membranes of the aforementioned O(2)-sensitive organisms and allows DiBAC(4)(3) influx. Oxonol uptake thereby provides a sensitive and early indication of plasma-membrane perturbation by agents that may lead to cytotoxicity and eventually to cell death by necrotic or apoptotic pathways.

Mailaender C, Reiling N, Engelhardt H, Bossmann S, Ehlers S, Niederweis M (2004) The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis. Microbiology 150 :853-864


Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of Mycobacterium smegmatis was expressed in Mycobacterium tuberculosis and Mycobacterium bovis. Approximately 20 and 35 MspA molecules per microm(2) cell wall were observed in M. tuberculosis and M. bovis BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in M. tuberculosis was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of M. bovis BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of M. bovis BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both M. bovis BCG and M. tuberculosis to zwitterionic beta-lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, M. tuberculosis became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of mspA. These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of M. tuberculosis.

Maraha N, Backman A, Jansson JK (2004) Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry. FEMS Microbiol Ecol 51 :123-132


Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied on seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and in pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured on agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil.

Meberg BM, Paulson AL, Priyadarshini R, Young KD (2004) Endopeptidase penicillin-binding proteins 4 and 7 play auxiliary roles in determining uniform morphology of Escherichia coli. J Bacteriol 186 :8326-8336


The low-molecular-weight (LMW) penicillin-binding protein, PBP 5, plays a dominant role in determining the uniform cell shape of Escherichia coli. However, the physiological functions of six other LMW PBPs are unknown, even though the existence and enzymatic activities of four of these were established three decades ago. By applying fluorescence-activated cell sorting (FACS) to quantify the cellular dimensions of multiple PBP mutants, we found that the endopeptidases PBP 4 and PBP 7 also influence cell shape in concert with PBP 5. This is the first reported biological function for these two proteins. In addition, the combined loss of three DD-carboxypeptidases, PBPs 5 and 6 and DacD, also impaired cell shape. In contrast to previous reports based on visual inspection alone, FACS analysis revealed aberrant morphology in a mutant lacking only PBP 5, a phenotype not shared by any other strain lacking a single LMW PBP. PBP 5 removes the terminal D-alanine from pentapeptide side chains of muropeptide subunits, and pentapeptides act as donors for cross-linking adjacent side chains. As endopeptidases, PBPs 4 and 7 cleave cross-links in the cell wall. Therefore, overall cell shape may be determined by the existence or location of a specific type of peptide cross-link, with PBP 5 activity influencing how many cross-links are made and PBPs 4 and 7 acting as editing enzymes to remove inappropriate cross-links.

Melsheimer P, Vinokurova S, Wentzensen N, Bastert G, von Knebel Doeberitz M (2004) DNA aneuploidy and integration of human papillomavirus type 16 e6/e7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri. Clin Cancer Res 10 :3059-3063


PURPOSE : Increasingly deregulated expression of the E6-E7 oncogenes of high-risk human papillomaviruses (HR-HPVs) has been identified as the major transforming factor in the pathogenesis of cervical dysplasia and derived cancers. The expression of these genes in epithelial stem cells first results in chromosomal instability and induces chromosomal aneuploidy. It is speculated that this subsequently favors integration of HR-HPV genomes into cellular chromosomes. This in turn leads to expression of viral cellular fusion transcripts and further enhanced expression of the E6-E7 oncoproteins. Chromosomal instability and aneuploidization thus seems to precede and favor integration of HR-HPV genomes. EXPERIMENTAL DESIGN : To prove this sequential concept, we analyzed here the sequence of events of DNA aneuploidization and integration in a series of HPV-16-positive cervical dysplastic lesions and carcinomas. Eighty-five punch biopsies of HPV-16-positive cervical lesions (20 CIN1/2, 50 CIN3, and 15 CxCa) were analyzed for DNA ploidy by DNA flow cytometry and for integration of HPV E6/E7 oncogenes using the amplification of papillomavirus oncogene transcripts assay, a reverse transcription-PCR method to detect integrate-derived human papillomavirus oncogene transcripts. RESULTS : DNA aneuploidy and viral genome integration were both associated with increasing dysplasia (P < 0.001, chi(2) test for trend). In addition, DNA aneuploidy was associated with increased viral integration (P < 0.01, Fisher’s exact test). Nineteen of 20 (95%) lesions with integrated viral genomes had aneuploid cell lines ; however, only 19 of 32 (59%) lesions with aneuploid cell lines had integrated viral genomes. CONCLUSIONS : These data support the hypothesis that aneuploidization precedes integration of HR-HPV genomes in the progression of cervical dysplasia. Accordingly, deregulated viral oncogene expression appears to result first in chromosomal instability and aneuploidization and is subsequently followed by integration of HR-HPV genomes in the affected cell clones.

Milner RJ, Horton JH, Crawford PC, O’Kelley J, Nguyen A (2004) Suppurative rhinitis associated with Haemophilus species infection in a cat. J S Afr Vet Assoc 75 :103-107


A young cat with signs of chronic rhinitis was evaluated for underlying anatomical, inflammatory, or infectious disease. Initial diagnostics were significant for the isolation of an unusual pathogen, Haemophilus species. Isolation using a human RapID NH system erroneously identified the isolate as H. segnis, a human pathogen. No database of veterinary pathogens (Haemophilus) are included in the system and animal pathogens will either be erroneously identified or yield a unique biocode not listed. Because of the unique nature of the pathogen we explored the possibility of immunosuppression as a contributory factor to infection. A variety of laboratory tests were employed to evaluate immune function. The clinical indications and utility of immune function testing are discussed. No immune dysfunction was identified.

Moayeri M, Ramezani A, Morgan RA, Hawley TS, Hawley RG (2004) Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells. Mol Ther 10 :892-902


Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation. Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen. An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment. Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks). Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified. Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A.

Moragues M, Comas-Riu J, Vives-Rego J (2004) Rapid G+ count and subpopulation assessment of the intestinal bacteria in Apodemus sylvaticus and Mus musculus by flow cytometry. Folia Microbiol (Praha) 49 :587-590


We report a novel application of calcein-acetomethyl ester in flow cytometry for rapid estimation of the number of G+-bacteria in rodent feces (Apodemus sylvaticus and Mus sp.f. muridae). We also use the combined application of flow cytometry and Syto-13 or Sypro Orange staining to count rapidly the total bacterial population and to describe bacterial subpopulations in the intestine.

Morgan CA, Bigeni P, Herman N, Gauci M, White PA, Vesey G (2004) Production of precise microbiology standards using flow cytometry and freeze drying. Cytometry A 62 :162-168


BACKGROUND : Quality control standards provide a quantity of microorganisms for routine use in microbiology to demonstrate the efficacy of testing methods and culture media. Standards are normally prepared by diluting a culture of microorganisms to obtain a suspension that contains an estimated number of colony-forming units per milliliter. The variability and inaccuracy of these standards increase the potential for false results. Flow cytometry has been used extensively to prepare precise standards of Cryptosporidium and Giardia that contain exact numbers of organisms in a volume of liquid (1). However, the same levels of accuracy have yet to be obtained for bacterial quality control standards. METHODS : A modification of a Becton Dickinson FACScalibur flow cytometer enabled 30 bacterial cells to be sorted into a single droplet, mixed with a cryoprotective solution within the droplet, and frozen in liquid nitrogen. The frozen droplets were then freeze dried for stable preservation of the viable bacterial cells. RESULTS : A freeze-dried sphere 3 mm in diameter was produced, which contained 30 microorganisms. The within-batch variation for these freeze-dried spheres was no greater than two standard deviations, and the between-batch variation was less than one standard deviation. CONCLUSIONS : Bacterial reference controls can now be produced with consistent accuracy and unparalleled precision, thus enabling harmonization across the microbiological testing industry.

Nasirudeen AM, Tan KS (2004) Isolation and characterization of the mitochondrion-like organelle from Blastocystis hominis. J Microbiol Methods 58 :101-109


Blastocystis hominis in an unusual protozoan parasite of the human intestinal tract. Previous studies have described the presence of mitochondrial-like structures despite the anaerobic nature of the organism. In this study, we describe a simple and rapid technique to isolate and characterize mitochondrion-like organelles (MLO) from B. hominis. The parasite was disrupted using glass beads and the MLO were collected and purified using a sucrose gradient. Negative staining and transmission electron microscopy of the isolated organelles showed mitochondrial-like structures. B. hominis cells were stained with rhodamine 123 and MitoLight to show the presence of transmembrane potential of the MLO. DAPI staining of the cells suggested the presence of DNA in the MLO. Though brief reports have been made in literature, this study is the first to describe a technique for the isolation of the MLO from this organism. Using this technique of isolation, major metabolic functions of the organelle, their associated macromolecules and intra-mitochondrial location can be extensively studied. The role of MLO in this anaerobic protozoan can be widely investigated using this protocol.

Nielsen JL, Schramm A, Bernhard AE, van den Engh GJ, Stahl DA (2004) Flow cytometry-assisted cloning of specific sequence motifs from complex 16S rRNA gene libraries. Appl Environ Microbiol 70 :7550-7554


A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.

Nitsche A, Fleischmann J, Klima KM, Radonic A, Thulke S, Siegert W (2004) Inhibition of cord blood cell expansion by human herpesvirus 6 in vitro. Stem Cells Dev 13 :197-203


To elucidate the role of human herpesvirus 6 (HHV-6) in hematopoiesis, the influence of HHV-6A and HHV-6B on the in vitro expansion and differentiation of cord blood (CB) progenitor cells was investigated in liquid culture. Nonfractionated CB mononuclear cells (CB-MNC) or MACS-enriched CD34+ CB cells were seeded in liquid culture under conditions allowing maximal expansion of nucleated cells. Cells were either incubated with HHV-6A- or HHV-6B-containing cell culture supernatants or a virus-free control. After 7, 14, and 21 days, cells were analyzed for growth by cell count, for differentiation by flow cytometry, and for HHV-6 infection by antigen detection or PCR. Expansion of CB-MNC was significantly inhibited by HHV-6A and HHV-6B for a period of 3 weeks, including reduced proportions of CD34+ and CD33+ cells in HHV-6-treated cultures on day 7. In contrast, when starting with enriched CD34+ cells, the expansion was only affected by HHV-6A. Inhibition of CD34 and CD33 cell development was less pronounced in these cultures compared to CB-MNC cultures. However, HHV-6 antigen and DNA was detectable in these cultures. In conclusion, although HHV-6A inhibited expansion of CD34 progenitor cells, HHV-6B inhibited growth of immature CB cells only in interaction with nonfractionated CB-MNC.

Nordstrom T, Blom AM, Forsgren A, Riesbeck K (2004) The emerging pathogen Moraxella catarrhalis interacts with complement inhibitor C4b binding protein through ubiquitous surface proteins A1 and A2. J Immunol 173 :4598-4606


Moraxella catarrhalis ubiquitous surface protein A2 (UspA2) mediates resistance to the bactericidal activity of normal human serum. In this study, an interaction between the complement fluid phase regulator of the classical pathway, C4b binding protein (C4BP), and M. catarrhalis mutants lacking UspA1 and/or UspA2 was analyzed by flow cytometry and a RIA. Two clinical isolates of M. catarrhalis expressed UspA2 at a higher density than UspA1. The UspA1 mutants showed a decreased C4BP binding (37.6% reduction), whereas the UspA2-deficient Moraxella mutants displayed a strongly reduced (94.6%) C4BP binding compared with the wild type. In addition, experiments with recombinantly expressed UspA1(50-770) and UspA2(30-539) showed that C4BP (range, 1-1000 nM) bound to the two proteins in a dose-dependent manner. The equilibrium constants (K(D)) for the UspA1(50-770) and UspA2(30-539) interactions with a single subunit of C4BP were 13 microM and 1.1 microM, respectively. The main isoform of C4BP contains seven identical alpha-chains and one beta-chain linked together with disulfide bridges, and the alpha-chains contain eight complement control protein (CCP) modules. The UspA1 and A2 bound to the alpha-chain of C4BP, and experiments with C4BP lacking CCP2, CCP5, or CCP7 showed that these three CCPs were important for the Usp binding. Importantly, C4BP bound to the surface of M. catarrhalis retained its cofactor activity as determined by analysis of C4b degradation. Taken together, M. catarrhalis interferes with the classical complement activation pathway by binding C4BP to UspA1 and UspA2.

Ogino M, Yoshimatsu K, Ebihara H, Araki K, Lee BH, Okumura M, Arikawa J (2004) Cell fusion activities of Hantaan virus envelope glycoproteins. J Virol 78 :10776-10782


Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.

O’Sullivan A, Chang HC, Yu Q, Kaplan MH (2004) STAT4 is required for interleukin-12-induced chromatin remodeling of the CD25 locus. J Biol Chem 279 :7339-7345


Signal transducer and activator of transcription 4 (STAT4) is a critical mediator of interleukin-12 (IL-12)-stimulated inflammatory immune responses. Despite extensive analysis of the immune responses of STAT4-deficient mice, there is still very little understood about STAT4-dependent gene induction. IL-12 stimulated increases in IL-2 receptor alpha chain gene (CD25) mRNA levels and surface expression require STAT4. In this report, we utilize chromatin immunoprecipitation assays to analyze IL-12-stimulated and STAT4-dependent changes in chromatin remodeling of the CD25 gene. Gene activation requires binding of STAT4 to the PRRIII upstream regulatory element, the recruitment of the CREB-binding protein (CBP), and chromatin remodeling including increased acetylation and decreased methylation of histones within the CD25 promoter. Evidence suggests that STAT4 also facilitates binding of other factors to the CD25 promoter including c-Jun. Thus, these results provide a model for STAT4-dependent gene induction and a mechanism for cytokine-induced expression of the CD25 gene.

Ottiger C, Regeniter A, Kochli HP, Huber AR (2004) [Standardized counting of particles in the urine : a comparison between flow cytometry, cell chamber counting and traditional sediment analysis]. Praxis (Bern 1994) 93 :15-21


We compared the number of particles in the urine with flow cytometry and manual methods : cell chamber and standardised sediment analysis. The correlation (r) between the KOVA-cell chamber and the flow cytometer UF-100 was 0.966 for erythrocytes, 0.935 for leukocytes and 0.902 for squamous epithelial cells. Similar results were obtained by a standardised preparation of the sediment. Today, the estimation of the cell number in the sediment analysis is still common. The KOVA cell chamber system is a cheap alternative for microscopy, whereas automation with flow cytometry is only used for large laboratories. Reference values were established under optimal conditions (erythrocytes < 14/microliter, leukocytes < 16/microliter) with a cut-off of 20/microliter.

Palkova Z, Vachova L, Valer M, Preckel T (2004) Single-cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure-driven chip-based system. Cytometry A 59 :246-253


BACKGROUND : Cytomics aims at understanding the function of cellular systems by analysis of single cells. Recently, there has been a growing interest in single cell measurements being performed in microfluidic systems. These systems promise to integrate staining, measurement, and analysis in a single system. One important aspect is the limitation of allowable cell sizes due to microfluidic channel dimensions. Here we want to demonstrate the broad applicability of microfluidic chip technology for the analysis of many different cell types. METHODS : We have developed a microfluidic chip and measurement system that allows flow cytometric analysis of fluorescently stained cells from different organisms. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by fluorescence detection. RESULTS : We have successfully applied the system to develop a methodology to detect viable and dead cells in yeast cell populations. Also, we have measured short interfering RNA (siRNA) mediated silencing of protein expression in mammalian cells. In addition, we have characterized the infection state of Magnaportae grisea fungal spores. CONCLUSIONS : Results obtained with the microfluidic system demonstrate a broad applicability of microfluidic flow cytometry to measurement of various cell types.

Park MT, Hwang SJ, Lee GM (2004) Flow cytometric application of helper adenovirus (HAd) containing GFP gene flanked by two parallel loxP sites to evaluation of 293 cre-complementing cell line and monitoring of HAd in Gutless Ad production. Biotechnol Prog 20 :913-920


Gutless adenoviruses (GAds), namely, all gene-deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end-point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.

Pearce HR, Kalia N, Bardhan KD, Atherton JC, Brown NJ (2004) Effects of Helicobacter pylori on endothelial cell proliferation and chemotaxis. Digestion 69 :201-210


BACKGROUND/AIM : Helicobacter pylori is associated with an increased risk of peptic ulcer disease development and recurrence. Ulcer healing is dependent upon angiogenesis, requiring endothelial cell (EC) proliferation and chemotaxis. This study determined whether extracts of H. pylori affected EC proliferation and/or chemotaxis in vitro. METHODS : The effects of water and broth extracts of three genotypically different strains of H. pylori and of single strains of Campylobacter jejuni and Escherichia coli on human dermal microvascular EC (HuDMEC) and human umbilical vein EC (HUVEC) were assessed. Tetrazolium dye (MTT) proliferation, dual staining cell viability, flow cytometry, and microchemotaxis assays were performed. RESULTS : H. pylori (all strains) and C. jejuni inhibited HuDMEC (p < 0.01) and HUVEC (p < 0.01) proliferation and decreased the proportion of HUVECs in the S phase of the cell cycle. E. coli had no effect on EC proliferation. The levels of vascular endothelial growth factor stimulated chemotaxis were significantly greater (p < 0.01) than the levels of basal migration for both control and extract-treated ECs. However, none of the bacterial extracts affected EC basal migration or chemotaxis. CONCLUSION : H. pylori extracts inhibit HuDMEC and HUVEC proliferation in vitro by a cytostatic, strain-independent mechanism. A similar antiproliferative effect of C. jejuni was observed. Our findings suggest that both H. pylori and C. jejuni have the ability to inhibit one of the key stages of angiogenesis which may have implications in peptic ulcer disease.

Penders J, Fiers T, Dhondt AM, Claeys G, Delanghe JR (2004) Automated flow cytometry analysis of peritoneal dialysis fluid. Nephrol Dial Transplant 19 :463-468


BACKGROUND : Recently, the Sysmex UF-100 flow cytometer has been developed to automate urinalysis. We have evaluated this instrument to explore the possibilities of flow cytometry in the analysis of peritoneal dialysis fluid (PD) and have compared the obtained data with those of counting chamber techniques, biochemical analysis and bacterial culture. METHODS : UF-100 data were correlated with microscopy and biochemical data in 135 PD samples. Microbiological analysis was performed in 63 suspected cases of peritonitis. RESULTS : Good agreement (P < 0.001) was obtained between UF-100 and microscopy data for leukocytes (r = 0.825). UF-100 bacterial count correlated (P < 0.001) with UF-100 leukocyte count (r = 0.549). UF-100 bacterial counts were unreliable in samples where interference by blood platelets was observed. Another major problem was the UF-100 ’bacterial’ background signal in sterile PD samples. Yeast cells were detected by the flow cytometer in spiked samples. CONCLUSIONS : Flow cytometry of PD with the UF-100 offers a rapid and reliable leukocyte count. Sensitivity of the ’bacterial’ channel count in predicting positive culture exceeds the sensitivity of conventional Gram stain. Furthermore, additional semi-quantitative information is provided regarding the presence of yeasts.

Perfetto SP, Ambrozak DR, Roederer M, Koup RA (2004) Viable infectious cell sorting in a BSL-3 facility. Methods Mol Biol 263 :419-424


With the increase in demand for high-speed cell sorting of viable infectious and now therapeutic cell samples, safety concerns for the protection of flow cytometer operators have increased. This chapter describes a quick, sensitive, and reproducible procedure to assure sample containment before sorting these samples. This procedure includes aerosol containment, physical barriers, environmental controls, and personal protection. An aerosol management system produces a negative pressure within the sort chamber where aerosols are vacuumed directly into a HEPA filter. Physical barriers include the manufacturer’s standard plastic shield and panels. The flow cytometer is contained in a BSL-3 laboratory for maximum environmental control and the operator is protected using a respiratory system. Containment is measured using highly fluorescent Glo-Germ particles under the same conditions as the cell sort but with the sorter adjusted to produce large amounts of aerosols. These aerosols are collected by a vacuum air sampling system for 10 min in three locations onto a glass slide and examined microscopically. With this system in place, aerosol containment can be measured quickly and efficiently, therefore reducing the risk to the operator when sorting viable infectious cells.

Perotti CG, Del Fante C, Viarengo G, Papa P, Rocchi L, Bergamaschi P, Bellotti L, Marchesi A, Salvaneschi L (2004) A new automated cell washer device for thawed cord blood units. Transfusion 44 :900-906


BACKGROUND : The current available techniques to wash out DMSO from thawed umbilical cord blood (UCB) units are based essentially on standard centrifugation in an open system with various degrees of cell loss. STUDY DESIGN AND METHODS : We evaluated the capacity of a new automated closed device (Cytomate, Baxter, IL) to wash out the DMSO from thawed UCB units, saving at the same time the progenitor and accessory cells in terms of CD34+ cells and MNCs. We modified the standard software of the device and calculated the cell recovery on 25 UCB units. Moreover, we set up a new gas chromatographic method to exactly detect the DMSO removal rate. RESULTS : To evaluate the efficiency of the Cytomate device, we considered the postthawing (prewashing) versus postwashing cell recovery. The average recovery (%) in terms of total nucleated cells was 63.30 (range, 40.12-89.00), CD34+ cells was 70.20 (range, 11.51-89.01), CD3+ cells was 61.01 (range, 28.80-87.08), CD4+ cells was 62.53 (range, 30.62-96.73), CD8+ cells was 57.4 (range, 26.87-94.72), CD19+ cells was 63.33 (range, 39.10-90.33), CD16+/56+ cells was 70.67 (range, 8.91-98.40), CFU-GM was 74.33 (range, 20.23-98.60), total CFUs was 82.34 (range, 14.83-247.12), and viability was 89.67(range, 70.74-98.30). The total working time required was, on average, 15 minutes (range, 7-20). CONCLUSIONS : The Cytomate device demonstrated a satisfying efficiency in cell recovery and in maintaining the clonogenic power of the UCB graft. The removal rate of DMSO was practically complete with evident advantages for the recipient. Finally, the entire manipulation performed in a closed system revealed to be safe, maintaining the sterility of the graft.

Petit L, Pierard- Franchimont C, Uhoda E, Vroome V, Cauwenbergh G, Pierard GE (2004) Coping with mild inflammatory catamenial acne : a clinical and bioinstrumental split-face assessment. Skin Res Technol 10 :278-282


BACKGROUND : Acne is a multifactorial disease exhibiting distinct clinical presentations. Among them, the catamenial type is a matter of concern for young women. Some oral contraceptives may help without, however, clearing the skin condition. AIM : The present open study aimed at evaluating the effect of overnight applications of a paste made of petrolatum,15% zinc oxide and 0.25% miconazole nitrate. METHOD : The split-face trial was conducted in 35 women. A non-medicated cream was used as control. Clinical evaluations and biometrological assessments on cyanoacrylate follicular biopsies were performed monthly for 3 months. Comedometry and the density in autofluorescent follicular casts were used as analytical parameters. In addition, the five most severe cases at inclusion were tested at the completion of the study for follicular bacterial viability using dual flow cytometry. RESULTS : Compared with baseline and to the control hemi-face, the medicated paste brought significant improvement of acne. The number of papules and their redness were reduced beginning with the first treatment phase. A reduction in the follicular fluorescence was yielded beginning with the second treatment phase. The ratios between injured and dead bacteria, on the one hand, and live bacteria, on the other hand were significantly increased at completion of the study. CONCLUSION : A miconazole paste applied for 1 week at the end of the ovarian cycle has a beneficial effect on catamenial acne.

Philippe J, De Logi E, Baele G (2004) Comparison of five different citrated tubes and their in vitro effects on platelet activation. Clin Chem 50 :656-658


Pina-Vaz C, Goncalves Rodrigues A, Pinto E, Costa-de-Oliveira S, Tavares C, Salgueiro L, Cavaleiro C, Goncalves MJ, Martinez-de-Oliveira J (2004) Antifungal activity of Thymus oils and their major compounds. J Eur Acad Dermatol Venereol 18 :73-78


The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungals have stimulated the search for therapeutic alternatives. Essential oils have been used empirically. The essential oils of Thymus (Thymus vulgaris, T. zygis subspecies zygis and T. mastichina subspecies mastichina) have often been used in folk medicine. The aim of the present study was to evaluate objectively the antifungal activity of Thymus oils according to classical bacteriological methodologies - determination of the minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) - as well as flow cytometric evaluation. The effect of essential oils upon germ tube formation, an important virulence factor, was also studied. The mechanism of action was studied by flow cytometry, after staining with propidium iodide. The chemical composition of the essential oils was investigated by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). The antifungal activity of the major components (carvacrol, thymol, p-cymene and 1,8-cineole) and also possible interactions between them were also investigated. The essential oils of T. vulgaris and T. zygis showed similar antifungal activity, which was greater than T. mastichina. MIC and MLC values were similar for all the compounds tested. At MIC values of the essential oils, propidium iodide rapidly penetrated the majority of the yeast cells, indicating that the fungicidal effect resulted primarily from an extensive lesion of the cell membrane. Concentrations below the MIC values significantly inhibited germ tube formation. This study describes the potent antifungal activity of the essential oils of Thymus on Candida spp., warranting future therapeutical trials on mucocutaneous candidosis.

Porter J (2004) Flow cytometry and environmental microbiology. Curr Protoc Cytom Chapter 11 :Unit 11 12


This survey unit discusses many of the issues involved for flow cytometry in the field of microbiology, particularly the preparative procedures, which are far more stringent than many other procedures using larger cells. For instance, it is often necessary to filter laboratory agents multiple times to obtain the true particle-free solutions needed for flow cytometry of microbes. It is difficult enough to recognize bacteria in cell extracts from soil, sediment, or sludge given the background of same-size particles. This unit provides an excellent overview of a potentially large application area in flow cytometry and is written by one of the most respected scientists in the field.

Prigione V, Lingua G, Marchisio VF (2004) Development and use of flow cytometry for detection of airborne fungi. Appl Environ Microbiol 70 :1360-1365


Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.

Qin ZL, Zhao P, Zhang XL, Yu JG, Cao MM, Zhao LJ, Luan J, Qi ZT (2004) Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells. Biochem Biophys Res Commun 324 :1186-1193


Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.

Ramirez-Arcos S, Greco V, Douglas H, Tessier D, Fan D, Szeto J, Wang J, Dillon JR (2004) Conserved glycines in the C terminus of MinC proteins are implicated in their functionality as cell division inhibitors. J Bacteriol 186 :2841-2855


Alignment of 36 MinC sequences revealed four completely conserved C-terminal glycines. As MinC inhibits cytokinesis in Neisseria gonorrhoeae and Escherichia coli, the functional importance of these glycines in N. gonorrhoeae MinC (MinC(Ng)) and E. coli MinC (MinC(Ec)) was investigated through amino acid substitution by using site-directed mutagenesis. Each mutant was evaluated for its ability to arrest cell division and to interact with itself and MinD. In contrast to overexpression of wild-type MinC, overexpression of mutant proteins in E. coli did not induce filamentation, indicating that they lost functionality. Yeast two-hybrid studies showed that MinC(Ec) interacts with itself and MinD(Ec) ; however, no interactions involving MinC(Ng) were detected. Therefore, a recombinant MinC protein, with the N terminus of MinC(Ec) and the C terminus of MinC(Ng), was designed to test for a MinC(Ng)-MinD(Ng) interaction. Each MinC mutant interacted with either MinC or MinD but not both, indicating the specificity of glycine residues for particular protein-protein interactions. Each glycine was mapped on the C-terminal surfaces (A, B, and C) of the solved Thermotoga maritima MinC structure. We found that MinC(Ec) G161, residing in close proximity to the A surface, is involved in homodimerization, which is essential for MinC function. Glycines corresponding to MinC(Ec) G135, G154, and G171, located within or adjacent to the B-C surface junction, are critical for MinC-MinD interactions. Circular dichroism revealed no gross structural perturbations of the mutant proteins, although the contribution of glycines to protein flexibility and stability cannot be discounted. Using molecular modeling, we propose that exposed conserved MinC glycines interact with exposed residues of the alpha-7 helix of MinD.

Ramon R, Feliu JX, Cos O, Montesinos JL, Berthet FX, Valero F (2004) Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris. Biotechnol Lett 26 :1447-1452


The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastoris based on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensor’s interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g x l(-1) and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.

Raoult D, Fournier PE, Drancourt M (2004) What does the future hold for clinical microbiology ? Nat Rev Microbiol 2 :151-159


In the past decade, clinical microbiology laboratories have undergone important changes with the introduction of molecular biology techniques and laboratory automation. In the future, there will be a need for more rapid diagnoses, increased standardization of testing and greater adaptability to cope with new threats from infectious microorganisms, such as agents of bioterrorism and emerging pathogens. The combination of the new tools that are now being developed in research laboratories, the general reorganization of clinical laboratories and improved communication between physicians and clinical microbiologists should lead to profound changes in the way that clinical microbiologists work.

Ren B, Szalai AJ, Hollingshead SK, Briles DE (2004) Effects of PspA and antibodies to PspA on activation and deposition of complement on the pneumococcal surface. Infect Immun 72 :114-122


Streptococcus pneumoniae infection is a frequent cause of pneumonia, otitis media, meningitis, and septicemia. Pneumococcal surface protein A (PspA) is an important virulence factor on the pathogen surface, and it is known to interfere with complement activation. In this study, flow cytometry was used to study the effects of PspA and antibodies to PspA on the deposition of complement C3 on the surface of a capsular type 3 strain, WU2, and its PspA- mutant, JY1119. Using naive mouse serum as a complement source, measurable deposition of C3 was observed within 4 min on PspA- pneumococci, and the amount of surface-bound C3 accumulated rapidly as the amount of serum was increased. In contrast, very little C3 was deposited on the PspA+ strain. In nonimmune mouse serum, the classical pathway was the dominant activation pathway triggered by PspA- pneumococci. Accordingly, EGTA blocked almost all of the complement activation. Moreover, a significant amount of C3 was still deposited on the PspA- strain when serum from factor B-deficient mice was used. This deposition was not observed on the PspA+ pneumococci, indicating that PspA may inhibit complement deposition via the classical pathway. Furthermore, under the conditions we tested, PspA also inhibited C3 deposition when the classical pathway was initiated by antibodies to capsular polysaccharide. Antibodies to PspA could overcome the anticomplementary effect of PspA, allowing for increased complement activation and C3 deposition onto PspA+ bacteria.

Robinson JP (2004) Overview of flow cytometry and microbiology. Curr Protoc Cytom Chapter 11 :Unit 11 11


Although in recent years flow cytometry has become commonplace in hematology and immunology laboratories, application of the technology to microbiology remains largely unrealized. This overview presents the historical background, discusses applications in various areas of the field, and speculates on the directions of future developments. The availability of high-quality methods should be a prime factor in convincing microbiologists that flow cytometry may have certain advantages over traditional methods and that it does indeed have much to contribute to microbiology.

Rocha A, Ruiz S, Tafalla C, Coll JM (2004) Conformation- and fusion-defective mutations in the hypothetical phospholipid-binding and fusion peptides of viral hemorrhagic septicemia salmonid rhabdovirus protein G. J Virol 78 :9115-9122


Fourteen single and two double point mutants in the highly conserved region (positions 56 to 159) of the G gene of viral hemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were selected and obtained in plasmids by site-directed mutagenesis. Fish cell monolayers transfected with the mutant plasmids were then assayed for protein G (pG) expression, conformation-dependent monoclonal antibody (MAb) reactivity, and cell-cell fusion. Some mutations located in the phospholipid-binding p2 peptide (positions 82 to 110 ; mutants P86A, A96E, G98A, and R107A) abolished both MAb recognition and fusion activity, while others (P79A, L85S, and R103A) abolished MAb recognition but retained fusion at similar or lower pHs compared to those for the wild type. Phospholipid-binding assays of p2-derived synthetic peptides suggested that phosphatidylserine binding was not affected by the mutations studied. On the other hand, three (P79A, L85S, and T135E) of the four mutants retaining fusion activity mapped around two locations showing amino acid variation in 22 VHSV isolates and in neutralizing MAb-resistant mutants described previously. Mutations located in the hypothetical fusion peptide (positions 142 to 159 ; mutants F147K, P148K, and W154K) abolished both MAb recognition and fusion activity. The existence of mutants with altered conformation and defective fusion in both p2 and fusion peptides provides further evidence in favor of the participation of these and adjacent regions in some of the steps of the VHSV fusion processes, as suggested by previous studies. In addition, because the studied region induced strong immunological responses in trout, some of the mutants described here might be used to design attenuated VHSV vaccines.

Rochet V, Rigottier-Gois L, Rabot S, Dore J (2004) Validation of fluorescent in situ hybridization combined with flow cytometry for assessing interindividual variation in the composition of human fecal microflora during long-term storage of samples. J Microbiol Methods 59 :263-270


This work was conducted to assess the accuracy of in situ hybridization to show differences in human microflora composition between volunteers and to optimize the storage of fecal samples to allow delayed analysis of gut microflora composition in humans. Fecal samples from 25 healthy subjects (14 women, 11 men aged 24-51) were collected. The samples were fixed in 4% Paraformaldehyde (PFA) solution at 4 degrees C overnight and stored at -70 degrees C. Twenty samples were analysed to quantify the variation due to interindividual differences in the composition of fecal microflora. The five remaining samples were stored either after PFA fixation or directly frozen at -70 degrees C and were monitored on a 12-month period. The fecal microflora was analysed by in situ hybridization combined with flow cytometry detection. Ribosomal RNA-targeted probes were used to assess the relative proportions of four phylogenetic groups : Clostridium coccoides-Eubacterium rectale (Erec 482), Bacteroides (Bac 303), Faecalibacterium prausnitzii (Fprau 645) and Bifidobacterium (Bif 164). Our results demonstrated that the method used is adapted to detect significant differences in fecal microflora composition in humans. Moreover, samples stored in PFA solution demonstrated a stable composition even after 8 months of storage. Conversely, frozen samples were less stable as the Bifidobacterium and C. coccoides-E. rectale groups showed significant differences after 2 months of storage. In conclusion, the fecal microflora composition can be analysed up to 8 months after 4% PFA fixation and storage at -70 degrees C. It represents an extended time compared with the 2-month period currently recommended. This will give more flexibility for applying this technology in epidemiological studies including a large number of samples.

Rohde KH, Dyer DW (2004) Analysis of haptoglobin and hemoglobin-haptoglobin interactions with the Neisseria meningitidis TonB-dependent receptor HpuAB by flow cytometry. Infect Immun 72 :2494-2506


Neisseria meningitidis expresses a two-component TonB-dependent receptor, HpuAB, which mediates heme-iron (Hm-Fe) acquisition from hemoglobin and hemoglobin-haptoglobin complexes. Due to genetic polymorphisms in the human haptoglobin gene, haptoglobin (and hemoglobin-haptoglobin) exists as three structurally distinct phenotypes. In this study, we examined the influence of the haptoglobin phenotype on the interactions of HpuAB with apo-haptoglobin and hemoglobin-haptoglobin. Growth assays confirmed that HpuAB utilizes hemoglobin-haptoglobin more efficiently than hemoglobin as an Fe source and revealed a preference for human-specific, polymeric 2-2 or 2-1 hemoglobin-haptoglobin complexes. We developed a flow cytometry-based assay to measure the binding kinetics of fluorescein-labeled ligands to HpuAB on live, intact meningococci. The binding affinity of HpuAB for hemoglobin-haptoglobin phenotypes correlated well with the ability of each ligand to support Neisseria meningitidis growth, with higher affinities exhibited for types 2-2 and 2-1 hemoglobin-haptoglobin. Saturable binding of Hb and apo-haptoglobin suggested that HpuAB-mediated utilization of hemoglobin-haptoglobin involves specific interactions with both components. In contrast to previous studies, we detected binding of HpuB expressed alone to hemoglobin, apo-haptoglobin, and hemoglobin-haptoglobin of all three phenotypes. However, in the absence of HpuA, the binding capacity and/or affinity of the receptor was reduced and the dissociation of hemoglobin was impaired. We did not detect binding of HpuA alone to hemoglobin, apo-haptoglobin, or hemoglobin-haptoglobin ; however, the lipoprotein is crucial for optimal recognition and use of ligands by the receptor. Finally, this study confirmed the integral role of TonB and the proton motive force in the binding and dissociation of Hb and hemoglobin-haptoglobin from HpuAB.

Rong J, Xu X, Ewen C, Bleackley RC, Kane KP (2004) Isolation and characterization of novel single-chain Fv specific for human granzyme B. Hybrid Hybridomics 23 :219-231


Granzyme B, a neutral serine protease, has been demonstrated to be a pivotal molecule for protective immunity against viral infection and cellular malignant transformation. To facilitate monitoring of granzyme B levels, we have recently applied phage display technology to produce single-chain Fv antibodies specific for granzyme B, as versatile alternatives and complementary reagents to currently available monoclonal antibodies. Through four rounds of panning on purified human granzyme B-coated on solid phase, three unique clones were isolated. Expressed soluble scFv antibodies demonstrated specific immunological applications including ELISA, Western blotting, immunoprecipitation and intracellular staining. Based on sequence analyses and structural modeling, one scFv, Fv17, may have overlapping antigen binding specificity with monoclonal antibodies 2C5/F5 and GB11. Owing to the availability of its DNA sequence and large scale production capability, Fv17 should be a superior reagent for monitoring granzyme B expression in natural killer cells and antigen specific CD8+ T cell immunity.

Rose JJ, Foley JF, Murphy PM, Venkatesan S (2004) On the mechanism and significance of ligand-induced internalization of human neutrophil chemokine receptors CXCR1 and CXCR2. J Biol Chem 279 :24372-24386


It is well established that leukocyte chemotactic receptors, a subset of G protein-coupled receptors, undergo endocytosis after stimulation by ligand. However, the significance of this phenomenon to cell motility and other important leukocyte functions induced by chemoattractants has not been clearly defined. Here we show that in primary human neutrophils, the threshold levels of agonist required for endocytosis of the chemotactic receptors CXCR1 and CXCR2 were approximately 10-fold or higher than those needed for maximal chemotactic and calcium flux responses. Moreover, when stimulated by agonists at concentrations that are high enough for chemotaxis but too low for receptor endocytosis, neutrophil CXCR1 and CXCR2 could be reactivated in response to repeated application of the same agonist. Both receptors were excluded from Triton X-100-insoluble lipid rafts, and at high agonist concentrations were rapidly endocytosed by a clathrin/rab5/dynamin-dependent pathway. These data support the conclusion that neutrophil migration in response to CXCR1 or CXCR2 agonists is not dependent on endocytosis of CXCR1 or CXCR2. Rather than being integral to the process of cell migration, receptor endocytosis may be a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest.

Roy S, Mir MA, Anand SP, Niederweis M, Ajitkumar P (2004) Identification and semi-quantitative analysis of Mycobacterium tuberculosis H37Rv ftsZ gene-specific promoter activity-containing regions. Res Microbiol 155 :817-826


The cytokinetic protein FtsZ plays a pivotal role in regulation of cell division in bacteria. Multiple promoters regulate transcription of the ftsZ gene in Escherichia coli, Streptomyces and Bacillus species. In order to identify promoter activity-containing regions of the ftsZ gene of Mycobacterium tuberculosis H37Rv (MtftsZ) in vivo, different regions upstream of MtftsZ, namely, the ftsQ-ftsZ intergenic region, the ftsQ open reading frame (ORF), and different regions of ftsQ ORF, were analyzed in a gfp reporter plasmid in Mycobacterium smegmatis mc(2)155 cells. Flow cytometric analysis of mid-logarithmic M. smegmatis mc(2)155 cells containing these transcription fusion constructs revealed GFP expression in the cells harboring the ftsQ-ftsZ intergenic region (172 bp), the entire ftsQ ORF (945 bp), and 5’ 467-bp and 3’ 217-bp regions of ftsQ ORF. RT-PCR analyses on RNA from M. smegmatis mc(2)155 cells, transformed with the entire ftsQ ORF-ftsQ-ftsZ intergenic region-containing construct, as well as on RNA from M. tuberculosis, confirmed that the regions identified indeed elicit promoter activity. Semi-quantitative RT-PCR analyses of gfp transcripts driven by cloned MtftsZ promoter regions in M. smegmatis cells showed threefold higher promoter activity from ftsQ ORF than from the ftsQ-ftsZ intergenic region. Expression from the individual 5’ and 3’ regions of ftsQ ORF was almost equivalent to that from the ftsQ-ftsZ intergenic region. RT-PCR analyses on RNA from M. tuberculosis quantitatively confirmed these promoter activities. Thus, at least three independent regions in the immediate upstream sequence of MtftsZ contain promoter activity, with the major contribution coming from ftsQ ORF.

Santos A, Marquina D (2004) Ion channel activity by Pichia membranifaciens killer toxin. Yeast 21 :151-162


The cytocidal effect of Pichia membranifaciens killer toxin on Candida boidinii cells was studied. The halotolerant yeast P. membranifaciens CYC 1106 produces a unique 18 kDa killer toxin that exerts its killer activity against C. boidinii IGC 3430 only in the presence of NaCl. Metabolic events associated with the loss of C. boidinii IGC 3430 viability were quantitatively identical to those known to occur with K1 killer toxin-treated sensitive strains of Saccharomyces cerevisiae. The death of sensitive cells was characterized by a leakage of potassium, an influx of sodium and a decrease in intracellular pH. These effects occurred prior to and concomitantly with cell death, indicating that they were primary effects of the action of the toxin. Here we report that this protein forms ion-permeable channels in liposome membranes. These channels are freely permeable to common physiological ions. We suggest that channel formation is the cytotoxic mechanism of action of P. membranifaciens killer toxin. The channels described here are sufficiently non-selective to mediate cell death through a discharge of cellular membrane potential and changes in ionic homeostasis. No specific effects against killer toxin-treated sensitive cells were observed when the cell cycle was analysed.

Schickli JH, Thackray LB, Sawicki SG, Holmes KV (2004) The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells. J Virol 78 :9073-9083


Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.

Schubert A, Zakikhany K, Pietrocola G, Meinke A, Speziale P, Eikmanns BJ, Reinscheid DJ (2004) The fibrinogen receptor FbsA promotes adherence of Streptococcus agalactiae to human epithelial cells. Infect Immun 72 :6197-6205


Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates. During the course of infection, S. agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood. The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells. Deletion of the fbsA gene in various S. agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes. The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin. Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells. Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells. Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S. agalactiae to epithelial cells.

Schwickerath O, Brouns G, Thrasher A, Kinnon C, Roes J, Casimir C (2004) Enhancer-deleted retroviral vectors restore high levels of superoxide generation in a mouse model of CGD. J Gene Med 6 :603-615


BACKGROUND : Retroviral vectors possess many advantages for use in gene therapy protocols, especially within the haematopoietic system. A number of attendant problems, however, still limit their safety in clinical application. The effects of the enhancer present in the retroviral long terminal repeat (LTR) are a major concern for the clinical usage of such vectors, as they can exert a powerful regulatory influence on the genes that surround them. METHODS : To improve the safety and widen the applicability of retroviral vectors for use in gene therapy we have developed an enhancer-deleted (Delta-LTR) retroviral vector that retained high titre and demonstrated transcriptional activity in myeloid cells. RESULTS : When used to correct a mouse model of autosomal recessive chronic granulomatous disease, the Delta-LTR vectors gave acceptable levels of gene transfer to mouse bone marrow cells. Evidence for a slight preferential expression in myeloid cells was obtained with all the vectors studied. Nitroblue tetrazolium assay of superoxide generation in mouse bone marrow derived haematopoietic colonies revealed that transduction with Delta-LTR vectors could restore functional NADPH oxidase to cells from these animals. Superoxide assay of peripheral blood confirmed that, although relatively low numbers of cells were transduced, the Delta-LTR vector was capable of reconstituting very high levels of oxidase activity, comparable to that obtained from normal cells. CONCLUSIONS : The Delta-LTR vector described here could provide the basis for a new generation of retroviral vectors with improved safety.

Sekar R, Fuchs BM, Amann R, Pernthaler J (2004) Flow sorting of marine bacterioplankton after fluorescence in situ hybridization. Appl Environ Microbiol 70 :6210-6219


We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

Siennicka J (2004) Detection of CMV infected cells by flow cytometry—evaluation of MAbs CCH2 and AAC10 directed against early and late CMV antigens. Pol J Microbiol 53 :127-129


In this work evaluation of usefulness of monoclonal antibodies (MAbs) CCH2 and AAC10 directed against early—pUL44(DB52) and late—ppUL83(pp65) CMV antigens, utilized in Department of Virology, NIH for routine diagnosis of CMV infection by shell vial and pp65 antigenemia assay, for determination of CMV antigens by flow cytometry in human leucocytes, isolated, infected and cultivated in vitro was presented.

Singh BN, Lucas JJ, Hayes GR, Kumar I, Beach DH, Frajblat M, Gilbert RO, Sommer U, Costello CE (2004) Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells. Infect Immun 72 :4151-4158


Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISA(PLUS) assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo.

Singh SP, McDonald D, Hope TJ, Prabhakar BS (2004) Upon thyrotropin binding the thyrotropin receptor is internalized and localized to endosome. Endocrinology 145 :1003-1010


To study the fate of TSH receptor (TSHR) on TSH binding, we constructed a chimeric cDNA that encodes a yellow fluorescent protein (YFP) fused to the carboxyl terminus of human TSHR. The protein expression in transfected cells was confirmed using flow cytometry. The functionality of the chimeric protein was determined by its ability to transduce signal leading to activation of cAMP in a TSH dose-dependent manner. The levels of cAMP produced by these cells were comparable with the levels seen in cells transfected with unfused TSHR without the YFP. Using deconvolution microscopy, we observed that the receptor is largely expressed on the cell surface, but on addition of TSH, some of the receptors were rapidly internalized. This conclusion was supported by several independent observations involving different cells expressing either native or recombinant TSHR. On TSH treatment, we observed internalization of human TSHR-YFP and human TSHR, expressed on 293 and CHO cells, respectively. This was further substantiated when we observed colocalization of rhodamine-labeled TSH with TSHR-YFP within the cell and by the uptake of radiolabeled TSH. Furthermore, shortly after ligand binding, there was a profound change in the morphology of the cells and some of the receptors accumulated in the perinuclear region of the cell. The TSHR-YFP was colocalized with RhoB-cyan fluorescent protein, indicating that it accumulated within the endosomes. These results indicate that the receptor internalization might in part be responsible for TSHR desensitization on TSH binding.

Srikumar R, Mikael LG, Pawelek PD, Khamessan A, Gibbs BF, Jacques M, Coulton JW (2004) Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae. Microbiology 150 :1723-1734


From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding ; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

Steinmetz J, Henny J, Gueguen R (2004) [Reference limits for urine sediments performed on Sysmex UF-50]. Ann Biol Clin (Paris) 62 :671-680


The Sysmex UF-50 is an automated flow cytometer for urine sediments analysis. Interpretation of the results needs establishment of reference limits for the different constituents measured. First of all, we checked precision of measurements and stability of urines during transportation. Then, urine samples from 4 to 95 year old subjects were examined with the UF-50, by visual microscopy and test strips. Distributions of results for erythrocytes, leukocytes, epithelial cells, casts, bacteria and conductivity were described in a sample of 680 subjects (364 men and 316 women), with creatininemia below 140 micromol/L, consuming no drugs and for women without intra uterine device and apart from menstruation period. Then, the results were compared with those obtained in groups selected on microscopic analysis and test strip results. UF-50 sensitivity and specificity were 77.5% and 88% for 15 erythrocytes/microL in reference to microscopic urinalysis, they were 91.3% and 87.3% for 15 leucocytes/microL. The reference sample was defined with negative microscopic results. The upper reference limits (centile 97.5) were 16 red blood cells/microL for men 14.5 for women, 13.5 and 33 leucocytes/microL, 8 and 19 epithelial cells/microL, 1,3 and 0,4 casts/microL, 5 500 et 7 700 bacteria/microL, 36,2 et 34,6 conductivity mS/cm. The Sysmex UF-50 is a suitable analyser for urinary sediments. Reference limits may be different from usual reference limits due to variability in performances of other methods.

Stevenson RA, Huang JA, Studdert MJ, Hartley CA (2004) Sialic acid acts as a receptor for equine rhinitis A virus binding and infection. J Gen Virol 85 :2535-2543


Equine rhinitis A virus (ERAV) is a member of the genus Aphthovirus, family Picornaviridae, and causes respiratory disease in horses worldwide. To characterize the putative receptor molecule(s) of the ERAV isolate 393/76 (ERAV.393/76) on the surface of Vero and other cells, an assay was developed to measure the binding of purified biotinylated ERAV.393/76 virions to cells by flow cytometry. Using this assay, the level of binding to different cell types correlated with the relative infectivity of ERAV in each cell type. In particular, equine fetal kidney cells, mouse fibroblast cells, rabbit kidney-13 and Crandell feline kidney cells bound virus at high levels and produced high virus yields (> or =10(7) TCID50 ml(-1)). Madin-Darby bovine kidney and baby hamster kidney cells showed little or no binding of virus, producing yields of < or =10(1.8) TCID50 ml(-1). Treatment of Vero and other cells with sodium periodate and the metabolic inhibitors tunicamycin, benzyl N-acetyl-alpha-D-galactosamide, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and proteases indicated that part of the receptor-binding and entry complex for ERAV.393/76 is on N-linked carbohydrates and that the carbohydrate is likely to be present on a protein rather than a lipid backbone. The effect of carbohydrate-specific lectins and neuraminidases on ERAV.393/76 binding and infection of Vero and other cell types implicated alpha2,3-linked sialic acid residues on the carbohydrate complex in the binding and infection of ERAV.

Sundstrom H, Wallberg F, Ledung E, Norrman B, Hewitt CJ, Enfors SO (2004) Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes. Biotechnol Lett 26 :1533-1539


In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml(-1)) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml(-1) dropped to approximately 10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml(-1) probably reflects a loss of cell division capability rather than cell death.

Szaniszlo P, Wang N, Sinha M, Reece LM, Van Hook JW, Luxon BA, Leary JF (2004) Getting the right cells to the array : Gene expression microarray analysis of cell mixtures and sorted cells. Cytometry A 59 :191-202


BACKGROUND : Most biological samples are cell mixtures. Some basic questions are still unanswered about analyzing these heterogeneous samples using gene expression microarray technology (MAT). How meaningful is a cell mixture’s overall gene expression profile (GEP) ? Is it necessary to purify the cells of interest before microarray analysis, and how much purity is needed ? How much does the purification itself distort the GEP, and how well can the GEP of a small cell subset be recovered ? METHODS : Model cell mixtures with different cell ratios were analyzed by both spotted and Affymetrix MAT. GEP distortion during cell purification and GEPs of purified cells were studied. CD34+ cord blood cells were purified and analyzed by MAT. RESULTS : GEPs for mixed cell populations were found to mirror the cell ratios in the mixture. Over 75% pure samples were indistinguishable from pure cells by their overall GEP. Cell purification preserved the GEP. The GEPs of small cell subsets could be accurately recovered by cell sorting both from model cell mixtures and from cord blood. CONCLUSIONS : Purification of small cell subsets from a mixture prior to MAT is necessary for meaningful results. Even completely hidden GEPs of small cell subpopulations can be recovered by cell sorting.

Tada Y, Mori T, Shinogi T, Yao N, Takahashi S, Betsuyaku S, Sakamoto M, Park P, Nakayashiki H, Tosa Y, Mayama S (2004) Nitric oxide and reactive oxygen species do not elicit hypersensitive cell death but induce apoptosis in the adjacent cells during the defense response of oat. Mol Plant Microbe Interact 17 :245-253


Nitric oxide (NO) acts as a signaling molecule in many cellular responses in plants and animals. Oat plants (Avena sativa L.) evoke the hypersensitive response (HR), which shares morphological and biochemical features with mammalian apoptosis, such as DNA laddering and heterochromatin condensation, in response to the avirulent crown rust fungus (Puccinia coronata f. sp. avenae). We examined the role of NO and reactive oxygen species (ROS) in the initiation of hypersensitive cell death, which is induced by direct contact with the pathogen, and apoptotic cell death in the adjacent cells. Cytofluorimetric analysis using the fluorescent NO probe DAF and the H2O2 probe DCF demonstrated that NO and H2O2 were generated simultaneously in primary leaves at an early stage of the defense response. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) markedly enhanced H2O2 accumulation detected by 3,3-diaminobenzidine staining and DCF, whereas treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) strongly suppressed it. Superoxide dismutase (SOD) increased NO accumulation, suggesting that endogenous NO may modulate the level of H2O2 by interacting with O2- in the HR lesion. Cytological observation showed that administration of cPTIO, SNAP, or SOD had no effect on elicitation of hypersensitive cell death, but clearly reduced heterochromatin condensation in the nearby cells and DNA laddering. These findings indicate that NO and ROS are not essential mediators for the initiation of hypersensitive cell death. However, NO and O2- but not H2O2 are required for the onset of apoptotic cell death in the adjacent cells, where excess NO may exert its anti-apoptotic function by regulating cellular redox state.

Taylor RM, Burritt JB, Baniulis D, Foubert TR, Lord CI, Dinauer MC, Parkos CA, Jesaitis AJ (2004) Site-specific inhibitors of NADPH oxidase activity and structural probes of flavocytochrome b : characterization of six monoclonal antibodies to the p22phox subunit. J Immunol 173 :7349-7357


The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22(phox) subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22(phox) by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22(phox)-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22(phox). Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22(phox).

Trentmann O, Khatri NK, Hoffmann F (2004) Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol Prog 20 :1766-1775


A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates ; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.

Troussellier M, Got P, Bouvy M, M’Boup M, Arfi R, Lebihan F, Monfort P, Corbin D, Bernard C (2004) Water quality and health status of the Senegal River estuary. Mar Pollut Bull 48 :852-862


The Senegal River estuary was sampled in May 2002 to get the first data on both the trophic and sanitary status of the water of the main river of the northwest African coast. Several physical, chemical and microbiological variables were measured twice along a transect. Inorganic nutrient concentrations were low while phytoplanktonic abundances (0.58-1.8 x 10(5) cells ml(-1)), bacterial abundances (0.27-8.1 x 10(7) cells ml(-1)), activity (22-474 pmol l(-1) h(-1)), were among the highest recorded in such ecosystems. Microbiological variables revealed a eutrophicated status for this estuary. Largest abundances of fecal contamination bacterial indicators were only detected in localized areas (Saint-Louis city and surrounding areas). The apparent good survival of fecal indicator bacteria in the estuarine waters despite a long residence time (4-5 days) has been evaluated by complementary survival experiments. Exposed to a salinity gradient, a local Escherichia coli strain showed a significantly better survival than those of an E. coli reference strain.

Tsukiyama-Kohara K, Tone S, Maruyama I, Inoue K, Katsume A, Nuriya H, Ohmori H, Ohkawa J, Taira K, Hoshikawa Y, Shibasaki F, Reth M, Minatogawa Y, Kohara M (2004) Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells. J Biol Chem 279 :14531-14541


Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.

Veach RA, Liu D, Yao S, Chen Y, Liu XY, Downs S, Hawiger J (2004) Receptor/transporter-independent targeting of functional peptides across the plasma membrane. J Biol Chem 279 :11425-11431


Targeting of peptides, proteins, and other functional cargo into living cells is contingent upon efficient transport across the plasma membrane barrier. We have harnessed the signal sequence hydrophobic region (SSHR) to deliver functional cargoes to cultured cells and to experimental animals. We now report evidence that two chirally distinct forms of SSHR composed of all l or all d amino acids showed similar membrane-translocating activity as assessed by confocal microscopy, flow cytometry, and direct fluorescence measurement. An attached nuclear localization sequence ferried by the SSHR enantiomers displayed similar intracellular function by inhibiting inducible nuclear import of transcription factor nuclear factor kappa B and suppressing nuclear factor kappa B-dependent gene expression of cytokines. A nuclear localization sequence comprised of a positively charged cluster of amino acids was rapidly translocated by SSHR enantiomers to the interior of unilamellar phospholipid vesicles. These findings indicate that the SSHR translocates functional peptides directly through the plasma membrane phospholipid bilayer without involving chirally specific receptor/transporter mechanisms. This mechanism of SSHR translocation is suitable for facile delivery of biologically active peptides for cell-based and animal-based functional proteomic studies.

Venkaiah B, Viswanathan P, Habib S, Hasnain SE (2004) An additional copy of the homologous region (hr1) sequence in the Autographa californica multinucleocapsid polyhedrosis virus genome promotes hyperexpression of foreign genes. Biochemistry 43 :8143-8151


The Autographa californica multinucleocapsid nuclear polyhedrosis virus genome contains nine homologous region (hr1, hr1a, hr2, hr2a, hr3, hr4a, hr4b, hr4c, and hr5) sequences that are thought to be involved in viral replication and activation of transcription. Our results show that the 750 bp hr1 sequence is capable of functioning as an enhancer of transcription of foreign genes from the homologous late polyhderin gene promoter and the heterologous Drosophila heat shock protein (hsp70) promoter in insect cells. Introduction of an additional copy of the complete hr1 element downstream to the polyhedrin locus in the viral genome, while not affecting the stability of the recombinant virus for at least 30 serial passages, led to hyperexpression of reporter genes. The enhancement in the expression levels of foreign genes varied from 40 to 90-fold depending on the promoter used.

Vielma SA, Mironova M, Ku JR, Lopes-Virella MF (2004) Oxidized LDL further enhances expression of adhesion molecules in Chlamydophila pneumoniae-infected endothelial cells. J Lipid Res 45 :873-880


Chlamydophila pneumoniae is a common respiratory pathogen that has been shown to be associated with coronary artery disease. Recent studies have shown that one of the possible mechanisms of the atherogenicity of C. pneumoniae is overexpression of cell adhesion molecules (CAMs) in infected endothelial cells. We investigated whether exposure of C. pneumoniae-infected endothelial cells to oxidized LDL (oxLDL) leads to further upregulation of CAMs. Flow cytometry and immunoblot analysis of human aortic endothelial cells (HAECs) was performed for intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. ICAM-1 was expressed in 78.7% of C. pneumoniae-infected HAECs. The addition of oxLDL (100 microg/ml) to infected HAECs increased the proportion of ICAM-1-positive cells to 92%. VCAM-1 was only observed in 9.3% of infected HAECs, and the addition of oxLDL had no further effect on the surface expression of VCAM-1. C. pneumoniae also upregulated the surface expression of E-selectin on 52.2% of the cells, and incubation with oxLDL further increased the proportion of positive cells to 63.64%. In conclusion, C. pneumoniae upregulated the expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin on HAECs. The addition of oxLDL to the infected cells further enhanced the surface expression of ICAM-1 and E-selectin.

Wang Y, Xie J, Yarber FA, Mazurek C, Trousdale MD, Medina-Kauwe LK, Kasahara N, Hamm-Alvarez SF (2004) Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland. Gene Ther 11 :970-981


Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in >80% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UV-inactivated Ad, carbachol-stimulated release of bulk protein and beta-hexosaminidase were significantly (P< or =0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.

Wong SK, Li W, Moore MJ, Choe H, Farzan M (2004) A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem 279 :3197-3201


The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC(50) of less than 10 nm, whereas the IC(50) of the S1 domain was approximately 50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.

Yang JW, Wang FI (2004) Impairment of oxidative burst in porcine neutrophils induced by pseudorabies virus. Vet Immunol Immunopathol 101 :123-130


Industrial swine production is affected by several serious viral diseases, such as pseudorabies, hog cholera, porcine reproductive and respiratory syndrome, which are frequently complicated with the increased incidence of bacterial complications such as Actinobacillus pleuropneumoniae (APP). This clinical observation is suggestive of a virus-bacteria synergism on the pathogenesis. One hypothesis is that viruses induce polymorphonuclear cell (PMNs, primarily neutrophils) dysfunction resulting in defective antibacterial resistance. The purpose of this study was to use the pseudorabies virus (PrV) as a model to explore the possibility of virus-induced PMN dysfunctions in pigs. The goals were to evaluate, in ex vivo settings, the oxidative burst (OB) function of pig PMNs, and to evaluate whether PrV could affect these responses to APP. We found that PrV served as a mild OB stimulant (2-fold) to pig PMNs, which also launched a significant burst to phorbol 12-myristate 13-diacetate (PMA ; 61-fold), to non-opsonized, heat-killed and formaldehyde-fixed APP (8-fold), and to normal pig serum-opsonized APP (34-fold). Interestingly, the PMA-induced OB could be reduced 50-70% by preincubating PMNs with PrV, and the critical target was not likely the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase itself. Inactivated PrV was as efficient as viable PrV at exerting the inhibitory effect. On the other hand, PrV exerted a primarily additive effect on APP-induced OB, when the cytotoxic effect of APP on PMNs was avoided. The current finding suggests the possibility that activated PMNs are susceptible to PrV-induced dysfunction, and that the PrV-APP synergism may require upstream stimuli of PMNs to be initiated.

Yuan JP, Li T, Chen HB, Li ZH, Yang GZ, Hu BY, Shi XD, Tong SQ, Li YX, Guo XK (2004) Analysis of gene expression profile in gastric cancer cells stimulated with Helicobacter pylori isogenic strains. J Med Microbiol 53 :965-974


To understand the biological processes within host cells induced by VacA, isogenic strains of Helicobacter pylori (NCTC 11638 or 11638-DeltavacA) were used to stimulate gastric cancer cells SGC7901, and differentially expressed genes in host cells were identified using cDNA microarray technology. More than 300 genes were found to alter their mRNA expression at different time points, among which 68 were related to the cytoskeleton, 87 were associated with cell cycle, cell death and proliferation, IL8 expression was also found to be up-regulated. Cells co-cultured with broth-culture supernatant (BCS) of NCTC 11638 showed more alteration in microtubule cytoskeleton morphology, as observed by laser scanning confocal microscopy, and a lower apoptosis rate, detected by flow cytometry, compared with those co-cultured with BCS of 11638-DeltavacA. The supernatants of cells co-cultured with NCTC 11638 showed significantly higher IL8 expression than those co-cultured with 11638-DeltavacA. It is concluded that VacA disrupts cytoskeletal architecture by influencing the expression of cytoskeleton-associated genes. VacA breaks the balance between cell proliferation and cell death by inducing the maladjustment of genes related to cell cycle. VacA is also able to induce the inflammatory response.

Zhang HK, Zhang X, Mao BZ, Li Q, He ZH (2004) Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice. Cell Res 14 :27-33


Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPH-oxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also provides a simple eliciting tool for studying apoptosis in the plant cell.

Zhao X, Oh SH, Cheng G, Green CB, Nuessen JA, Yeater K, Leng RP, Brown AJ, Hoyer LL (2004) ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin ; functional comparisons between Als3p and Als1p. Microbiology 150 :2415-2428


The ALS (agglutinin-like sequence) gene family of Candida albicans encodes eight cell-surface glycoproteins, some of which are involved in adherence to host surfaces. A mutational analysis of each ALS gene is currently being performed to deduce the functions of the encoded proteins and to better understand the role of these proteins in C. albicans biology and pathogenesis. This paper describes construction of an als3/als3 mutant and comparison of its phenotype to an als1/als1 strain. Efforts to disrupt ALS3 indicated that the gene could be deleted in two transformation steps, suggesting that the gene is encoded by a single locus and that the ALS3-like locus, ALS8, does not exist. Strains lacking ALS3 or ALS1 did not exhibit a defect in germ tube formation when grown in RPMI 1640 medium, but the als1/als1 mutant formed significantly fewer germ tubes in Lee medium. Analysis of ALS3 and ALS1 promoter activity using green fluorescent protein (GFP) reporter strains and flow cytometry showed that when cells are placed into medium that promotes germ tube formation, ALS1 is transcribed prior to ALS3. Comparison of the mutant strains in adhesion assays showed that the als3/als3 strain was defective in adhesion to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC), but not to fibronectin-coated plastic plates. In contrast, the als1/als1 strain showed decreased adherence to HUVEC, but adherence to BEC and fibronectin were the same as wild-type controls. Inoculation of the buccal reconstituted human epithelium (RHE) model of oral candidiasis with the mutant strains showed nearly a total lack of adhesion and epithelial destruction by the als3/als3 mutant while the als1/als1 strain showed only a slightly reduced degree of epithelial destruction compared to the wild-type control. Adhesion data presented here suggest that, in the assays performed, loss of Als3p affects C. albicans adhesion more than loss of Als1p. Collectively, these results demonstrate functional similarities and differences between Als1p and Als3p, and suggest the potential for more complex interrelationships between the ALS genes and their encoded proteins.

Zhong SS, Zhang ZS, Wang JD, Lai ZS, Wang QY, Pan LJ, Ren YX (2004) Competitive inhibition of adherence of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027. World J Gastroenterol 10 :1630-1633


AIM : To observe competitive inhibition of adherence of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC) and Clostridium difficile (C. difficile) to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027 (B. ado 1027). METHODS : The binding of bacteria to intestinal epithelial cell line Lovo was counted by adhesion assay. The inhibition of adherence of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo by purified adhesin of B. ado 1027 was evaluated quantitatively by flow cytometry. RESULTS : The purified adhesin at the concentration of 10 microg/mL, 20 microg/mL and 30 microg/mL except at 1 microg/mL and 5 microg/mL could inhibit significantly the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo. Moreover, we observed that a reduction in bacterial adhesion was occurred with increase in the concentration of adhesin, and MFI (Mean fluorescent intensity) was decreased with increase in the concentration of adhesin. CONCLUSION : The purified adhesin of B. ado 1027 can inhibit the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo in a dose-dependent manner.