Auguet JC, Montanie H, Hartmann HJ, Lebaron P, Casamayor EO, Catala P, Delmas D (2009) Potential effect of freshwater virus on the structure and activity of bacterial communities in the Marennes-Oleron Bay (France). Microb Ecol 57 :295-306
Batch culture experiments using viral enrichment were conducted to test the response of a coastal bacterial community to autochthonous (i.e., co-existing) or allochthonous riverine viruses. The effects of viral infections on bacterial dynamics and activity were assessed by epifluorescence microscopy and thymidine incorporation, respectively, whereas the effect of viral infection on bacterial community composition was examined by polymerase chain reaction-single strand conformation polymorphism 16S ribosomal RNA fingerprinting. The percentages of high nucleic acid-containing cells, evaluated by flow cytometry, were significantly correlated (r2=0.91, n=12, p<0.0001) to bacterial production, making this value a good predictor of active cell dynamics along the study. While confinement and temperature were the two principal experimental factors affecting bacterial community composition and dynamics, respectively, additions of freshwater viruses had significant effects on coastal bacterial communities. Thus, foreign viruses significantly reduced net bacterial population increase as compared to the enrichment treated with inactivated virus. Moreover, freshwater viruses recurrently and specifically affected bacterial community composition, as compared to addition of autochthonous viruses. In most cases, the combined treatment viruses and freshwater dissolved organic matter helped to maintain or even enhance species richness in coastal bacterial communities in agreement to the ’killing the winner’ hypothesis. Thus, riverine virus input could potentially influence bacterial community composition of the coastal bay albeit with modest modification of bulk bacterial growth.
Bahl MI, Oregaard G, Sorensen SJ, Hansen LH (2009) Construction and use of flow cytometry optimized plasmid-sensor strains. Methods Mol Biol 532 :257-268
Determining the stability of plasmids in bacterial populations is traditionally performed by isolating a large number of clones followed by screening for the presence of plasmids by replica transfer to plasmid-selective agar plates. This is often a laborious task, especially when the intrinsic stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid-harboring cells in a bacterial population facilitates a very high throughput of cells and thus provides excellent sensitivity and statistics toward detecting even very low levels of plasmid instability.
Bergmann S, Lang A, Rohde M, Agarwal V, Rennemeier C, Grashoff C, Preissner KT, Hammerschmidt S (2009) Integrin-linked kinase is required for vitronectin-mediated internalization of Streptococcus pneumoniae by host cells. J Cell Sci 122 :256-267
By interacting with components of the human host, including extracellular matrix (ECM) proteins, Streptococcus pneumoniae has evolved various strategies for colonization. Here, we characterized the interaction of pneumococci with the adhesive glycoprotein vitronectin and the contribution of this protein to pneumococcal uptake by host cells in an integrin-dependent manner. Specific interaction of S. pneumoniae with the heparin-binding sites of purified multimeric vitronectin was demonstrated by flow cytometry analysis. Host-cell-bound vitronectin promoted pneumococcal adherence to and invasion into human epithelial and endothelial cells. Pneumococci were trapped by microspike-like structures, which were induced upon contact of pneumococci with host-cell-bound vitronectin. Alphavbeta3 integrin was identified as the major cellular receptor for vitronectin-mediated adherence and uptake of pneumococci. Ingestion of pneumococci by host cells via vitronectin required a dynamic actin cytoskeleton and was dependent on integrin-linked kinase (ILK), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt), as demonstrated by gene silencing or in inhibition experiments. In conclusion, pneumococci exploit the vitronectin-alphavbeta3-integrin complex as a cellular receptor for invasion and this integrin-mediated internalization requires the cooperation between the host signalling molecules ILK, PI3K and Akt.
Bergquist PL, Hardiman EM, Ferrari BC, Winsley T (2009) Applications of flow cytometry in environmental microbiology and biotechnology. Extremophiles http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19301090
Flow cytometry (FCM) is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It uses the principles of light scattering, light excitation and the emission from fluorescent molecules to generate specific multiparameter data from particles and cells. The cells are hydrodynamically focussed in a sheath solution before being intercepted by a focused light source provided by a laser. FCM has been used primarily in medical applications but is being used increasingly for the examination of individual cells from environmental samples. It has found uses in the isolation of both culturable and hitherto non-culturable bacteria present infrequently in environmental samples using appropriate growth conditions. FCM lends itself to high-throughput applications in directed evolution for the analysis of single cells or cell populations carrying mutant genes. It is also suitable for encapsulation studies where individual bacteria are compartmentalised with substrate in water-in-oil-in-water emulsions or with individual genes in transcriptional/translational mixtures for the production of mutant enzymes. The sensitivity of the technique has allowed the examination of gene optimisation by a procedure known as random or neutral drift where screening and selection is based on the retention of some predetermined level of activity through multiple rounds of mutagenesis.
Bisha B, Brehm-Stecher BF (2009) Simple adhesive-tape-based sampling of tomato surfaces combined with rapid fluorescence in situ hybridization for Salmonella detection. Appl Environ Microbiol 75 :1450-1455
A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.
Brown KN, Barratt-Boyes SM (2009) Surface phenotype and rapid quantification of blood dendritic cell subsets in the rhesus macaque. J Med Primatol http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19344375
Background The study of dendritic cell (DC) biology in the rhesus macaque is becoming increasingly important but is limited by incomplete characterization and the lack of a rapid assay to quantify cells. Methods We characterized the surface phenotype of myeloid (mDC) and plasmacytoid DC (pDC) subsets in healthy rhesus macaque blood and developed a flow cytometry-based assay for absolute DC determinations. Results Rhesus CD11c(+) mDC were CD16(+) CD11b(+) CD56(lo) CD8(-) CD1c(-) whereas CD123(+) pDC lacked expression of these markers. Precise DC determinations were performed using a rapid two-step assay combining the analysis of whole blood and peripheral blood leukocytes (PBL). Conclusions Antibodies to CD11b, CD56 and CD16 must be omitted from the lineage antibody cocktail to prevent inadvertent gating-out of DC when analyzing rhesus blood. The combined whole-blood/PBL quantification assay will be invaluable for the rapid and repeated monitoring of blood DC counts in this species.
Chan CM, Tse H, Wong SS, Woo PC, Lau SK, Chen L, Zheng BJ, Huang JD, Yuen KY (2009) Examination of seroprevalence of coronavirus HKU1 infection with S protein-based ELISA and neutralization assay against viral spike pseudotyped virus. J Clin Virol http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19342289
BACKGROUND : Human coronavirus HKU1 (HCoV-HKU1) is a recently identified coronavirus with a global distribution and known to cause mainly respiratory infections. OBJECTIVES : To investigate the seroepidemiology of HKU1 infections in our local population. STUDY DESIGN : An ELISA-based IgG antibody detection assay using recombinant HCoV-HKU1 nucleocapsid and spike (S) proteins (genotype A) were developed for the diagnosis of CoV-HKU1 infections, Additionally, a neutralization antibody assay using the HCoV-HKU1 pseudotyped virus was developed to detect the presence of neutralizing antibodies in serum with antibody positivity in an S protein-based ELISA. RESULTS : Results of the recombinant S protein-based ELISA were validated with Western blot, immunofluorescence analysis and flow cytometry. The coupled results demonstrated good correlation with Spearmen’s coefficient of 0.94. Seroepidemiological study in a local hospital-based setting using this newly developed ELISA showed steadily increasing HCoV-HKU1 seroprevalence in childhood and early adulthood, from 0% in the age group of <10 years old to a plateau of 21.6% in the age group of 31-40 years old. CONCLUSIONS : Our study demonstrated the usefulness of the S-based ELISA which could be applied to future epidemiological studies of HCoV-HKU1 in other localities.
Chanchamroen S, Kewcharoenwong C, Susaengrat W, Ato M, Lertmemongkolchai G (2009) Human polymorphonuclear neutrophil responses to Burkholderia pseudomallei in healthy and diabetic subjects. Infect Immun 77 :456-463
The major predisposing factor for melioidosis is diabetes mellitus, but no immunological mechanisms have been investigated to explain this. In this study, polymorphonuclear neutrophil (PMN) responses to Burkholderia pseudomallei, the causative agent of melioidosis, in healthy and diabetic Thai subjects were determined by flow cytometry. The results showed that B. pseudomallei displayed reduced uptake by PMNs compared to Salmonella enterica serovar Typhimurium and Escherichia coli. Additionally, intracellular survival of B. pseudomallei was detected throughout a 24-h period, indicating the intrinsic resistance of B. pseudomallei to killing by PMNs. Moreover, PMNs from diabetic subjects displayed impaired phagocytosis of B. pseudomallei, reduced migration in response to interleukin-8, and an inability to delay apoptosis. These data show that B. pseudomallei is intrinsically resistant to phagocytosis and killing by PMNs. These observations, together with the impaired migration and apoptosis in diabetes mellitus, may explain host susceptibility in melioidosis.
Cubillos FA, Vasquez C, Faugeron S, Ganga A, Martinez C (2009) Self-fertilization is the main sexual reproduction mechanism in native wine yeast populations. FEMS Microbiol Ecol 67 :162-170
Saccharomyces cerevisiae is a model eukaryotic organism for classical genetics and genomics, and yet its ecology is still largely unknown. In this work, a population genetic analysis was performed on five yeast populations isolated from wine-making areas with different enological practices using simple sequence repeats and restriction fragment length polymorphism of mitochondrial DNA as molecular markers on 292 strains. In accordance with other studies, genome size estimation suggests that native S. cerevisiae strains are mainly homothallic and diploids. Analysis of mtDNA data showed that yeast populations from nonindustrial areas have 40% higher genetic diversity than populations isolated from industrial areas, demonstrating that industrial enological practices are likely to affect native yeast populations negatively by reducing its biodiversity. On the other hand, genetic differentiation analysis based on their microsatellite showed no correlation between genetic and geographic distance and a nonsignificant value when a Mantel test was applied. Finally, in the five populations studied, positive inbreeding (F(is)) values from 0.4 to 0.75, a low but significant level of linkage disequilibrium and a high number of multilocus genotypes were detected. These results strongly advocate that sexual reproduction is frequent enough to erase clonal signature in natural populations and that self-fertilization is the main mating system.
Denlinger LC, Shi L, Guadarrama A, Schell K, Green D, Morrin A, Hogan K, Sorkness RL, Busse WW, Gern JE (2009) Attenuated P2X7 pore function as a risk factor for virus-induced loss of asthma control. Am J Respir Crit Care Med 179 :265-270
RATIONALE : Upper respiratory tract infection is a guideline accepted risk domain for the loss of asthma control. The ionotrophic nucleotide receptor P2X(7) regulates compartmentalized acute inflammation and the immune response to airway pathogens. OBJECTIVES : We hypothesized that variability in P2X(7) function contributes to neutrophilic airway inflammation during a cold and thereby is linked to acute asthma. METHODS : Research volunteers with asthma were enrolled at the onset of a naturally occurring cold and monitored through convalescence, assessing symptoms, lung function, and airway inflammation. P2X(7) pore activity in whole blood samples was measured using a genomically validated flow cytometric assay. MEASUREMENTS AND MAIN RESULTS : Thirty-five participants with mild to moderate allergic asthma were enrolled and 31 completed all visits. P2X(7) pore function correlated with the change in nasal lavage neutrophil counts during the cold (R(s) = 0.514, P = 0.004) and was inversely related to the change in asthma symptoms (R(s) = -0.486, P = 0.009). The change in peak expiratory flow recordings, precold use of inhaled corticosteroids, and P2X(7) pore function were multivariate predictors of asthma symptoms (P = 0.001, < 0.001 and = 0.003 respectively). Attenuated P2X(7) activity was associated with the risk of losing asthma control (crude odds ratio, 11.0 ; 95% confidence interval, 1.1-106.4) even after adjustment for inhaled corticosteroids and rhinovirus (odds ratio, 15.0). CONCLUSIONS : A whole blood P2X(7) pore assay robustly identifies participants with loss-of-function genotypes. Using this assay as an epidemiologic tool, attenuated P2X(7) pore activity may be a novel biomarker of virus-induced loss of asthma control.
Detje CN, Meyer T, Schmidt H, Kreuz D, Rose JK, Bechmann I, Prinz M, Kalinke U (2009) Local type I IFN receptor signaling protects against virus spread within the central nervous system. J Immunol 182 :2297-2304
Several neurotropic viruses such as vesicular stomatitis virus (VSV) induce peripheral neutralizing Ab responses and still can infect cells within the CNS. To address whether local type I IFN receptor (IFNAR) triggering plays a role in controlling virus replication within the brain, we generated mice with a cell type-specific IFNAR deletion in neuroectodermal cells of the CNS (NesCre(+/-)IFNAR(flox/flox)). Intranasal VSV infection with 10(3) PFU was well tolerated by wild-type mice, whereas conventional IFNAR(-/-) mice died within 2-3 days. In contrast, brain-specific NesCre(+/-)IFNAR(flox/flox) mice survived until day 5-6 and then became hemiplegic and died. Terminally ill NesCre(+/-)IFNAR(flox/flox) mice showed 10- to 100-fold higher virus loads in the brain than IFNAR(-/-) mice, whereas little or no virus was found in other organs. In wild-type animals, virus could be reisolated only from the olfactory bulb until day 6 where also STAT1 activation as a measure of IFNAR triggering was detected. Virus infection was found exclusively in glomerular structures of the olfactory bulb, whereas surrounding cells that showed STAT1 phosphorylation as a measure of IFNAR trigging were free of virus. Our data indicate that upon intranasal VSV instillation, early and localized IFNAR triggering in the glomerular layer of the olfactory bulb is critically required to prevent viral spread over the entire CNS and thus confers survival.
Enstrom AM, Lit L, Onore CE, Gregg JP, Hansen RL, Pessah IN, Hertz-Picciotto I, Van de Water JA, Sharp FR, Ashwood P (2009) Altered gene expression and function of peripheral blood natural killer cells in children with autism. Brain Behav Immun 23 :124-133
Immune related abnormalities have repeatedly been reported in autism spectrum disorders (ASD), including evidence of immune dysregulation and autoimmune phenomena. NK cells may play an important role in neurodevelopmental disorders such as ASD. Here we performed a gene expression screen and cellular functional analysis on peripheral blood obtained from 52 children with ASD and 27 typically developing control children enrolled in the case-control CHARGE study. RNA expression of NK cell receptors and effector molecules were significantly upregulated in ASD. Flow cytometric analysis of NK cells demonstrated increased production of perforin, granzyme B, and interferon gamma (IFNgamma) under resting conditions in children with ASD (p<0.01). Following NK cell stimulation in the presence of K562 target cells, the cytotoxicity of NK cells was significantly reduced in ASD compared with controls (p<0.02). Furthermore, under similar stimulation conditions the presence of perforin, granzyme B, and IFNgamma in NK cells from ASD children was significantly lower compared with controls (p<0.001). These findings suggest possible dysfunction of NK cells in children with ASD. Abnormalities in NK cells may represent a susceptibility factor in ASD and may predispose to the development of autoimmunity and/or adverse neuroimmune interactions during critical periods of development.
Foresta C, De Toni L, Di Mambro A, Garolla A, Ferlin A, Zuccarello D (2009) The PDE5 inhibitor sildenafil increases circulating endothelial progenitor cells and CXCR4 expression. J Sex Med 6 :369-372
INTRODUCTION : Endothelial progenitor cells (EPC) are a specific subtype of progenitor cells that can be isolated from circulating mononuclear cells, able to migrate from the bone marrow to the peripheral circulation where they contribute to vascular repair. CXC-motif chemochine receptor 4 (CXCR4) receptor seems to play a critical role in this process. AIM : To assess the effects of sildenafil (a type 5 phosphodiesterase [PDE5] inhibitor) administration in 20 healthy young men. METHODS : Evaluation of CXCR4 expression in circulating EPC before and 4 hours after in vivo administration of 100 mg sildenafil by flow cytometry and colony-forming unit. RESULTS : We found that sildenafil increases circulating EPC number, the relative expression of CXCR4 on these cells and the ability to generate colonies in vitro. CONCLUSIONS : These data allow us to suppose an involvement of PDE5 in bone marrow release and peripheral homing of EPC.
Gekara NO, Dietrich N, Lyszkiewicz M, Lienenklaus S, Weiss S (2009) Signals triggered by a bacterial pore-forming toxin contribute to toll-like receptor redundancy in gram-positive bacterial recognition. J Infect Dis 199 :124-133
BACKGROUND : Toll-like receptor (TLR) 2 is the principal recognition receptor for gram-positive microbes. However, in some gram-positive bacterial infections, TLR2 is dispensable. One of the outstanding questions regarding host-bacteria interactions is why TLR2 is essential in some infections but dispensable in others. METHODS : We used a combination of bacterial plating, flow cytometry, enzyme-linked immunosorbent assay, and reverse-transcriptase polymerase chain reaction to analyze the inflammatory responses induced by Listeria monocytogenes and its toxin listeriolysin O (LLO) in vitro and in vivo. We analyzed wild-type, TLR2(-/-)-, TLR4(-/-)-, MyD88(-/-)-, interleukin (IL)-1beta(-/-)-, and IL-18(-/-)-deficient mice and the bone marrow-derived mast cells obtained from these respective groups. RESULTS : TLR2(-/-) mice had unaltered L. monocytogenes clearance and did not experience impairment of cytokine/chemokine induction and neutrophil mobilization by L. monocytogenes or purified LLO, but they were unresponsive to the LLO-deficient mutant L. monocytogenes (LmDeltahly). We show that L. monocytogenes and LLO mediate such responses in part via interleukin (IL)-1beta and IL-18-MyD88 pathways. CONCLUSIONS : The results illustrate that signals triggered by LLO contribute to TLR2 redundancy in recognition of L. monocytogenes. Under normal conditions, multiple and, sometimes, redundant pathways cooperate to induce a rapid antimicrobial defense. When one signaling pathway-in this case, TLR2-is removed from the system, the other pathways are still capable of mounting a sufficient response to ensure survival of the host.
George NP, Ymele-Leki P, Konstantopoulos K, Ross JM (2009) Differential binding of biofilm-derived and suspension-grown Staphylococcus aureus to immobilized platelets in shear flow. J Infect Dis 199 :633-640
BACKGROUND : The adhesion of Staphylococcus aureus to platelets is a critical first step in endovascular infection. S. aureus is known to form biofilms and detached cells are likely responsible for initiating bloodborne secondary infections. Although several previous studies have evaluated the mechanisms of S. aureus-platelet binding, standard procedures have used suspension-grown cells, which are known to differ in their adhesion properties from biofilm-derived cells. METHODS : This study used both shake flask-grown cells (hereafter, "suspension-grown cells") and cells derived from growing biofilms to compare the level and mechanisms of adhesion to immobilized platelets under physiologically relevant shear conditions. Of specific interest were the roles of clumping factor A (ClfA) and plasma proteins in supporting adhesion. RESULTS : S. aureus cells collected after 24 h of biofilm growth demonstrate significantly reduced levels of binding to immobilized platelets in the presence of exogenous plasma proteins, in comparison with suspension-grown cells. These adhesion results correlate directly with ClfA expression levels for the different cell populations. CONCLUSIONS : The results presented herein demonstrate that the mode of growth, temporal adhesin expression pattern, and hydrodynamic shear govern S. aureus adhesion to immobilized platelets. ClfA was identified as the critical binding adhesin, regardless of the mode or phase of growth.
Georgiev V, Weber J, Bley T, Pavlov A (2009) Improved procedure for nucleus extraction for DNA measurements by flow cytometry of red beet (Beta vulgaris L.) hairy roots. J Biosci Bioeng 107 :439-441
Three often cited systems for the extraction of plant nuclei for flow cytometric measurement (CyStain PI, Partec GmbH, Munster, Germany, the method of Arumuganathan and Earle, and LB01 buffer) failed, when applied to the hairy roots of red beet (Beta vulgaris). By combining these systems and introducing a centrifugation step, the extraction, staining, and analysis of nuclei from this tissue were performed successfully.
Gunther S, Trutnau M, Kleinsteuber S, Hause G, Bley T, Roske I, Harms H, Muller S (2009) Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4’,6’-diamidino-2-phenylindole) and tetracycline labeling. Appl Environ Microbiol 75 :2111-2121
Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.
Heine F, Stahl F, Strauber H, Wiacek C, Benndorf D, Repenning C, Schmidt F, Scheper T, von Bergen M, Harms H, Muller S (2009) Prediction of flocculation ability of brewing yeast inoculates by flow cytometry, proteome analysis, and mRNA profiling. Cytometry A 75 :140-147
The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively good beer. Flocculation involves two main cell wall structures, which are the flocculation proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice, the flocculation ability may get lost after several repitches. Flow cytometry was employed to analyze glucose and mannose structures of the cell surface by application of fluorescent lectins. Validation of the expression of the flocculin genes Lg-FLO1, FLO1, FLO5, and FLO9 was carried out using microarray techniques. SDS-PAGE, western blot, and ESI-MS/MS analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling with fluorescent lectins allowed differentiating powdery and flocculent yeast cells under laboratory conditions. Using microarray techniques and proteomics, the four flocculation genes Lg-FLO1, FLO1, FLO5, FLO9, and the protein Lg-Flo1p were identified as factors of major importance for flocculation. The expression of the genes was several times higher in flocculent yeast cells than in powdery ones. Flow cytometry is a fast and simple method to quantify the proportions of powdery and flocculent yeast cells in suspensions under defined cultivation conditions. However, differentiation under industrial conditions will require mRNA and protein expression profiling.
Hilmi HT, Hakkila K, Saris PE (2009) Isolation of sensitive nisin-sensing GFP(uv) bioassay Lactococcus lactis strains using FACS. Biotechnol Lett 31 :119-122
Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFP(uv) nisin bioassay. It harbors the GFP(uv) encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFP(uv) fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml(-1)) for nisin than the parent strain (10 pg ml(-1)). This showed that mutants of LAC275 could successfully be isolated using FACS.
Horst D, van Leeuwen D, Croft NP, Garstka MA, Hislop AD, Kremmer E, Rickinson AB, Wiertz EJ, Ressing ME (2009) Specific targeting of the EBV lytic phase protein BNLF2a to the transporter associated with antigen processing results in impairment of HLA class I-restricted antigen presentation. J Immunol 182 :2313-2324
EBV persists for life in the human host while facing vigorous antiviral responses that are induced upon primary infection. This persistence supports the idea that herpesviruses have acquired dedicated functions to avoid immune elimination. The recently identified EBV gene product BNLF2a blocks TAP. As a result, reduced amounts of peptides are transported by TAP from the cytoplasm into the endoplasmic reticulum (ER) lumen for binding to newly synthesized HLA class I molecules. Thus, BNLF2a perturbs detection by cytotoxic T cells. The 60-aa-long BNLF2a protein prevents the binding of both peptides and ATP to TAP, yet further mechanistic insight is, to date, lacking. In this study, we report that EBV BNLF2a represents a membrane-associated protein that colocalizes with its target TAP in subcellular compartments, primarily the ER. In cells devoid of TAP, expression levels of BNLF2a protein are greatly diminished, while ER localization of the remaining BNLF2a is retained. For interactions of BNLF2a with the HLA class I peptide-loading complex, the presence of TAP2 is essential, whereas tapasin is dispensible. Importantly, we now show that in B cells supporting EBV lytic replication, the BNLF2a protein is expressed early in infection, colocalizing and associating with the peptide-loading complex. These results imply that, during productive EBV infection, BNLF2a contributes to TAP inhibition and surface HLA class I down-regulation. In this way, EBV BNLF2a-mediated evasion from HLA class I-restricted T cell immunity contributes to creating a window for undetected virus production.
Imbeault S, Gauvin LG, Toeg HD, Pettit A, Sorbara CD, Migahed L, DesRoches R, Menzies AS, Nishii K, Paul DL, Simon AM, Bennett SA (2009) The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells. BMC Neurosci 10 :13
BACKGROUND : Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. RESULTS : We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. CONCLUSION : Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.
Jarzembowski T, Wisniewska K, Jozwik A, Witkowski J (2009) Heterogeneity of Methicillin-Resistant Staphylococcus aureus Strains (MRSA) Characterized by Flow Cytometry. Curr Microbiol http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19330377
We used fluorescent penicillin Bocillin FL for characterization of control methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to one of four heterogenic classes and comparing them with clinical MRSA isolates. Significant differences in percentage of fluorescent cells and reduction of Bocillin FL binding after incubation with methicillin between control strains from classes I and IV were observed, whereas the strains from classes II and III were differed after incubation with methicillin. According to this criteria, 55.8% of the clinical isolates population were similar to the strain of class IV or homogenic resistant, 11.8% was found as I, and 32.3% were categorized as class II or III. However, continuous diversity of measured features was also discussed.
Ji S, Shin JE, Kim YS, Oh JE, Min BM, Choi Y (2009) Toll-like receptor 2 and NALP2 mediate induction of human beta-defensins by fusobacterium nucleatum in gingival epithelial cells. Infect Immun 77 :1044-1052
We previously reported that infection by Fusobacterium nucleatum strongly induced the expression of both human beta-defensin 2 (HBD-2) and HBD-3 by gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptors (PRRs) and regulatory mechanisms involved in the induction of HBD-2 and -3 expression by F. nucleatum in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with live or heat-killed F. nucleatum, and the expression of HBDs and interleukin-8 (IL-8) was examined by real-time reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Live, but not heat-killed, F. nucleatum invaded HOK-16B cells, as seen by confocal microscopy and flow cytometry. Live F. nucleatum induced both HBD-2 and -3 efficiently, whereas heat-killed bacteria induced only HBD-3 at a reduced level. Knockdown of NACHT-LRR- and pyrin domain-containing protein 2 (NALP2), the most abundant intracellular PRR in HOK-16B cells, by RNA interference (RNAi) significantly reduced the induction of HBD-3 but not HBD-2 and IL-8. In addition, knockdown of Toll-like receptor 2 (TLR2) by RNAi reduced the upregulation of HBD-2 and -3 but not IL-8. Heat-killed F. nucleatum was hindered in its ability to activate TLR2 and JNK signaling pathways. Theses data show that TLR2 and NALP2 mediate the induction of HBDs by F. nucleatum in gingival epithelial cells, but thresholds for the two HBD genes are different.
Johnson DR, Czechowska K, Chevre N, van der Meer JR (2009) Toxicity of triclosan, penconazole and metalaxyl on Caulobacter crescentus and a freshwater microbial community as assessed by flow cytometry. Environ Microbiol http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19239485
Biocides are widely used for domestic hygiene, agricultural and industrial applications. Their widespread use has resulted in their introduction into the environment and raised concerns about potential deleterious effects on aquatic ecosystems. In this study, the toxicity of the biocides triclosan, penconazole and metalaxyl were evaluated with the freshwater bacterium Caulobacter crescentus and with a freshwater microbial community using a combination of single- and double-stain flow cytometric assays. Growth of C. crescentus and the freshwater community were repressed by triclosan but not by penconazole or metalaxyl at concentrations up to 250 muM. The repressive effect of triclosan was dependent on culture conditions. Caulobacter crescentus was more sensitive to triclosan when grown with high glucose at high cell density than when grown directly in sterilized lake water at low cell density. This suggests that the use of conventional growth conditions may overestimate biocide toxicity. Additional experiments showed that the freshwater community was more sensitive to triclosan than C. crescentus, with 10 nM of triclosan being sufficient to repress growth and change the phylogenetic composition of the community. These results demonstrate that isolate-based assays may underestimate biocide toxicity and highlight the importance of assessing toxicity directly on natural microbial communities. Because 10 nM of triclosan is within the range of concentrations observed in freshwater systems, these results also raise concerns about the risk of introducing triclosan into the environment.
Ju SY, Cho KA, Cho SJ, Jung YJ, Woo SY, Seoh JY, Han HS, Ryu KH (2009) Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver. Cell Biol Int 33 :31-35
Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1alpha was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl(4)-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1alpha, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl(4) damaged liver.
Kim KJ, Sung WS, Suh BK, Moon SK, Choi JS, Kim JG, Lee DG (2009) Antifungal activity and mode of action of silver nano-particles on Candida albicans. Biometals 22 :235-242
In this study, the antifungal effects of silver nano-particles (nano-Ag) and their mode of action were investigated. Nano-Ag showed antifungal effects on fungi tested with low hemolytic effects against human erythrocytes. To elucidate the antifungal mode of action of nano-Ag, flow cytometry analysis, a glucose-release test, transmission electron microscopy (TEM) and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest nano-Ag may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. The present study indicates nano-Ag has considerable antifungal activity, deserving further investigation for clinical applications.
King LB, Swiatlo E, Swiatlo A, McDaniel LS (2009) Serum resistance and biofilm formation in clinical isolates of Acinetobacter baumannii. FEMS Immunol Med Microbiol 55 :414-421
Acinetobacter baumannii has few known virulence factors and yet causes a variety of opportunistic infections. Many gram-negative bacteria are directly killed by complement, but we hypothesized that A. baumannii would be resistant to serum killing. A serum bactericidal assay assessed the resistance of seven A. baumannii isolates to serum killing, and C2-deficient serum was used to examine its activation of the alternative pathway. Flow cytometry was utilized to determine whether complement regulator factor H (FH) was bound by A. baumannii, and to assay C3 deposition on cells. A microtiter biofilm assay compared biofilm production among isolates. Of seven isolates, four were serum sensitive and three were serum resistant. The C2-deficient serum demonstrated that A. baumannii can activate the alternative pathway. None of the isolates bound FH. Serum-resistant strains accumulated little C3 when exposed to human serum, while sensitive strains had a high amount of surface C3 deposition. Biofilm production varied extensively among strains. Most serum-resistant isolates formed a substantial amount of biofilm, while sensitive isolates produced negligible amounts of biofilm. Our data indicate that some strains of A. baumannii are resistant to serum killing and produce biofilms and by understanding the resistance mechanisms used by this bacterium, we can further elucidate its complex pathogenicity.
Kumagai Y, Kumar H, Koyama S, Kawai T, Takeuchi O, Akira S (2009) Cutting Edge : TLR-Dependent viral recognition along with type I IFN positive feedback signaling masks the requirement of viral replication for IFN-alpha production in plasmacytoid dendritic cells. J Immunol 182 :3960-3964
Plasmacytoid dendritic cells (pDCs) recognize RNA virus infection via TLRs and consequently produce vast amounts of type I IFN. Because nucleic acid-sensing TLRs reside in the intracellular membrane compartment, it is presumable that pDCs do not require cytoplasmic viral replication to recognize the infection. By checking Newcastle disease virus (NDV) RNA abundance in GFP(+) and GFP(-) pDCs from Ifna6gfp mice, we found that NDV replication was not detected in IFN-producing pDCs. GFP(+) pDC was induced in response to replication-incompetent NDV. In contrast, the replication-incompetent NDV failed to induce IFN-producing pDCs in type I IFNR-deficient mice. The lack of IFNR signaling led to the replication of NDV and the subsequent RIG-I-like helicase-dependent IFN-alpha production in pDCs. These results showed that detection of viruses via TLRs together with a type I IFN feedback system circumvents the requirement for viral replication-dependent recognition in pDCs.
Law WS, Li SF, Kricka LJ (2009) Detection of enteropathogenic Escherichia coli by microchip capillary electrophoresis. Methods Mol Biol 509 :169-179
There is always a need to detect the presence of microorganisms, either as contaminants in food and pharmaceutical industries or bioindicators for disease diagnosis. Hence, it is important to develop efficient, rapid, and simple methods to detect microorganisms. Traditional culturing method is unsatisfactory due to its long incubation time. Molecular methods, although capable of providing a high degree of specificity, are not always useful in providing quick tests of presence or absence of microorganisms. Microchip elec-trophoresis has been recently employed to address problems associated with the detection of microorganisms due to its high versatility, selectivity, sensitivity, and short analysis times. In this work, the potential of PDMS-based microchip electrophoresis in the identification and characterization of microorganism was evaluated. Enteropathogenic E. coli (EPEC) was selected as the model microorganism. To obtain repeat-able separations, sample pretreatment was found to be essential. Microchip electrophoresis with laser-induced fluorescence detection could potentially revolutionize certain aspects of microbiology involving diagnosis, profiling of pathogens, environmental analysis, and many others areas of study.
Li W, Sofi MH, Wei DG, Du W, Gervay-Hague J, Renukaradhya GJ, Brutkiewicz RR, Chang CH (2009) MHC class II-expressing thymocytes suppress invariant NKT cell development. Immunol Cell Biol 87 :186-189
Natural killer T (NKT) cells are positively selected on cortical thymocytes expressing the non-classical major histocompatibility complex (MHC) class I CD1d molecules. However, it is less clear how NKT cells are negatively selected in the thymus. In this study, we investigated the role of MHC class II expression in NKT cell development. Transgenic mice expressing MHC class II on thymocytes and peripheral T cells had a marked reduction in invariant NKT (iNKT) cells. Reduced numbers of iNKT cells correlated with the absence of in vivo production of cytokines in response to the iNKT cell agonist alpha-galactosylceramide. Using mixed bone marrow chimeras, we found that MHC class II-expressing thymocytes suppressed the development of iNKT cells in trans in a CD4-dependent manner. Our observations have significant implications for human iNKT cell development as human thymocytes express MHC class II, which can lead to an inefficient selection of iNKT cells.
Lin X, Patel S, Litvintseva AP, Floyd A, Mitchell TG, Heitman J (2009) Diploids in the Cryptococcus neoformans serotype A population homozygous for the alpha mating type originate via unisexual mating. PLoS Pathog 5 :e1000283
The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the alpha mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual alpha population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (alpha-alpha) also occurs in nature as evidenced by the existence of natural diploid alphaADalpha hybrids that arose by fusion between two alpha cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, approximately 500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the alpha MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct alpha cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens.
Margarit I, Rinaudo CD, Galeotti CL, Maione D, Ghezzo C, Buttazzoni E, Rosini R, Runci Y, Mora M, Buccato S, Pagani M, Tresoldi E, Berardi A, Creti R, Baker CJ, Telford JL, Grandi G (2009) Preventing bacterial infections with pilus-based vaccines : the group B streptococcus paradigm. J Infect Dis 199 :108-115
We recently described the presence of 3 pilus variants in the human pathogen group B streptococcus (GBS ; also known as Streptococcus agalactiae), each encoded by a distinct pathogenicity island, as well as the ability of pilus components to elicit protection in mice against homologous challenge. To determine whether a vaccine containing a combination of proteins from the 3 pilus types could provide broad protection, we analyzed pili distribution and conservation in 289 clinical isolates. We found that pilus sequences in each island are conserved, all strains carried at least 1 of the 3 islands, and a combination of the 3 pilus components conferred protection against all tested GBS challenge strains. These data are the first to indicate that a vaccine exclusively constituted by pilus components can be effective in preventing infections caused by GBS, and they pave the way for the use of a similar approach against other pathogenic streptococci.
Matteoli FP, d’Avila-Levy CM, Santos LO, Barbosa GM, Holandino C, Branquinha MH, Santos AL (2009) Roles of the endosymbiont and leishmanolysin-like molecules expressed by Crithidia deanei in the interaction with mammalian fibroblasts. Exp Parasitol 121 :246-253
Crithidia deanei is an insect trypanosomatid that harbors a bacterial endosymbiont in its cytoplasm. In this work, we have demonstrated the influence of the endosymbiont on the interaction of C. deanei with mammalian fibroblasts, also implicating the surface leishmanolysin-like molecules of C. deanei in this process. The wild strain of C. deanei expressed a higher amount (2-fold) of leishmanolysin-like molecules in the parasite surface than the aposymbiotic strain. The treatment of parasites with anti-leishmanolysin antibodies or the fibroblasts with purified leishmanolysin-like molecules from C. deanei significantly reduced the association index. The aposymbiotic strain of C. deanei presented interaction rates about 2- and 3-fold lower with fibroblasts than the endosymbiont-bearing counterpart after 1 and 2h, respectively. However, the association indexes were similar after 3 and 4h of interaction. Additionally, we observed a 2-fold increase in the association index after 24-96 h of parasite-fibroblast interaction when compared to the interaction process performed for 4h, irrespective to the presence of the endosymbiont, suggesting that fibroblasts support multiplication and survival of C. deanei. Both parasite strains were able to induce fibroblast lysis. Interestingly, the wild strain led to a 2-fold increase in fibroblasts death in comparison to the aposymbiotic strain after 48-96 h. We also showed that both wild and aposymbiotic biotinylated live parasites recognized the same receptor in the fibroblast cells.
Mayeed MS, Al-Mekhnaqi AM, Auner GW, Newaz GM (2009) A surface accumulator of Escherichia coli in water flow. Comput Methods Biomech Biomed Engin 12 :109-112
The objective of this research is to design and optimise a mini/micro-channel based surface accumulator of Escherichia coli to be detected by acoustic wave biosensors. A computational research has been carried out using the state of the art software, CFD-ACE with water as bacteria bearing fluid. E. coli bacteria have been modelled as random discrete particles tracked by solving the Lagrangian equations. The design challenges are to achieve low shear force (pico-N), high concentration at accumulation and high enough Reynolds number to avoid bacteria swimming. A range of low Reynolds number (Re) from 28.2 to 58.3 has been considered along with the effects of particle-boundary interactions, gravity, Saffman lift and Magnus lift. About four orders of magnitude higher concentration at accumulation than the inlet concentration and lower shear force in the order of less than pico-N have been achieved in the optimised design with particles accumulating at a specific location under random particle-boundary interactions.
Megnekou R, Hviid L, Staalsoe T (2009) Variant-specific immunity to Plasmodium berghei in pregnant mice. Infect Immun 77 :1827-1834
We have investigated the immunological basis of pregnancy-related Plasmodium berghei recrudescence in immune mice with substantial preexisting immunity. Specifically, we examined the relevance of this experimental model to the study of pregnancy-associated malaria (PAM) caused by P. falciparum in women with substantial preexisting protective immunity. We used mice with immunity induced prior to pregnancy and employed flow cytometry to assess their levels of immunoglobulin G (IgG) recognizing antigens on the surfaces of infected erythrocytes (IEs) in plasma. After immunization, the mice did not possess IgG specific for antigens on IEs obtained during pregnancy-related recrudescence but they acquired recrudescence-specific IgG over the course of several pregnancies and recrudescences. In contrast, levels of antibodies recognizing IEs from nonpregnant mice did not increase with increasing parity. Furthermore, maternal hemoglobin levels increased and pregnancy-related parasitemia decreased with increasing parity. Finally, parasitemic mice produced smaller litters and pups with lower weights than nonparasitemic mice. Taken together, these observations suggest that levels of antibodies specific for recrudescence-type IEs are related to the protection of pregnant mice from maternal anemia, low birth weight, and decreased litter size. We conclude that the model replicates many of the key parasitological and immunological features of PAM, although the P. berghei genome does not encode proteins homologous to the P. falciparum erythrocyte membrane protein 1 adhesins, which are of key importance in P. falciparum malaria. The study of P. berghei malaria in pregnant, immune mice can be used to gain significant new insights regarding malaria pathogenesis and immunity in general and regarding PAM in particular.
Moon JJ, Chu HH, Hataye J, Pagan AJ, Pepper M, McLachlan JB, Zell T, Jenkins MK (2009) Tracking epitope-specific T cells. Nat Protoc 4 :565-581
The tracking of antigen-specific T cells in vivo is a useful approach for the study of the adaptive immune response. This protocol describes how populations of T cells specific for a given peptide-major histocompatibility complex (pMHC) epitope can be tracked based solely on T-cell receptor (TCR) specificity as opposed to other indirect methods based on function. The methodology involves the adoptive transfer of TCR transgenic T cells with defined epitope specificity into histocompatible mice and the subsequent detection of these cells through the use of congenic or clonotypic markers. Alternatively, endogenous epitope-specific T cells can be tracked directly through the use of pMHC tetramers. Using magnetic bead-based enrichment and advanced multiparameter flow cytometry, populations as small as five epitope-specific T cells can be detected from the peripheral lymphoid organs of a mouse. The adoptive transfer procedure can be completed within 3 h, whereas analysis of epitope-specific cells from mice can be completed within 6 h.
Moran XA, Calvo-Diaz A (2009) Single-cell vs. bulk activity properties of coastal bacterioplankton over an annual cycle in a temperate ecosystem. FEMS Microbiol Ecol 67 :43-56
The connections between single-cell activity properties of heterotrophic planktonic bacteria and whole community metabolism are still poorly understood. Here, we show flow cytometry single-cell analysis of membrane-intact (live), high nucleic acid (HNA) content and actively respiring (CTC+) bacteria with samples collected monthly during 2006 in northern Spain coastal waters. Bulk activity was assessed by measuring 3H-Leucine incorporation and specific growth rates. Consistently, different single-cell relative abundances were found, with 60-100% for live, 30-84% for HNA and 0.2-12% for CTC+ cells. Leucine incorporation rates (2-153 pmol L(-1) h(-1)), specific growth rates (0.01-0.29 day(-1)) and the total and relative abundances of the three single-cell groups showed marked seasonal patterns. Distinct depth distributions during summer stratification and different relations with temperature, chlorophyll and bacterial biovolume suggest the existence of different controlling factors on each single-cell property. Pooled leucine incorporation rates were similarly correlated with the abundance of all physiological groups, while specific growth rates were only substantially explained by the percentage of CTC+ cells. However, the ability to reduce CTC proved notably better than the other two single-cell properties at predicting bacterial bulk rates within seasons, suggesting a tight linkage between bacterial individual respiration and biomass production at the community level.
Nakagawa H, Yuno T, Itho K (2009) [Clinical usefulness of urine formed elements’ information obtained from bacteria detection by flow cytometry method that uses the nucleic acid staining]. Rinsho Byori 57 :221-227
Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.
Naushad H, Choi WW, Page CJ, Sanger WG, Weisenburger DD, Aoun P (2009) Mantle cell lymphoma with flow cytometric evidence of clonal plasmacytic differentiation : a case report. Cytometry B Clin Cytom 76 :218-224
BACKGROUND : Plasmacytic differentiation in mantle cell lymphoma (MCL) occurs rarely. However, no flow cytometric studies that demonstrate plasmacytic (PC) differentiation in MCL have been reported. Herein, we report a case of MCL with PC differentiation identified by flow cytometry. METHODS : Morphologic review was performed by hematoxylin and eosin (H&E) stained sections from paraffin-embedded lymph node, colon and bone marrow specimens, and Wright-Geimsa stained bone marrow aspirate smears and touch imprints. Immunohistochemical stains using antibodies against CD3, CD5, CD20, and cyclin-D1, and in-situ hybridization for kappa and lambda light chains were reviewed. Multicolor flow cytometry analysis was performed on the bone marrow aspirate with monoclonal antibodies to CD3, CD4, CD5, CD8, CD14, CD19, CD20, CD23, CD38, CD45, CD56, CD138, and kappa and lambda light chains. FISH analysis for t(11 ;14)(q13 ;q32) was performed on interphase cells. RESULTS : The neoplastic cells had the cytologic features of MCL with nodal, bone marrow, and colonic involvement. In-situ hybridization for kappa and lambda light chains demonstrated clonal plasma cells in the lymph node and bone marrow biopsies. In addition, flow cytometric studies of the bone marrow aspirate showed three populations of neoplastic cells : a clonal B-cell population with typical MCL phenotype, a similar B-cell population in transition to plasma cells, and a clonal plasma cell population. The plasma cells retained CD5 expression and had the same light chain restriction as the clonal B-cells. CONCLUSIONS : Multi-parameter flow cytometry can be useful in demonstrating clonal PC differentiation in MCL and distinguishing from a concurrent but unrelated plasma cell dyscrasia.
Nielsen TH, Sjoholm OR, Sorensen J (2009) Multiple physiological states of a Pseudomonas fluorescens DR54 biocontrol inoculant monitored by a new flow cytometry protocol. FEMS Microbiol Ecol 67 :479-490
A new fluorescence staining and flow cytometry protocol was developed to monitor several physiological states in biocontrol strain Pseudomonas fluorescens DR54 during storage survival in a stationary-phase culture, preparation of clay carrier for seed formulation, and establishment in a sugar beet spermosphere. The high load of impurities in the environmental samples was dealt with by adding a density-gradient purification step to the staining protocol. Staining by SYBR Green, combined with either propidium iodide or ethidium bromide (EB)+DiBAC((4))3, was used to quantify the total cell population and further divide this population into : (1) intact cells with an unaffected membrane and energy metabolism. (2) De-energized cells unable to maintain membrane export (EB exclusion). (3) Depolarized cells unable to maintain membrane potential. (4) Permeabilized cells with a damaged membrane. During both stationary-phase storage and steps for preparation of formulation carrier, loss of intact P. fluorescens DR54 cells was quantitatively accounted for by depolarized and permeabilized states. Surviving inoculum cells subsequently proliferated on the germinating seeds, but with a surprisingly high abundance of de-energized cells. The new protocol is the first for flow cytometry to include a recording of both intact and several subpopulations of physiologically affected bacteria in complex, environmental samples with high impurity loads.
osshard F, Berney M, Scheifele M, Weilenmann HU, Egli T (2009) Solar disinfection (SODIS) and subsequent dark storage of Salmonella typhimurium and Shigella flexneri monitored by flow cytometry. Microbiology 155 :1310-1317
Pathogenic enteric bacteria are a major cause of drinking water related morbidity and mortality in developing countries. Solar disinfection (SODIS) is an effective means to fight this problem. In the present study, SODIS of two important enteric pathogens, Shigella flexneri and Salmonella typhimurium, was investigated with a variety of viability indicators including cellular ATP levels, efflux pump activity, glucose uptake ability, and polarization and integrity of the cytoplasmic membrane. The respiratory chain of enteric bacteria was identified to be a likely target of sunlight and UVA irradiation. Furthermore, during dark storage after irradiation, the physiological state of the bacterial cells continued to deteriorate even in the absence of irradiation : apparently the cells were unable to repair damage. This strongly suggests that for S. typhimurium and Sh. flexneri, a relatively small light dose is enough to irreversibly damage the cells and that storage of bottles after irradiation does not allow regrowth of inactivated bacterial cells. In addition, we show that light dose reciprocity is an important issue when using simulated sunlight. At high irradiation intensities (>700 W m(-2)) light dose reciprocity failed and resulted in an overestimation of the effect, whereas reciprocity applied well around natural sunlight intensity (<400 W m(-2)).
Palenik B, Ren Q, Tai V, Paulsen IT (2009) Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity. Environ Microbiol 11 :349-359
The extent to which cultured strains represent the genetic diversity of a population of microorganisms is poorly understood. Because they do not require culturing, metagenomic approaches have the potential to reveal the genetic diversity of the microbes actually present in an environment. From coastal California seawater, a complex and diverse environment, the marine cyanobacteria of the genus Synechococcus were enriched by flow cytometry-based sorting and the population metagenome was analysed with 454 sequencing technology. The sequence data were compared with model Synechococcus genomes, including those of two coastal strains, one isolated from the same and one from a very similar environment. The natural population metagenome had high sequence identity to most genes from the coastal model strains but diverged greatly from these genomes in multiple regions of atypical trinucleotide content that encoded diverse functions. These results can be explained by extensive horizontal gene transfer presumably with large differences in horizontally transferred genetic material between different strains. Some assembled contigs showed the presence of novel open reading frames not found in the model genomes, but these could not yet be unambiguously assigned to a Synechococcus clade. At least three distinct mobile DNA elements (plasmids) not found in model strain genomes were detected in the assembled contigs, suggesting for the first time their likely importance in marine cyanobacterial populations and possible role in horizontal gene transfer.
Pariseau J, Saint-Louis R, Delaporte M, El Khair MA, McKenna P, Tremblay R, Davidson TJ, Pelletier E, Berthe FC (2009) Potential link between exposure to fungicides chlorothalonil and mancozeb and haemic neoplasia development in the soft-shell clam Mya arenaria : A laboratory experiment. Mar Pollut Bull 58 :503-514
The aetiology of haemic neoplasia (HN) is unknown, so far but many causative factors are suggested such as viral, pollution and genetics. The aim of this study was to determine if, under chronic exposure, two major pesticides (chlorothalonil and mancozeb) which are used in potato production could induce HN in soft-shell clams (Mya arenaria). Short-term experiments with acute exposure were also performed. Clams were collected from an epizootic site (North River, PEI) and from a site free of the disease (Magdalen Islands, Quebec). The tetraploid level of haemocytes was assessed by flow cytometry for each clam to determine the HN status. The bioaccumulation of pesticides in tissues was quantified by gas chromatography/mass spectrometry (GC/MS) for chlorothalonil while mancozeb and manganese were quantified by inductively coupled plasma-mass spectrometer (ICP/MS). Long term exposure to fungicide Bravo 500((R)) did not induce high tetraploid levels on negative calm from North River and the analysis of the digestive gland and the mantle did not reveal any detectable level of chlorothalonil. In the Manzate 200 DF((R)), some clams revealed high level of tetraploid cells but no difference were observed between the treatments and the control. The analysis of the digestive gland and the mantle for manganese did not highlight any significant difference in tissue concentration (p=0.05). For the acute exposure, chlorothalonil analysis showed that the active ingredient is distributed between four chlorinated compounds : 99.5% for chlorothalonil isomers, 0.4% for pentachlorothalonil and 0.1% for trichlorothalonil isomers. For a 72h experiment, the accumulation was within 4h ; the higher tissue concentration of chlorothalonil was 59.2mugg(-1) in the mantle after 48h, following by a decrease to an undetectable level at the end. For the manganese, the accumulation was detected after 4h ; the higher tissue concentration was 48.8mugg(-1) in the mantle after 24h and, over the following 48h, the accumulation decreased until the end of the trial. Based on the data, the accumulation of these fungicides seems to be transitory. Chlorothalonil and mancozeb are both oxidative-stress promoters and could have induced cell dysfunction while in the tissue. Study on the effect of these fungicides on the p53 protein system is an example of strategy that would provide information on cellular events promoting neoplasia.
Pedersen AL, Nybroe O, Winding A, Ekelund F, Bjornlund L (2009) Bacterial Feeders, the Nematode Caenorhabditis elegans and the Flagellate Cercomonas longicauda, have different Effects on Outcome of Competition among the Pseudomonas Biocontrol Strains CHA0 and DSS73. Microb Ecol 57 :501-509
How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the selective feeding flagellate Cercomonas longicauda versus the non-selective feeding nematode Caenorhabditis elegans) influence the abundance of two bacteria that compete for resources in simple model communities. Microcosms consisted of either one gfp-tagged bacterial strain (Pseudomonas fluorescens DSM50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured by flow cytometry. In the presence of flagellates, CHA0 increased its abundance as compared to the other biocontrol strain DSS73 or to DSM50090, which were both eaten by the flagellates. In contrast, the number of CHA0 declined as compared to DSS73 when the model community was subjected to nematode predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator.
Rivers AR, Jakuba RW, Webb EA (2009) Iron stress genes in marine Synechococcus and the development of a flow cytometric iron stress assay. Environ Microbiol 11 :382-396
Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient. To understand their capacity to respond to Fe stress, we screened picoplankton genomes and the Global Ocean Survey metagenome for known Fe stress genes. Many open ocean strains of Synechococcus lack most known genes for Fe stress, while coastal and upwelling strains contain many, suggesting that maintaining multiple Fe limitation compensation strategies is not a selective advantage in the open ocean. All genomes contained iron deficiency-induced protein A (IdiA) and its complementary Fe(3+) transport proteins. The ubiquity of IdiA was exploited to develop an in situ Fe stress bioassay based on immunolabelling and flow cytometry. As a test of field applicability, we used the assay on natural Synechococcus populations from one station in the Costa Rica Upwelling Dome where total Fe ranged from <0.08 to 0.14 nM in the upper water column. The bioassay found Fe stress in 5-54% of the population. Based on our findings, we believe that when reactive strains are present this assay can reveal environmental and clade-specific differences in the response of Synechococcus to Fe stress.
Rodriguez-Blanco A, Ghiglione JF, Catala P, Casamayor EO, Lebaron P (2009) Spatial comparison of total vs. active bacterial populations by coupling genetic fingerprinting and clone library analyses in the NW Mediterranean Sea. FEMS Microbiol Ecol 67 :30-42
Spatial distributions of both total (i.e. 16S rDNA-based fingerprints) and active (i.e. 16S rRNA-based fingerprints) bacterial populations, together with total bacterial activity measured by 3H-leucine incorporation, were studied along a 98 km transect in the NW Mediterranean Sea. Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) fingerprinting was coupled to a clone library, allowing CE-SSCP peaks identification and the monitoring of the spatial variation of bacterial phylotypes. Up to 80% of the community peaks matched those obtained from clone library sequences, accounting for 86.7% of the total fingerprinting area. A good agreement was found between the relative abundance of Prochlorococcus in the CE-SSCP fingerprints and flow cytometry counts (r2=0.66, P<0.05). The largest differences between total and active bacterial populations distribution were found at depths with higher bacterial activity (i.e. surface and deep chlorophyll maximum, DCM). SAR11 at the surface and Gammaproteobacteria at the DCM were the most abundant groups on the 16S rDNA-based fingerprints. However, their ratio of relative importance between rRNA : rDNA was <1 in most cases. Conversely, ratios observed for Prochlorococcus, were consistently >1 both at the surface and at the DCM. These results emphasize the need for combining rDNA- and rRNA-based analyses to better understand the functional role of individual bacterial populations in situ.
Russo I, Oksman A, Vaupel B, Goldberg DE (2009) A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development. Proc Natl Acad Sci U S A 106 :1554-1559
Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.
Sabharwal V, Ram S, Figueira M, Park IH, Pelton SI (2009) Role of complement in host defense against pneumococcal otitis media. Infect Immun 77 :1121-1127
Strategies to limit complement deposition on Streptococcus pneumoniae are established as virulence features for invasive disease, but their role in respiratory tract infection requires further analysis. We evaluated complement C3 protein deposition on discordant S. pneumoniae isolates of the same serotype (6A) and their capacity to cause nasopharyngeal (NP) colonization and experimental otitis media (EOM) in an animal model. We compared C3 binding to five 6A isolates from asymptomatic NP carriers with five 6A strains that caused invasive disease, and we observed less C3 ( approximately 10-fold less fluorescence) binding to invasive isolates. We selected two high-level C3-binding carriage and two low-level C3-binding invasive 6A isolates for further study. In the EOM model, 11/12 (92%) ears challenged with a low-level C3-binding 6A strain became infected. Only 2/8 (25%) ears challenged with the discordant high-level C3-binding 6A isolate developed disease (P = 0.005). Results with the second discordant 6A isolate pair were comparable. Cobra venom factor (CoVF) treatment, which depletes C3 and consumes complement, restored virulence of the high-level C3-binding strain ; 8/8 (100%) ears in CoVF-treated animals developed EOM compared to only 25% of ears in naive animals (P = 0.007). These studies demonstrate the critical role for complement evasion in pneumococcal EOM. Colonization with carriage isolates that bound high levels of C3 caused EOM in fewer animals compared to low-level C3-binding invasive strains. Thus, limiting C3 deposition on the surface of S. pneumoniae correlates with increased incidence of EOM following NP colonization and barotrauma in the animal model.
Sachs K, Itani S, Carlisle J, Nolan GP, Pe’er D, Lauffenburger DA (2009) Learning signaling network structures with sparsely distributed data. J Comput Biol 16 :201-212
Flow cytometric measurement of signaling protein abundances has proved particularly useful for elucidation of signaling pathway structure. The single cell nature of the data ensures a very large dataset size, providing a statistically robust dataset for structure learning. Moreover, the approach is easily scaled to many conditions in high throughput. However, the technology suffers from a dimensionality constraint : at the cutting edge, only about 12 protein species can be measured per cell, far from sufficient for most signaling pathways. Because the structure learning algorithm (in practice) requires that all variables be measured together simultaneously, this restricts structure learning to the number of variables that constitute the flow cytometer’s upper dimensionality limit. To address this problem, we present here an algorithm that enables structure learning for sparsely distributed data, allowing structure learning beyond the measurement technology’s upper dimensionality limit for simultaneously measurable variables. The algorithm assesses pairwise (or n-wise) dependencies, constructs "Markov neighborhoods" for each variable based on these dependencies, measures each variable in the context of its neighborhood, and performs structure learning using a constrained search.
Shachaf CM, Elchuri SV, Koh AL, Zhu J, Nguyen LN, Mitchell DJ, Zhang J, Swartz KB, Sun L, Chan S, Sinclair R, Nolan GP (2009) A novel method for detection of phosphorylation in single cells by surface enhanced Raman scattering (SERS) using composite organic-inorganic nanoparticles (COINs). PLoS ONE 4 :e5206
BACKGROUND : Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. METHODOLOGY/PRINCIPAL FINDINGS : To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. CONCLUSIONS/SIGNIFICANCE : Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.
Shao SW, Wu WB, Bian ZQ, Yu JG, Zhao P, Zhao LJ, Zhu SY, Qi ZT (2009) Hepatitis C virus F protein inhibits cell apoptosis by activation of intracellular NF-kappaB pathway. Hepatol Res 39 :282-289
Aim : To observe the influence of HCV F protein on apoptosis of HepG2 cells, and explore the association between F protein and NF-kappaB signal pathway. Methods : HCV 1b F gene containing HepG2-F cells and HCV 1b C gene containing HepG2-C cells were treated with 100 IU/mL TNF-alpha, and analyzed by flow cytometry, Western blotting, and dual luciferase reporter assay. Empty plasmid pcDNA3.1(+) containing HepG2-3.1 cells were used as control. Results : (i) With the treatment of TNF-alpha for 18 h, the apoptosis rates (AR) of HepG2-F and HepG2-3.1 cells were 0.41% (+/- 0.11%) and 37.43% (+/- 2.03%) respectively, while that of HepG2-C was 4.07% (+/- 0.18%). At 36 h after TNF-alpha treatment, the AR of HepG2-F and HepG2-3.1 cells were 10.03% (+/- 0.41%) and 44.63% (+/- 3.37%), and that of HepG2-C was 14.95% (+/- 0.85%). (ii) After the treatment of TNF-alpha for 0.5-18 h, the p65 contents in the whole cells of HepG2-F and HepG2-3.1 showed no significant difference (P = 0.34, t = 1.08), while the p65 contents in the nucleus of HepG2-F and HepG2-3.1 cells were 3.8-1.9 times and 1.8-1.0 times higher than that in the non-treated cells (P = 0.013, t = 4.25). (iii) The relative luciferase unit (RLU) of the HepG2 cells, co-transfected with pcDNA3.1-F and pNF-kappaB-luc, and then treated with TNF-alpha (100 IU/mL) for 18 h, showed a pcDNA3.1-F dose-dependent increase. Conclusion : HCV F protein can over-activate NF-kappaB signal pathway, which makes HepG2-F cells able to resist TNF-alpha induced apoptosis.
Shelley LK, Balfry SK, Ross PS, Kennedy CJ (2009) Immunotoxicological effects of a sub-chronic exposure to selected current-use pesticides in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 92 :95-103
Many current-use pesticides (CUPs) are found at increasing concentrations in aquatic environments, yet relatively little is known about their effects on the immune system of fish. We examined the in vivo effects of three pesticides (chlorothalonil, cypermethrin and pentachlorophenol) on the immune system of juvenile rainbow trout (Oncorhynchus mykiss) by assessing a suite of innate immune function tests, as well as a host resistance test using Listonella anguillarum. Increased activity of phagocytic leukocytes, as evidenced using flow cytometry, was observed following 28-day exposures to pentachlorophenol (1 microg/L and 2 microg/L), but not for cypermethrin or chlorothalonil, although a trend of increasing activity was noted for chlorothalonil. No recovery was observed during a 14-day post-exposure chlorothalonil experiment, as evidenced by continued elevation of respiratory burst and percent phagocytic cells at the lowest exposure concentrations (100 ng/L and 200 ng/L), suggesting a prolonged CUP-induced impact on the immune system. No effects of any pesticide on body weights, direct lethality, serum lysozyme or relative leukocyte differential were observed, suggesting that modulation of the cellular responses of the innate immune system represents a sensitive sub-lethal endpoint for these three pesticides. However, a lack of detectable effects of these CUPs on host resistance to L. anguillarum in our study may reflect a dose-response range that did not elicit an effect on those immune responses responsible for control and clearance of this particular pathogen. Additional research may provide more insight into the immunomodulatory effects of these and other CUPs, and the implications for host resistance against a variety of bacterial, viral and macroparasitic pathogens.
Shinoda K, Wyatt LS, Irvine KR, Moss B (2009) Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization. Virol J 6 :28
BACKGROUND : The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun. RESULTS : The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus. CONCLUSION : Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine.
Strovas TJ, Lidstrom ME (2009) Population heterogeneity in Methylobacterium extorquens AM1. Microbiology http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19383691
Heterogeneity of cells within exponentially-growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter (PmxaF) and gfpuv was used with flow cytometry to analyze the distribution of gene expression in populations grown either on succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing. Through the use of flow cytometry, it was demonstrated that a significant percentage of the total population did not respond to carbon shift. Additionally, these experiments demonstrated that a small subset of the total population was significantly brighter than the rest of the population and dominated fluorimetry data. These results were corroborated with a continuous flow through system and laser scanning microscopy, confirming that subpopulations, not discernable in the population average, dominate population response. These results demonstrate that the combination of flow cytometry and microscopic single cell analysis can be effectively used to determine the dynamics of subpopulations in population response. In addition, they support the concept that physiological diversity in isogenic population can poise some proportion of the population to respond appropriately to changing conditions.
Suzuki M, Endo M, Shinohara F, Echigo S, Rikiishi H (2009) Enhancement of cisplatin cytotoxicity by SAHA involves endoplasmic reticulum stress-mediated apoptosis in oral squamous cell carcinoma cells. Cancer Chemother Pharmacol http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19280190
PURPOSE : The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances cisplatin [cis-diammine dichloroplatinum (II)] (CDDP)-induced apoptosis in the oral squamous cell carcinoma (OSCC) cell line by complex, multifunctional mechanisms. We investigated the role of endoplasmic reticulum (ER) stress in the enhancing effect of SAHA on CDDP, compared with the ER stressor thapsigargin. METHODS : We chose OSCC cell line HSC-3 to ascertain the mechanism of SAHA-enhanced cytotoxicity among various cell lines. HSC-3 cells were incubated with CDDP/SAHA for 48 h, followed by the assessment of cell chemosensitivity to CDDP with MTT and TUNEL assays. Western blot analysis was used to detect the expressions of ER-related molecules, and flow cytometry was used to monitor caspase activity. RESULTS : Treatment with CDDP/SAHA potently induced apoptosis in HSC-3 cells with a significant increase in caspase-4 and -12 functions. For example, 60% of cells became apoptotic after 48 h of treatment with CDDP/SAHA. In addition, SAHA alone rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)alpha, which is up-regulated during ER stress. Inhibition of ER stress by salubrinal, an inhibitor of eIF2alpha dephosphorylation, abrogated SAHA’s enhancement of CDDP cytotoxicity. Levels of phospho-Akt are decreased in SAHA-treated cells, and this is in turn associated with increased activity of protein phosphatase 1 (PP1) by SAHA, the phosphatase upstream of Akt. CONCLUSION : These data indicate that up-regulation of specific-ER stress-associated events is an integral part of the mechanism by which SAHA enhances CDDP-induced apoptosis, and PP1 up-regulation followed by Akt dephosphorylation plays an important role in SAHA-enhanced CDDP apoptosis.
Torrero MN, Larson D, Hubner MP, Mitre E (2009) CD200R surface expression as a marker of murine basophil activation. Clin Exp Allergy 39 :361-369
BACKGROUND : Basophils are increasingly recognized as playing important roles in the immune responses of allergic diseases and helminth infections. One of the main obstacles to studying basophils has been the lack of a simple and rapid assay to measure basophil activation in mice. OBJECTIVE : The purpose of this study was to develop an assay to measure murine basophil activation. METHODS : Mouse blood cells were stained with various combinations of positive and negative markers for basophils—sorted and then assessed for basophil purity by May-Grunwald staining of cytospins. Once a flow cytometric strategy for staining basophils was determined, basophil surface expression of CD200R was assessed by multi-colour flow cytometry after stimulation of whole blood with anti-IgE, ionomycin or N-formyl MetLeuPhe (fMLP). Confirmation of basophil activation was assessed by concomitant staining of cells for intracellular IL-4. To test the ability of flow cytometric basophil CD200R measurements to assess for antigen-specific IgE-mediated activation of basophils, surface CD200R expression in response to in vitro stimulation with media alone, helminth antigen or ovalbumin was measured on basophils obtained from control mice, mice infected with helminths and mice sensitized to ovalbumin. RESULTS : Using anti-IgE-FITC as a positive marker and a combination of anti-CD4-PERCP and anti-B220-PERCP as negative markers resulted in a well-separated basophil population. Additional staining with anti-CD200R-PE demonstrated that (1) basophil CD200R expression increases in response to anti-IgE, ionomycin and fMLP, (2) most CD200R-positive basophils also stain positively for IL-4 and (3) CD200R expression increases after antigen-specific activation of basophils in murine models of helminth disease and allergy. CONCLUSION : We developed a multi-colour flow cytometry assay that measures murine basophil activation by utilizing CD200R as an activation marker. This assay is straightforward and rapid, taking approximately half a day for obtaining blood, in vitro stimulation and flow cytometric analysis.
Wang M, Harhaji L, Lamberth K, Harndahl M, Buus S, Heegaard NH, Claesson MH, Nissen MH (2009) Modified human beta 2-microglobulin (desLys(58)) displays decreased affinity for the heavy chain of MHC class I and induces nitric oxide production and apoptosis. Scand J Immunol 69 :203-212
Beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex class I (MHC-I) molecules, and is a prerequisite for the binding of peptides to the heavy chain and their presentation to CD8+ T cells. beta2m can be modified in vivo and in vitro by proteolytic cleavage by complement C1 and subsequent carboxypeptidase B-like activity—processes that lead to the generation of desLys(58) beta2m (dbeta2m). This work aims to study the effect of dbeta2m on peptide binding to MHC-I, the influence of dbeta2m on the binding of beta2m to the MHC-I heavy chain and the biological activity of dbeta2m. Both beta2m and dbeta2m are able to support the generation of MHC-I/peptide complexes at 18 degrees C, but complexes formed in the presence of dbeta2m destabilize at 37 degrees C. Moreover, a 250 times higher concentration of dbeta2m than of beta2m is needed to displace MHC-I associated beta2m from the cell surface. In addition, only beta2m is able to restore MHC-I/peptide complex formation on acid-treated cells whereas dbeta2m appears to bind preferentially to denatured MHC-I heavy chains. In cell cultures, exogenously added dbeta2m, but not beta2m, induces apoptotic cell death in monocytic leukaemic cell lines but spares other kinds of leukaemic cells. Additionally, the presence of dbeta2m, and to a lesser extent beta2m, enhances IFN-gamma-induced NO production by monocytic leukaemic cells. In conclusion, these data show that dbeta2m is not able to support the formation of a stable tri-molecular MHC-I complex at physiological temperature and that dbeta2m exerts other biological functions compared to beta2m when bound to cells.
Want A, Thomas OR, Kara B, Liddell J, Hewitt CJ (2009) Studies related to antibody fragment (Fab) production in Escherichia coli W3110 fed-batch fermentation processes using multiparameter flow cytometry. Cytometry A 75 :148-154
Microbiology is important to industry therefore rapid and statistically representative measurements of cell physiological state, proliferation, and viability are essential if informed decisions about fermentation bioprocess optimization or control are to be made, because process performance will depend largely on the number of metabolically active viable cells. Samples of recombinant Escherichia coli W3110, containing the gene for the D1.3 anti-lysozyme Fab fragment under the control of the lac-based expression system, were taken at various stages from fed-batch fermentation processes and stained with a mixture of bis-(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI/BOX). Where appropriate, measurements of dissolved oxygen tension (DOT), OD600nm and Fab concentration were made. Depending on time of induction the maximum amount of Fab accumulating in the supernatant varied quite markedly from 1 to 4 microg ml(-1) as did subsequent cell physiological state with respect to PI/BOX staining with a concomitant drop in maximum biomass concentration. Depending on point of induction a fourfold increase in Fab production could be achieved accompanied by a approximately 50% drop in maximum biomass concentration but with a higher proportion of viable cells as measured by multiparameter flow cytometry.
Wills E, Mou F, Baines JD (2009) The U L 31 and U L 34 gene products of herpes simplex virus 1 are required for optimal localization of viral glycoproteins D and M to the inner nuclear membranes of infected cells. J Virol 83 :4800-4809
U(L)31 and U(L)34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pU(L)31, pU(L)34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a U(L)34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the U(L)34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on U(L)34 (P < 0.0001). pU(L)34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pU(L)34/gD interaction in recruiting gD to the NM. The effects of U(L)34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pU(L)31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, U(L)31 and U(L)34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.