(2001) Flow cytometry in the marine environment. Proceedings from the 20th ISAC Congress. Montpellier, France, 20-25 May 2000. Cytometry 44 :163-271
Adachi H, Saito I, Horiuchi M, Ishii J, Nagata Y, Mizuno F, Nakamura H, Yagyu H, Takahashi K, Matsuoka T (2001) Infection of human lung fibroblasts with Epstein-Barr virus causes increased IL-1beta and bFGF production. Exp Lung Res 27 :157-171
An association between Epstein-Barr virus (EBV) infection and fibroblast proliferation in the interstitial spaces of the lung has been suggested in idiopathic interstitial pneumonia. In this study we show that EBV can infect human lung fibroblasts in vitro. A primary-cultured human lung fibroblast cell line, designated CCD-32Lu, expressed EBV nuclear antigen 1 after coculture with lethally irradiated EBV producing cells. The infection further induced CCD-32Lu cells to produce the fibrogenic cytokines basic fibroblast growth factor (bFGF) and interleukin-1beta. These findings indicate that lung fibroblasts may be a target for EBV infection and suggest that EBV may play a role in increased production of these cytokines and induce fibroblast proliferation in idiopathic interstitial pneumonia.
Alessandri G, Girelli M, Taccagni G, Colombo A, Nicosia R, Caruso A, Baronio M, Pagano S, Cova L, Parati E (2001) Human vasculogenesis ex vivo : embryonal aorta as a tool for isolation of endothelial cell progenitors. Lab Invest 81 :875-885
SUMMARY : Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11- to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1]/vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34+/CD31-), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34+/CD31+ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34+/CD31- endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering.
Allam B, Ashton-Alcox KA, Ford SE (2001) Haemocyte parameters associated with resistance to brown ring disease in Ruditapes spp. clams. Dev Comp Immunol 25 :365-375
Brown ring disease (BRD) is a shell disease caused by Vibrio tapetis. This pathogen disturbs the periostracal lamina causing the appearance of a brown conchiolin deposit on the inner face of the shell, within the extrapallial space. Although differences in resistance to BRD have been documented, their relationship to possible defense functions has never been investigated. In this study, flow cytometry was used to analyze cellular parameters in asymptomatic and experimentally infected Ruditapes philippinarum from France and the west coast of the USA. Parallel analyses were made on Ruditapes decussatus, the native European clam, which is highly resistant to BRD. In the haemolymph and extrapallial fluid of animals without BRD, total haemocyte counts, the percentage of granulocytes, and the phagocytic activity against latex beads or V. tapetis by the haemocytes were significantly higher in American R. philippinarum than in French R. philippinarum. In most cases, levels in R. decussatus were the highest of all three groups. Four weeks following challenge with V. tapetis, BRD prevalence reached 52 in American clams and 100% in French specimens, but only 37% in R. decussatus. In symptomatic animals, phagocytosis of V. tapetis increased significantly in the resistant species of clam, R. decussatus, was unchanged in US clams, and decreased significantly in FR specimens when compared to asymptomatic individuals from each population. Ingestion of V. tapetis by haemocytes in the extrapallial fluid, which is in contact with the periostracal lamina, could be the main defense mechanism used to counter the pathogen. Our results suggest that resistance to BRD may well be related to the concentration of granular haemocytes and the phagocytic activity of haemocytes.
Alugupalli KR, Michelson AD, Barnard MR, Leong JM (2001) Serial determinations of platelet counts in mice by flow cytometry. Thromb Haemost 86 :668-671
Elucidation of the pathophysiological basis of platelet disorders in murine models requires a reliable method for the frequent determinations of platelet counts in individual mice. Here, we present a rapid, reproducible and accurate flow cytometric method for enumeration of platelets that involves fluorescent staining of platelets in whole blood with specific antibody and the addition of known numbers of fluorescent beads for standardization of the sample volume. Analysis of platelets obtained by tail bleeding indicated that this sampling procedure did not activate platelets, and that only five microliters of blood were required for platelet counting. Using this method, we followed platelet counts in mice infected with the relapsing fever spirochete Borrelia turicatae for 26 days, and found that this bacterium induces thrombocytopenia, a common manifestation of human relapsing fever. Therefore, this method can be used to follow the number and the activation state of circulating platelets from individual mice over extended periods of time and is applicable to a wide range of murine models of platelet disorders.
Andreasen CB, Akunda JK, Kramer TT (2001) Comparison of heterophil phagocytosis for heterophil-adapted Salmonella enteritidis (HASE) and wild-type Salmonella enteritidis (SE). Avian Dis 45 :432-436
Serial passage of Salmonella enteritidis (SE) yields heterophil-adapted SE (HASE) strains that have resulted in decreased shedding of SE in feces and reduced egg contamination. Additionally, increasing the number of heterophil passages further reduced the number and frequency of fecal shedding. To evaluate SE and heterophil interaction, nine SE strains were fluorescein isothiocyanate-labeled when viable. There were six wild-types : SE TK 474, SE TK 584, SE TK 599, SE TK 600, SE TK 655, and SE TK 657 ; and three HASE strains : TK 499 heterophil adapted five times, TK 598 heterophil adapted six times, and TK 605 heterophil adapted 11 times. Trials were repeated seven times in duplicate with heterophils isolated from seven healthy chickens. Heterophils were incubated with the bacterial strains at 41 C for 15 min, and 10,000 heterophils were analyzed by flow cytometry. Percentage of phagocytosis and mean channel number of fluorescence were compared. Both parameters were significantly increased for all HASE-type strains compared with wild-type, nonadapted SE strains. Increased phagocytosis of HASE bacterial strains may be significant in processing and elimination of the HASE strains and may be related to the protective effect of HASE by decreased shedding of wild-type SE challenge strains.
Andreatta S, Wallinger MM, Posch T, Psenner R (2001) Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis. Cytometry 44 :218-225
BACKGROUND : Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. METHODS : A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements. RESULTS : Three clusters could be detected from the bacterial assemblage. Protozoan grazing had a strong impact on the bacterial community, which could be analyzed in detail at the level of individual subgroups. CONCLUSIONS : Investigation at the level of bacterial subgroups allowed a more detailed analysis than whole-community statistics and delivered essential and ecologically meaningful information. Image analysis proved to be an adequate tool to detect the subgroups without a priori knowledge.
Bernard L, Courties C, Duperray C, Schafer H, Muyzer G, Lebaron P (2001) A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems. Cytometry 43 :314-321
BACKGROUND : Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS : Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS : The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION : This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.
Blom K, Lundin BS, Bolin I, Svennerholm A (2001) Flow cytometric analysis of the localization of Helicobacter pylori antigens during different growth phases. FEMS Immunol Med Microbiol 30 :173-179
Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory. We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H. pylori antigens that may be important virulence factors. All four methods detected the lipopolysaccharide and the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H. pylori bacteria during culture of H. pylori in liquid broth for 11 days. The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H. pylori.
Boettiger D, Huber F, Lynch L, Blystone S (2001) Activation of alpha(v)beta3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of beta3. Mol Biol Cell 12 :1227-1237
Integrin receptors serve as mechanical links between the cell and its structural environment. Using alpha(v)beta3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between alpha(v)beta3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the alpha(v)beta3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the beta3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of alpha(v)beta3 integrin.
Bunthof CJ, Bloemen K, Breeuwer P, Rombouts FM, Abee T (2001) Flow cytometric assessment of viability of lactic acid bacteria. Appl Environ Microbiol 67 :2326-2335
The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells ; in exponential-phase cultures almost all cells were labeled, while 70 degrees C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.
Bunthof CJ, van Schalkwijk S, Meijer W, Abee T, Hugenholtz J (2001) Fluorescent method for monitoring cheese starter permeabilization and lysis. Appl Environ Microbiol 67 :4264-4271
A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.
Button DK, Robertson BR (2001) Determination of DNA content of aquatic bacteria by flow cytometry. Appl Environ Microbiol 67 :1636-1645
The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4’,6’-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells ("dims"). The dims could be formed from laboratory cultivars ; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell(-1) was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell(-1).
Calamita HG, Ehringer WD, Koch AL, Doyle RJ (2001) Evidence that the cell wall of Bacillus subtilis is protonated during respiration. Proc Natl Acad Sci U S A 98 :15260-15263
Several independent experiments suggest that cell walls of Bacillus subtilis are protonated during growth. When cells were grown in the presence of fluorescein-labeled dextran to saturate the cell walls, centrifuged, and suspended in PBS, fluorescence-activated cell sorter analyses revealed the bacteria were only poorly fluorescent. In contrast, when the bacteria were purged with N(2) to dissipate protonmotive force (pmf) fluorescence became intense. Upon reconstitution of the pmf with phenazine methosulfate, glucose, and oxygen, fluorescence declined. Another approach used pH-dependent chemical modification of cell walls. The walls of respiring B. subtilis cells were amenable to carboxylate modification by [(14)C]ethanolamine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The carbodiimide activation of carboxylate groups occurs only in acidic conditions. Upon dissipation of pmf the walls were refractory to chemical modification. Ammonium groups can be condensed with FITC in alkaline medium, but the condensation is very slow in acidic buffers. It was found that the derivatization of the walls with FITC could occur in the absence of pmf. The use of pH-dependent fluorophores and pH-dependent chemical modification reactions suggest that cell walls of respiring B. subtilis cells have a relatively low pH environment. This study shows a bacterium has a protonated compartment. Acidification of cell walls during growth may be one means of regulating autolytic enzymes.
Carro D, Pina B (2001) Genetic analysis of the karyotype instability in natural wine yeast strains. Yeast 18 :1457-1470
Yeast strains isolated from the wild may show high rates of changes in their karyotypes during vegetative growth. We analysed over 500 karyotypes from mitotic and meiotic derivatives of strain DC5, which has a chromosome rearrangement rate of 8.2 x 10(-3) changes/generation. About 70% of the meiotic derivatives of DC5 had low rearrangement rates, with an average of 5.8 x 10(-4) changes/generation, suggesting that karyotype instability behaved as a dominant phenotype. Diploid derivatives with low karyotype variability in mitosis also had low rates of chromosomal rearrangement during meiosis, suggesting that the two phenotypes may be linked. DC5 and some of its meiotic derivatives (both with high and low karyotype variability) had chromosome XII hypervariable bands. Their distribution among the meiotic products indicates that they are not indicators for genetic instability. To our knowledge, data in this paper are the first to indicate that karyotypically unstable yeast strains may give stable progeny at high rates. Understanding of the relevant mechanism(s) may allow the design of genetic strategies to stabilize karyotypes from natural and/or industrial wine yeasts with unacceptable karyotype rearrangement rates.
Chase JC, Dawson-Coates JA, Haddow JD, Stewart MH, Haines LR, Whitaker DJ, Ken ML, Olafson RW, Pearson TW (2001) Analysis of Kudoa thyrsites (Myxozoa : Myxosporea) spore antigens using monoclonal antibodies. Dis Aquat Organ 45 :121-129
A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites, K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to > 220 kDa, whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses, depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated, intact, permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting, indicating that these mAbs have potential for use in developing a field-based diagnostic test.
Chen F, Lu JR, Binder BJ, Liu YC, Hodson RE (2001) Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold. Appl Environ Microbiol 67 :539-545
A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.
Chen LY, Jiang HM, Wu GJ (2001) [Listeria monocytogenes induces thymocyte apoptosis in mice]. Hunan Yi Ke Da Xue Xue Bao 26 :305-308
The murine thymocyte apoptosis induced by Listeria monocytogenes(LM) was detected with morphology, FCM, and DNA electrophoresis. The results were that LM elicited typical morphological changes of thymocyte apoptosis ; the typical apoptosis peak was displayed with FCM, and typical "ladder pattern" with agarose gel electrophoresis. The apoptotic cells were found at 8 h after the mice had infected LM and reached climax at 48 h. The thymus weight significantly reduced at 16 h, and reached the lowest at 48 h after the mice had infected LM. The percentage of apoptotic cells was raised with the increasing of LM. These results suggest that LM induces thymocyte apoptosis in dose- and time-dependent manner.
Cho JW, Baek WK, Suh SI, Yang SH, Chang J, Sung YC, Suh MH (2001) Hepatitis C virus core protein promotes cell proliferation through the upregulation of cyclin E expression levels. Liver 21 :137-142
AIMS/BACKGROUND : The hepatitis C virus (HCV) core protein is known to play an important role in hepatocarcinogenesis. Recent studies have suggested that the increased proliferation rate of hepatocytes is a major risk factor for the development of hepatocellular carcinoma. In this study, we investigated whether the HCV core protein promotes the cell growth rate through the modulation of cyclin E expression levels. METHODS/RESULTS : HCV core stable transfectant Rat-1 cell lines showed a markedly increased proliferation rate compared to mock cells. Cyclin E expression and its associated kinase activities were remarkably increased in HCV core stable transfectants. Cyclin E mRNA levels were also upregulated in these cell lines. CONCLUSIONS : Our data suggest that the HCV core protein promotes cell proliferation through upregulation of the cyclin E expression levels, implying this property of HCV core protein plays an important role in hepatocarcinogenesis.
Cocca BA, Seal SN, D’Agnillo P, Mueller YM, Katsikis PD, Rauch J, Weigert M, Radic MZ (2001) Structural basis for autoantibody recognition of phosphatidylserine-beta 2 glycoprotein I and apoptotic cells. Proc Natl Acad Sci U S A 98 :13826-13831
Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.
Cohen N, Abramov S, Dror Y, Freeman A (2001) In vitro enzyme evolution : the screening challenge of isolating the one in a million. Trends Biotechnol 19 :507-510
The generation of large mutant libraries for in vitro enzyme evolution presents the challenge of effectively screening libraries of 10(4)-10(7) mutants on the basis of simultaneously assaying their biocatalytic activity. In this review, we highlight the main steps involved in this process, describe the alternative approaches to address this challenge, survey the state-of-the-art technology and assess achievements already made. It is anticipated that, as a result of the expected accomplishment of further improvements in high-throughput screening that will allow routine screening of whole libraries, the number of useful new and improved enzymes derived through in vitro enzyme evolution will expand rapidly in the near future.
Connell C, Rutter A, Hill B, Suller M, Lloyd D (2001) Encystation of Acanthamoeba castellanii : dye uptake for assessment by flow cytometry and confocal laser scanning microscopy. J Appl Microbiol 90 :706-712
AIMS : To develop rapid means of distinguishing between cysts and trophozoites of the opportunistic pathogen, Acanthamoeba castellanii, the causative agent of keratitis. METHODS AND RESULTS : Fluorescence of Congo Red, Calcoflor White was specific for the endocyst wall ; trophozoites did not become fluorescent. The anionic oxonol dye, DiBAC4(3), did not penetrate the cytoplasmic membrane after short-term (<5 min) exposure, whereas cysts are permeable and become fluorescent. Confocal scanning laser microscopy confirmed these properties and large populations of organisms were analysed by flow cytometry. CONCLUSION : These data provide a rapid alternative to traditional haemocytometer or plate counts for discrimination of trophozoites from cysts. SIGNIFICANCE AND IMPACT OF THE STUDY : Rapid and precise determination of the growth cycle of a dangerous ocular pathogen.
Coscoy L, Sanchez DJ, Ganem D (2001) A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition. J Cell Biol 155 :1265-1273
Kaposi’s sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion ; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.
Cossarizza A, Salvioli S (2001) Analysis of mitochondria during cell death. Methods Cell Biol 63 :467-486
Cox DL, Radolf JD (2001) Insertion of fluorescent fatty acid probes into the outer membranes of the pathogenic spirochaetes Treponema pallidum and Borrelia burgdorferi. Microbiology 147 :1161-1169
The authors examined the ability of octadecanoyl (C(18)), hexadecanoyl (C(16)) and dodecanoyl (C(12)) fatty acid (FA) conjugates of 5-aminofluorescein (OAF, HAF and DAF, respectively) to insert into the outer membranes (OMs) of Treponema pallidum, Borrelia burgdorferi and Escherichia coli. Biophysical studies have demonstrated that these compounds stably insert into phospholipid bilayers with the acyl chain within the hydrophobic interior of the apical leaflet and the hydrophilic fluorescein moiety near the phospholipid head groups. Consistent with the known poor intrinsic permeability of the E. coli OM to hydrophobic compounds and surfactants, E. coli was not labelled with any of the FA probes. OAF inserted more readily into OMs of B. burgdorferi than into those of T. pallidum, although both organisms were completely labelled at concentrations at or below 2 microg ml(-1). Intact spirochaetes were labelled with OAF but not with antibodies against known periplasmic antigens, thereby confirming that the probe interacted exclusively with the spirochaetal OMs. Separate experiments in which organisms were cooled to 4 degrees C (i.e. below the OM phase-transition temperatures) indicated that labelling with OAF was due to insertion of the probe into the OMs. B. burgdorferi, but not T. pallidum, was labelled by relatively high concentrations of HAF and DAF. Taken as a whole, these findings support the prediction that the lack of lipopolysaccharide renders T. pallidum and B. burgdorferi OMs markedly more permeable to lipophilic compounds than their Gram-negative bacterial counterparts. The data also raise the intriguing possibility that these two pathogenic spirochaetes obtain long-chain FAs, nutrients they are unable to synthesize, by direct permeation of their OMs.
Davis BH (2001) Diagnostic advances in defining erythropoietic abnormalities and red blood cell diseases. Semin Hematol 38 :148-159
The majority of clinical applications of flow cytometry begin with various approaches to remove red blood cells (RBCs) from the clinical sample. However, multiparameter cytometry has and will continue to contribute much to the understanding of the pathophysiology and diagnostic accuracy in the clinical evaluation of human diseases affecting erythroid cells. This review summarizes the diagnostic advances relating to erythroid cells in the areas of immunohematology, laboratory hematology, and infectious disease with particular emphasis on medical evaluation of the anemic patient and fetomaternal hemorrhage. Semin Hematol 38:148-159.
Delgado ML, O’Connor JE, Azorin I, Renau-Piqueras J, Gil ML, Gozalbo D (2001) The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins. Microbiology 147 :411-417
The authors show that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Saccharomyces cerevisiae, previously thought to be restricted to the cell interior, is also present in the cell wall. GAPDH activity, proportional to cell number and time of incubation, was detected in intact wild-type yeast cells. Intact cells of yeast strains containing insertion mutations in each of the three structural TDH genes (tdh1, tdh2 and tdh3) and double mutants (tdh1 tdh2 and tdh1 tdh3) also displayed a cell-wall-associated GAPDH activity, in the range of parental wild-type cells, although with significant differences among strains. A cell wall location of GAPDH was further confirmed in wild-type and tdh mutants by indirect immunofluorescence and flow cytometry analysis with a polyclonal antibody against S. cerevisiae GAPDH. By immunoelectron microscopy, the GAPDH protein was detected at the outer surface of the cell wall of wild-type cells, as well as in the cytoplasm. Western immunoblot analysis of cell wall extracts and cytosol showed that Tdh2 and Tdh3 polypeptides are present in the cell wall, as well as in the cytosol, of exponentially growing cells. Tdh1 is only detected in stationary-phase cells, again in both cytosol and cell wall extracts. The results incorporate the GAPDH of S. cerevisiae, encoded by TDH1-3, into the newly emerging family of multifunctional cell-wall-associated GAPDHs which retain their catalytic activity.
D’Haese E, Nelis HJ, Reybroeck W (2001) Determination of somatic cells in milk by solid phase cytometry. J Dairy Res 68 :9-14
The somatic cell count of milk is routinely determined by the fluoro-opto-electron method and sometimes by the direct epifluorescent filter technique (DEFT). This paper investigates the potential of solid phase cytometry (SPC), a novel technique combining aspects of both the fluoro-opto-electronic method and epifluorescence microscopy for somatic cell counting. In SPC, cells are retained on a membrane filter, fluorescently labelled and automatically detected on the entire membrane filter by means of a laser scanning instrument ChemScan). Fluorescent spots can be visually inspected by an epifluorescence microscope with a computer-driven moving stage. The performance of SPC was compared with that of the fluoro-opto-electronic method using a Fossomatic 360 instrument for 68 milk samples with varying somatic cell counts (10(3)-10(6)/ml). The sample throughput and repeatability of SPC were inferior to those of’ the Fossomatic method and statistical analysis of the method comparison data using the approach of J. M. Bland & D. G. Altman (The Lancet 1986 February 8 pp 307-310) revealed a poor comparability between the two methods. Moreover, problems of milk filterability and the interference of fluorescent particles presently hamper the routine application of SPC. Nevertheless, this method represents the first example of the application of SPC to milk.
Draper J, Mur LA, Jenkins G, Ghosh-Biswas GC, Bablak P, Hasterok R, Routledge AP (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol 127 :1539-1555
A new model for grass functional genomics is described based on Brachypodium distachyon, which in the evolution of the Pooideae diverged just prior to the clade of "core pooid" genera that contain the majority of important temperate cereals and forage grasses. Diploid ecotypes of B. distachyon (2n = 10) have five easily distinguishable chromosomes that display high levels of chiasma formation at meiosis. The B. distachyon nuclear genome was indistinguishable in size from that of Arabidopsis, making it the simplest genome described in grasses to date. B. distachyon is a self-fertile, inbreeding annual with a life cycle of less than 4 months. These features, coupled with its small size (approximately 20 cm at maturity), lack of seed-head shatter, and undemanding growth requirements should make it amenable to high-throughput genetics and mutant screens. Immature embryos exhibited a high capacity for plant regeneration via somatic embryogenesis. Regenerated plants display very low levels of albinism and have normal fertility. A simple transformation system has been developed based on microprojectile bombardment of embryogenic callus and hygromycin selection. Selected B. distachyon ecotypes were resistant to all tested cereal-adapted Blumeria graminis species and cereal brown rusts (Puccinia reconditia). In contrast, different ecotypes displayed resistance or disease symptoms following challenge with the rice blast pathogen (Magnaporthe grisea) and wheat/barley yellow stripe rusts (Puccinia striformis). Despite its small stature, B. distachyon has large seeds that should prove useful for studies on grain filling. Such biological characteristics represent important traits for study in temperate cereals.
Elvang AM, Westerberg K, Jernberg C, Jansson JK (2001) Use of green fluorescent protein and luciferase biomarkers to monitor survival and activity of Arthrobacter chlorophenolicus A6 cells during degradation of 4-chlorophenol in soil. Environ Microbiol 3 :32-42
The recently isolated novel species Arthrobacter chlorophenolicus A6 is capable of growth on and degradation of high concentrations of 4-chlorophenol (up to 350 microg ml(-1)) as the sole carbon and energy source. This strain shows promise for bioremediation of environmental sites contaminated with high levels of chlorophenols. In this study, green fluorescent protein (gfp) or luciferase (luc) genes were used as biomarkers for monitoring cell number and activity, respectively, during degradation of 4-chlorophenol by A. chlorophenolicus cells. The individual marked strains, Arthrobacter chlorophenolicus A6L (luc-tagged) and Arthrobacter chlorophenolicus A6G (gfp-tagged), were monitored during degradation of 250 microg ml(-1) 4-chlorophenol in pure culture and 175 microg g(-1) 4-chlorophenol in soil microcosms. Both gene-tagged strains were capable of cleaning up the contaminated soil during 9 d incubation. During the bioremediation experiments, the luc-tagged cells were monitored using luminometry and the gfp-tagged cells using flow cytometry, in addition to selective plate counting for both strains. The cells remained at high population levels in the soil (evidenced by GFP-fluorescent cell counts) and the A. chlorophenolicus A6L population was metabolically active (evidenced by luciferase activity measurements). These results demonstrate that the Arthrobacter chlorophenolicus A6 inoculum is effective for cleaning-up soil containing high concentrations of 4-chlorophenol.
Esen M, Schreiner B, Jendrossek V, Lang F, Fassbender K, Grassme H, Gulbins E (2001) Mechanisms of Staphylococcus aureus induced apoptosis of human endothelial cells. Apoptosis 6 :431-439
Staphylococcus aureus plays an important role in sepsis, pneumonia and wound infections. Here, we demonstrate that infection with several S. aureus strains results in apoptosis of human endothelial cells. S. aureus induced an activation of cellular caspases, the acid sphingomyelinase, a release of cytochrome c and a stimulation of Jun NH2-terminal kinase (JNK). The significance of these findings is indicated by a prevention of S. aureus triggered apoptosis of human cells deficient for ASM or upon genetic or pharmacological inhibition of JNK or caspases, respectively.
Fillion P, Desjardins A, Sayasith K, Lagace J (2001) Encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with fluid liposome-encapsulated antisense oligonucleotides. Biochim Biophys Acta 1515 :44-54
Antisense therapy for the treatment of bacterial infections is a very attractive alternative to overcome drug resistance problems. However, the penetration of antisense oligonucleotides into bacterial cells is a major huddle that has delayed research and application in this field. In the first part of this study, we defined efficient conditions to encapsulate plasmid DNA and antisense oligonucleotides in a fluid negatively charged liposome. Subsequently, we evaluated the potential of liposome-encapsulated antisense oligonucleotides to penetrate the bacterial outer membrane and to inhibit gene expression in bacteria. It was found that 48.9+/-12% and 43.5+/-4% of the purified plasmid DNA and antisense oligonucleotides were respectively encapsulated in the liposomes. Using fluorescence-activated cell sorting analysis, it was shown, after subtraction of the fluorescence values due to the aggregation phenomenon measured at 4 degrees C, that about 57% of bacterial cells had integrated the encapsulated antisense oligonucleotides whereas values for free antisenses were negligible. The uptake of the encapsulated anti-lacZ antisense oligonucleotides resulted in a 42% reduction of beta-galactosidase compared to 9% and 6% for the encapsulated mismatch antisense oligonucleotides and the free antisense oligonucleotides respectively. This work shows that it is possible to encapsulate relatively large quantities of negatively charged molecules in negative fluid liposomes and suggests that fluid liposomes could be used to deliver nucleic acids in bacteria to inhibit essential bacterial genes.
Franks DJ, Mroske C, Laneuville O (2001) A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells. Biol Proced Online 3 :54-63
In platelets, PGHS-1-dependant formation of thromboxane A(2) is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01 which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique utilizing immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein levels as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 x 10(-8) M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 levels and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein level, which shows a consistent increase over the entire course of differentiation, can be used as an additional and better index by which to monitor megakaryocyte differentiation.
Fuchs BM, Syutsubo K, Ludwig W, Amann R (2001) In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 67 :961-968
One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973-4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083(T) was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows : class I, 3% ; class II, 21% ; class III, 35% ; class IV, 18% ; class V, 16% ; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of E. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.
Funda DP, Tuckova L, Farre MA, Iwase T, Moro I, Tlaskalova-Hogenova H (2001) CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro : lipopolysaccharide activation of epithelial cells revisited. Infect Immun 69 :3772-3781
Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs). However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail. We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins. Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs. Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs. No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 microg/ml. Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs. Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 microg/ml) by interleukin 8 release. Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14. As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS. The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.
Garba ML, Frelinger JA (2001) Intracellular cytokine staining for TGF-beta. J Immunol Methods 258 :193-198
TGF-beta is a well-described immunoregulatory molecule that is produced by most cell types. Many studies have been aimed at investigating the role played by TGF-beta in different cell types and situations. Most methods of measuring TGF-beta have previously relied on enzyme-linked immunosorbent assay (ELISA) or assays of its anti-proliferative effects on various cell lines. Both assays, though useful, cannot be used to effectively identify the cells that are producing TGF-beta in a mixture of cells. It is especially important to know the source and dynamics of TGF-beta secretion in cell culture studies since most cell types are known to be capable of producing TGF-beta. We describe here a technique of qualitative and quantitative measurement of TGF-beta production using flow cytometry. Previous work by others has led to the production of polyclonal and monoclonal antibodies to human and other mammalian TGF-beta. We have developed an intracellular cytokine staining for human TGF-beta using a monoclonal antibody, TB21.
Glickstein L, Edelstein M, Dong JZ (2001) Gamma interferon is not required for arthritis resistance in the murine Lyme disease model. Infect Immun 69 :3737-3743
Lyme arthritis is the most common complication following infection of human individuals with Borrelia burgdorferi sensu stricto. In mice, B. burgdorferi infection leads to arthritis of the tibiotarsal joints. Arthritis severity in mice is under host genetic control, as BALB/c mice developed mild arthritis but C3H/He mice developed severe disease following B. burgdorferi infection. To study the role of gamma interferon (IFN-gamma) in arthritogenesis, targeted mutant mice lacking the IFN-gamma receptor (IFN-gammaR) were infected by inoculation with B. burgdorferi. IFN-gammaR(-/-) and parental 129/SvEv mice developed mild arthritis of similar severity, as determined both by weekly tibiotarsal joint measurements and histopathology at 2 and 5 weeks postinfection. Both strains of mice had the same spirochetal burden in the joints, suggesting that the IFN-gammaR(-/-) mice were not impaired in controlling spirochetal expansion in vivo. The wild-type mice mounted a Th1 response, with a predominance of CD4(+) IFN-gamma(+) T cells observed by flow cytometry. In contrast, the IFN-gammaR(-/-) mice mounted a Th2 response, with a predominance of CD4(+) IL-4(+) T cells. As expected given their cytokine profile, the IFN-gammaR(-/-) mice produced fewer CD8(+) IFN-gamma(+) and MAC-1(+) IL-12(+) cells and less immunoglobulin G2a (IgG2a) than their wild-type counterparts. These results strongly suggest that IFN-gamma is not required for arthritis resistance or as part of an effective immune response against B. burgdorferi.
Goossens PH, Havenga MJ, Pieterman E, Lemckert AA, Breedveld FC, Bout A, Huizinga TW (2001) Infection efficiency of type 5 adenoviral vectors in synovial tissue can be enhanced with a type 16 fiber. Arthritis Rheum 44 :570-577
OBJECTIVE : To obtain an adenoviral vector with increased infection efficiency in the synovial tissue compared with conventional vectors based on adenovirus serotype 5 (Ad5), without compromising the specificity of infection. METHODS : Coxsackie adenovirus receptor (CAR) expression was assessed in cultured synoviocytes. Chimeric adenoviruses based on Ad5 but carrying the DNA encoding the fiber of adenovirus from subgroup B (Adll, 16, 35) or D (Ad24, 28, 33, 45, or 47) were constructed and produced on PER.C6 cells. The gene transfer efficiency of these chimera was tested on cultured synoviocytes and peripheral blood mononuclear cells (PBMC). RESULTS : No surface expression of CAR protein was observed on synoviocytes. CAR messenger RNA expression of synoviocytes was found to be low. Of all fiber chimeric vectors tested, vectors carrying the fiber of Ad16 (Ad5.fib16) were most potent, yielding approximately150 times increased transgene expression in cultured synoviocytes compared with those of Ad5. Flow cytometry showed that the increase in transgene expression was caused by the transduction of higher percentages of synoviocytes and higher gene expression per synoviocyte. Experiments with 500 virus particles/cell of Ad5.GFP or Ad5.fib16.GFP resulted in an infection efficiency of 0.6% and 1% in PBMC and 43% and 76% in synoviocytes, respectively. CONCLUSION : Synoviocytes hardly express CAR, which hampers Ad5-mediated gene transfer. Ad5.fib16 is superior to Ad5 vectors for transducing synoviocytes, without compromising the specificity of infection. Our data suggest that Ad5.fib16-mediated gene transfer to synovial tissue improves the therapeutic window.
Gorbatyuk B, Marczynski GT (2001) Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene. Mol Microbiol 40 :485-497
Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle. Like most eubacteria, C. crescentus has a DnaA homologue that is presumed to initiate chromosome replication. However, the C. crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes. Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator. We therefore created a conditional dnaA C. crescentus strain. Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period. Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage. Cell division also stopped, but not swarmer to stalked cell differentiation. All cells became stalked cells that grew as long filaments. Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped. However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors. Interestingly, transcription from Cori Ps continued unaltered. Therefore, Ps transcription is not sufficient for chromosome replication. Approximately 6-8 h after blocked dnaA expression, cells lost viability exponentially. Coincidentally, beta-galactosidase was induced from one transcription reporter, suggesting an altered physiology. We conclude that C. crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.
Granoff DM, Moe GR, Giuliani MM, Adu-Bobie J, Santini L, Brunelli B, Piccinetti F, Zuno-Mitchell P, Lee SS, Neri P, Bracci L, Lozzi L, Rappuoli R (2001) A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis. J Immunol 167 :6487-6496
Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.
Gregori G, Citterio S, Ghiani A, Labra M, Sgorbati S, Brown S, Denis M (2001) Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining. Appl Environ Microbiol 67 :4662-4670
The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green ; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.
Grondahl G, Sternberg S, Jensen-Waern M, Johannisson A (2001) Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli. Equine Vet J 33 :670-675
Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice.
Gryllos I, Cywes C, Shearer MH, Cary M, Kennedy RC, Wessels MR (2001) Regulation of capsule gene expression by group A Streptococcus during pharyngeal colonization and invasive infection. Mol Microbiol 42 :61-74
Capsular polysaccharide production by group A Streptococcus (GAS) is controlled by transcription of the has operon that encodes the enzymes uniquely required for synthesis of the hyaluronic acid polysaccharide. To investigate the regulation of capsule gene expression during infection, we developed a reporter strain of GAS in which the has operon promoter directed transcription of green fluorescent protein (GFP). Gfp expression was triggered within minutes after introduction of the reporter strain into the peritoneal cavity of mice, as evidenced by the recovery of highly fluorescent GAS from the peritoneum 1 h after challenge. Capsule gene expression was also stimulated in the bloodstream of infected mice, as intensely fluorescent bacteria were observed in blood samples collected after either intraperitoneal or intravenous challenge. Using a similar approach, we also observed rapid induction of capsule gene expression in bacteria inoculated into the pharynx of baboons. Compared to the inoculum, increased green fluorescence was recorded in bacteria recovered from throat swabs collected 1 h after inoculation in all five animals studied. We conclude that introduction of GAS into the pharynx or into deep tissues results in rapid induction of has operon expression, a critical adaptive response that enhances GAS survival in the infected host.
Haidinger W, Szostak MP, Beisker W, Lubitz W (2001) Green fluorescent protein (GFP)-dependent separation of bacterial ghosts from intact cells by FACS. Cytometry 44 :106-112
BACKGROUND : E. coli and Salmonella ghost preparations, produced by applying the PhiX174 protein E-mediated lysis system, contain nonlysed bacteria at a very low percentage. To use the ghosts as vaccines, additional methods have to be identified to remove any viable cell, to end up in totally inactivated ghost fractions. Materials and Methods To increase the purity of ghost fractions, we established a green fluorescent protein (GFP)-dependent "in vivo staining" method to be combined with the E-mediated lysis system. Several gfp expression vectors were constructed, and the corresponding cellular fluorescence was analyzed. Bacterial fluorescence, exclusively preserved in nonlysed cells, was utilized to separate these cells from ghost preparations via flow cytometric sorting. RESULTS : High-level production of GFP prior to induction of the lysis system did not affect bacterial growth rates and caused no inhibitory effects on the subsequent protein E-mediated lysis of the cells. The population of reproductive or inactivated but nonlysed cells was highly fluorescent at mean intensities 215-fold higher than ghosts, which exhibited fluorescence at background level. Fluorescent cells could effectively be separated from ghost preparations via flow cytometric sorting. Cell sorting subsequent to protein E-mediated lysis reduced the number of viable cells within ghost preparations by a factor of 3 x 10(5). CONCLUSIONS : The presented procedure is compatible with the protein E-mediated lysis system, is highly effective in separation of nonlysed fluorescent cells, and may serve as a prototype for ghost-purification in applications where only a minimum number of viable cells within ghost preparations can be tolerated.
Hansen LH, Ferrari B, Sorensen AH, Veal D, Sorensen SJ (2001) Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry. Appl Environ Microbiol 67 :239-244
Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated P(tet) promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.
Harvey KJ, Lukovic D, Ucker DS (2001) Membrane-targeted green fluorescent protein reliably and uniquely marks cells through apoptotic death. Cytometry 43 :273-278
BACKGROUND : An understanding of the molecular processes that comprise the program of physiological cell death demands analytical techniques for the assessment of death events on the level of the individual cell, especially among transfectants and within heterogeneous populations. The utility of available transfection markers is limited by the variability of marker retention and discrimination as cells die. For example, soluble green fluorescent protein (GFP) leaks from dying cells and is not useful when fixation is required ; conversely, transfected beta-galactosidase can be visualized only after fixation and staining. METHODS : We have tested a GFP variant as a marker for the direct identification and visualization of transfected cells. We have explored the utility of this membrane-targeted GFP, the genetic fusion of the enhanced GFP and the farnesylation sequence of p21(Ras) (EGFP-F), in a variety of cell death assays. RESULTS : EGFP-F is retained reliably in unfixed dying cells, permitting numerous events of the cell death process to be analyzed in real time in marked cells. Moreover, the cell rounding and shrinkage associated with the loss of adhesion during cell death result in a characteristic condensed EGFP-F signal. CONCLUSIONS : EGFP-F serves to identify transfectants consistently, independent of their ultimate fate. Cellular condensation of EGFP-F provides a specific and quantitative measure of physiological cell death.
Heininger A, Ulrich M, Priebe G, Unertl K, Muller-Schauenburg A, Botzenhart K, Doring G (2001) The effect of human serum DNAases on the ability to detect antibiotic-killed Escherichia coli in blood by PCR. J Med Microbiol 50 :243-248
PCR has proved superior to conventional blood culture for diagnosing bacteraemia in the presence of antibiotics. Nevertheless, even PCR might yield false-negative results if the template DNA were to be cleaved by serum DNAases after antibiotics had induced bacterial death. To evaluate the cleavage of bacterial template DNA by human serum DNAase I, serum samples inoculated with purified Escherichia coli DNA were incubated with increasing amounts of recombinant human DNAase (rhDNAase) and then examined by a PCR specific for E. coli. As a prerequisite of potential DNAase attack, the release of E. coli DNA after antibiotic-induced bacterial death was quantified by fluorescence microscopy and flow cytometry. Finally, the influence of rhDNAase on the PCR-based detection of antibiotic-killed E. coli in serum was assessed. The results indicated that purified E. coli DNA is remarkably stable in human serum ; positive PCR results did not decrease significantly until the ratio of recombinant human DNAase I:E. coli rose to 106:1. As only 14.8-28.4% of the total E. coli DNA was released after antibiotic killing, the PCR-based detection of E. coli fell by only 10% when cefotaxime-killed E. coli were incubated with rhDNAase. It was concluded that human serum DNAases and antibiotic killing do not compromise the reliability of PCR examinations for bacteraemia.
Herault O, Binet C, Rico A, Degenne M, Bernard MC, Chassaigne M, Sensebe L (2001) Evaluation of performance of white blood cell reduction filters : an original flow cytometric method for detection and quantification of cell-derived membrane fragments. Cytometry 45 :277-284
BACKGROUND : Contamination of blood products by white blood cells leads to a risk of transmission of infectious agents, particularly abnormal prion protein, the probable causative agent of new-variant Creutzfeldt-Jakob disease. Blood product filtration could reduce this risk, but the filtration systems might generate potentially infectious membrane fragments. We developed an original flow cytometric method that allows the detection and quantification of membrane fragments in filtered products and the evaluation of the quantity of destroyed cells. METHODS : This method has four technical requirements : cytofluorometric acquisition of forward scatter parameters on a log scale, use of a fluorescent aliphatic reporter molecule (PKH26-GL) to identify membrane fragments, quantification with fluorescent beads, and the drawing up of a standard curve on the basis of cells destroyed by freezing/thawing to generate cell debris (i.e., quantity of membrane fragments measured versus quantity of destroyed cells). RESULTS AND CONCLUSIONS : This original method can be used to test new filtration devices and it allows optimization of the filtration process or comparison of different filtration systems. We tested the method with three commercial white cell removal filters. We demonstrated that it is possible to evaluate the filter quality, particularly the likelihood of fragment removal during the filtration process.
Hewitt CJ, Nebe-Von-Caron G (2001) An industrial application of multiparameter flow cytometry : assessment of cell physiological state and its application to the study of microbial fermentations. Cytometry 44 :179-187
BACKGROUND : When using traditional microbiological techniques to monitor cell proliferation and viability, stressed, sublethally injured, or otherwise "viable but nonculturable" cells often go undetected. Because of this, such cells often are not considered by mathematical models used to predict bioprocess performance on scale-up and inaccuracies result. Therefore, analytical techniques, decoupled from postsampling growth, are desirable to rapidly monitor individual cell physiologic states during microbial fermentations. METHODS : Microbial cells, including Escherichia coli, Rhodococus sp., and Sacharomyces cerevisiae, were taken at various stages from a range of fermentation processes and stained with one of three mixtures of fluorescent stains : rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxonol/ethidium bromide/propidium iodide. An individual cell’s physiologic state was assessed with a Coulter Epics Elite analyzer based on the differential uptakes of these fluorescent stains. RESULTS : It was possible to resolve an individual cell’s physiologic state beyond culturability based on the functionality of dye extrusion pumps and the presence or absence of an intact polarized cytoplasmic membrane, enabling assessment of population heterogeneity. This approach allows the simultaneous differentiation of at least four functional subpopulations in microbial populations. CONCLUSIONS : Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual microbial cells based on reproductive activity, metabolic activity, and membrane integrity. We have used these techniques extensively for monitoring the stress responses of microorganisms in such diverse areas as bioremediation, biotransformation, food processing, and microbial fermentation ; microbial fermentation is discussed in this article.
Hodzic E, McKisic M, Feng S, Barthold SW (2001) Evaluation of diagnostic methods for Helicobacter bilis infection in laboratory mice. Comp Med 51 :406-412
Disease-susceptible (C3H) and -resistant (B6) immunocompetent and immunodeficient (C3H-scid and B6-rag1) mice were examined up to 10 weeks after inoculation with Helicobacter bilis (a prototype species of proven virulence). Infection was monitored weekly by use of fecal culture, polymerase chain reaction (PCR) nucleic acid amplification, membrane extract enzyme-linked immunosorbent assay (ELISA), and histologic examination. All mice became infected by three to five weeks after inoculation, on the basis of results of culture and PCR analysis of feces. The PCR analysis was more sensitive than culture at determining infection status, particularly during early infection. None of the mice had evidence of disease by week 10. Immunoglobulin G seroconversion was detectable in C3H mice by week eight and in B6 mice by week nine. Results indicated that culture and PCR analysis are more sensitive than is membrane extract ELISA serologic testing for detecting early infection in individual mice, regardless of genotype or immune status. Results underscore the need for improved seroassays for this important group of murine pathogens.
Hopfer RL, Sparks SD, Cox GM (2001) The use of flow cytometry as a tool for monitoring filament formation of fungi. Med Mycol 39 :103-107
Flow cytometry (FC) has the ability to discriminate a variety of cell parameters including cell size and complexity, and fluorescence intensity. As yeast cells or fungal spores germinate they undergo a morphological transformation from round oval shaped cells to elongate filamentous forms. To date, monitoring these events has been performed using microscopic examination. Microscopic examination is a labor intensive process that examines a very small percentage of the total cell population. We have developed a method using FC that is rapid, simple to perform, and reproducible. The major advantages of FC include analysis of a larger number of cells, increased objectivity due to nonselective measurements of all cells in the population studied, and the computer related data analysis capability of the flow cytometer.
Hukuhara T, Wijonarko A (2001) Enhanced fusion of a nucleopolyhedrovirus with cultured cells by a virus enhancing factor from an entomopoxvirus. J Invertebr Pathol 77 :62-67
Fusion of Pseudaletia unipuncta nucleopolyhedrovirus with an armyworm cell line (SIE-MSH-805-F) was studied by means of three fluorescence assays that are based on the relief of fluorescence self-quenching of octadecylrhodamine B chloride (R18). A gradual increase in fluorescence intensity indicative of virus-cell fusion was observed by spectrofluorometry when R18-labeled polyhedron-derived virus was incubated with cultured cells. The fusion was enhanced by the virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus. Lysosomotropic agents had little effect on the virus-cell fusion. The percentage of positively fluorescent cells, as determined by flow cytometry, gradually increased after the addition of labeled virus and was higher in the presence of the EF than in its absence. Confocal microscopy of cultured cells that had been combined with labeled virus showed that the fluorescence appeared first on their surface. The plasma membrane of cultured cells had specific affinity to the EF, as revealed by indirect immunofluorescence microscopy.
Jack DL, Jarvis GA, Booth CL, Turner MW, Klein NJ (2001) Mannose-binding lectin accelerates complement activation and increases serum killing of Neisseria meningitidis serogroup C. J Infect Dis 184 :836-845
The capacity for different lipo-oligosaccharide (LOS) sialylation patterns of Neisseria meningitidis serogroup C to influence the binding and function of the innate humoral component, mannose-binding lectin (MBL), was investigated. By use of flow cytometry and immunogold electron microscopy, a clinical isolate with reduced endogenous LOS sialylation was found to bind more MBL than did strains with higher endogenous sialylation. MBL binding was reduced but not ablated if the same strain was allowed to exogenously sialylate its LOS structures after incubation with cytidine-5’-monophospho-neuraminic acid. MBL binding led to an increased rate of complement activation, with enhanced deposition of the complement components C4 and C5b-9, and this correlated with an increase in bactericidal activity. LOS sialylation appears to be an important determinant of MBL binding to N. meningitidis and can modulate complement-dependent killing of the bacterium. These findings could explain the observed susceptibility to meningococcal disease of individuals genetically deficient in MBL.
Jansen WT, Vakevainen-Anttila M, Kayhty H, Nahm M, Bakker N, Verhoef J, Snippe H, Verheul AF (2001) Comparison of a classical phagocytosis assay and a flow cytometry assay for assessment of the phagocytic capacity of sera from adults vaccinated with a pneumococcal conjugate vaccine. Clin Diagn Lab Immunol 8 :245-250
Antibody- and complement-mediated phagocytosis is the main defense mechanism against Streptococcus pneumoniae. A standardized, easy to perform phagocytosis assay for pneumococci would be a great asset for the evaluation of the potential efficacy of (experimental) pneumococcal vaccines. Such an assay could replace the laborious phagocytosis assay of viable pneumococci (classical killing assay). Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was compared with the conventional killing assay and enzyme-linked immunosorbent assay (ELISA), using sera obtained from adults pre- and postvaccination with either a bivalent conjugate, a tetravalent conjugate, or the 23-valent polysaccharide vaccine. Highly significant correlations were observed between flow assay phagocytosis titers, killing assay phagocytosis titers, and ELISA antibody titers for serotype 6B and 23F as well. For serotype 19F, strong correlations were only observed between killing assay and ELISA titers. A potential drawback of the flow assay might be the low sensitivity compared with that of the killing assay. The choice of what assay to use, however, will depend on the objectives of the assay. When speed, easy performance, sample throughput, improved worker safety, absence of influence of antibiotics, and absence of false positives are the major criteria, the flow assay is the method of choice. When higher sensitivity is the major requirement, the classical killing assay should be used.
Jao HC, Yang RC, Hsu HK, Hsu C (2001) The decrease of PKCalpha is associated with hepatic apoptosis at early and late phases of polymicrobial sepsis. Shock 15 :130-134
The present study investigates the relationship between the PKC-alpha and hepatic apoptosis during sepsis. Cecal ligation and puncture- (CLP) induced animal model of polymicrobial sepsis was used, with early and late sepsis referring to those animals sacrificed at 9 and 18 h, respectively, after CLP. The expressions of PKCalpha and Bcl-2 family proteins as well as poly(ADP-ribose) polymerase (PARP) cleavage were quantified to evaluate the possible factors involved in the hepatic cell death during sepsis. The apoptosis of hepatocytes under septic condition or hepatocytes treated with PKCalpha antisense was evaluated by gel electrophoresis and/or flow cytometry after Annexin-V-Fluos and propidium iodide staining. The results indicated that (1) the protein expression of membrane-associated PKCalpha was decreased at early (P < 0.05) and late (P < 0.01) sepsis ; (2) the protein expressions of Bcl-2 and Bcl-xL were decreased, whereas Bax expression was increased at late sepsis ; (3) the percentage of PARP cleavage was increased at early (P < 0.05) and late (P < 0.01) sepsis ; (4) severe DNA fragmentation was observed at late sepsis ; (5) the apoptotic cell population was increased at early and late sepsis ; and (6) the percentage of apoptotic cell population in PKCalpha antisense-treated cells was significantly higher than that in untreated cells. These results suggest that inactivation of PKCalpha may play an important role in modulating hepatic apoptosis during sepsis and the apoptosis is closely associated with the alterations of Bcl-2 family proteins.
Jimenez L (2001) Rapid methods for the microbiological surveillance of pharmaceuticals. PDA J Pharm Sci Technol 55 :278-285
The use of rapid microbiological methods in pharmaceutical laboratories has improved the quality control analysis of water, products, raw materials, and enhanced the antimicrobial effectiveness testing of pharmaceutical finished products. Rapid release of samples has resulted in the optimization of manufacturing, product testing, and release allowing high throughput and simultaneous analysis of pharmaceutical formulations. ATP Bioluminescence, Impedance, Direct Viable Counts, and Flow Cytometry determine the total microbial content in a given pharmaceutical sample while PCR and Immunoassays detect the presence or absence of specific microbial species. Rapid methods provide reliable and cost effective analysis for the microbiological evaluation of pharmaceutical environments. The dramatic reduction in detection times and analysis, e.g., from 30 hours to 90 minutes, by using rapid methods will ultimately lead the pharmaceutical industry closer to real time monitoring of processes and samples.
Johnson PE, Lund ML, Shorthill RW, Swanson JE, Kellogg JL (2001) Real time biodetection of individual pathogenic microorganisms in food and water. Biomed Sci Instrum 37 :191-196
The primary objective of this research is to examine the feasibility of using an innovative technique based on laser-induced fluorescence coupled with flow cytometry to detect pathogenic microorganisms in food or water in real time. Our initial application is the rapid detection of E. coli O157:H7 in ground beef. The research performed demonstrated conclusively that this approach is feasible, and that the technique has key advantages over current alternatives including : it is (1) able to totally examine a large volume of food or water in real time, (2) capable of detecting single microorganisms (alternative techniques require in excess of 10(4) microorganisms), (3) intrinsically automatic, and (4) sensitive only to the selected bacteria. We have demonstrated the feasibility of detecting individual E. coli bacteria with a breadboard system. The performance of this system allows for rapid detection of individual specific pathogenic microorganisms. Two of the most significant commercial applications of this technique are the detection of infectious microorganisms in contaminated food and water. Food-borne microbial pathogens account for approximately 7 million illnesses and 9,000 deaths in the U.S. annually, with an estimated economic loss of at least $6 billion . In addition, this method has the potential for a broad range of other commercial applications, including the detection of small numbers of molecules, such as the ultrasensitive detection of explosives and groundwater contaminants.
Joseph TD, Look DC (2001) Specific inhibition of interferon signal transduction pathways by adenoviral infection. J Biol Chem 276 :47136-47142
Adenoviral evolution has generated strategies to resist host cell antiviral systems, but molecular mechanisms for evasion of interferon (IFN) effects by adenoviruses during late-phase infection are poorly defined. In this study, we examined adenovirus type 5 (AdV) effects on IFN-gamma-dependent gene expression and Janus family kinase-signal transducer and activator of transcription signaling components in human tracheobronchial epithelial cells. We found that AdV infection specifically inhibited IFN-gamma-dependent gene expression in airway epithelial cells without evidence of epithelial cell injury or generation of a soluble extracellular inhibitor. Furthermore, infection with AdV for 18-24 h blocked phosphorylation/activation of the Stat1 transcription factor that regulates IFN-gamma-dependent genes. Although AdV also inhibited IFN-alpha-dependent phosphorylation of Stat1 and Stat2, interleukin-4-dependent phosphorylation of the related transcription factor Stat6 was not affected, indicating that the virus selectively affected specific signaling pathways. Our results indicate that AdV inhibition of the IFN-gamma signal transduction cascade occurs through loss of ligand-induced receptor complex assembly and consequent component phosphorylation and suggest that lack of complex assembly is due to decreased expression of the IFN-gammaR2 chain of the IFN-gamma receptor. IFN-gammaR2 is required at an early step in Janus family kinase-signal transducer and activator of transcription pathway activation and is expressed at low levels in airway epithelial cells, supporting the concept that adenoviral down-regulation of the level of this IFN-gamma receptor component allows for persistent modulation of IFN-gamma-dependent gene expression.
Kao CL, Wu MC, Chiu YH, Lin JL, Wu YC, Yueh YY, Chen LK, Shaio MF, King CC (2001) Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture. J Clin Microbiol 39 :3672-3677
Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.
Kim K, Eaton MS, Schubert W, Wu S, Tang J (2001) Optimized expression of green fluorescent protein in Toxoplasma gondii using thermostable green fluorescent protein mutants. Mol Biochem Parasitol 113 :309-313
Koch B, Worm J, Jensen LE, Hojberg O, Nybroe O (2001) Carbon limitation induces sigma(S)-dependent gene expression in Pseudomonas fluorescens in soil. Appl Environ Microbiol 67 :3363-3370
Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the sigma(S)-dependent Escherichia coli promoter P(fic) and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of beta-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing beta-galactosidase from P(fic). Changes in cell size and expression of beta-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of beta-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil beta-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted.
Kuhn JF, Godshall CJ, Scott MJ, Franklin GA, Rowe SA, Peyton JC, Cheadle WG (2001) Immediate and delayed leukocyte apoptosis in two models of peritonitis. Inflammation 25 :389-397
Leukocyte apoptosis is an energy-dependent process that facilitates resolution of the cellular inflammatory response. Levels of apoptosis can be accelerated or inhibited after exposure to various stimuli. To compare apoptosis in transmigrated leukocytes, two models of peritonitis in mice were used that both cause leukocyte influx into the peritoneal cavity : (1) intraperitoneal thioglycollate administration producing a sterile peritonitis and (2) cecal ligation and puncture (CLP) producing a polymicrobial bacterial peritonitis. Samples of blood and peritoneal exudate cells (PEC) were collected at multiple time points after induction of peritonitis. Leukocytes were either fixed immediately to determine an immediate apoptosis level or cultured for 24 h to determine a delayed apoptosis level. Apoptosis was assessed using terminal uridine-triphosphate nick-end labeling (TUNEL) assay, flow cytometry, and confocal microscopy. Leukocyte influx into the peritoneal cavity was confirmed in both models. At all time points, and in both models, there was increased immediate apoptosis in PEC compared with unmanipulated controls and this increase was maximal in CLP after 18 h, although it appeared to remain at a stable level in the sterile peritonitis model by 3 h. There was also an increase in PEC delayed apoptosis at early time points in both models, again maximal at 18 h for CLP, with the levels being significantly higher than the thioglycollate model at 6 h and 18 h. The mice had a relative peripheral neutropenia at 6 h after CLP, but not post thioglycollate injection, and this persisted until 42 h. Lung and liver MPO levels were elevated in CLP but did not increase after thioglycollate. There was no increase in immediate peripheral leukocyte apoptosis in either model, but an increase in delayed peripheral leukocyte apoptosis was observed by 18 h in both models. Peripheral leukocyte CD1lb expression, which is a marker of activation, was also persistently elevated in the CLP model, but not in sterile peritonitis. In conclusion, CLP is a more potent stimulus for apoptosis of leukocytes than their migration to the site of inflammation alone, as occurs in the thioglycollate model. Blood leukocyte apoptosis also appears not to be dependent on CD11b expression, and therefore activation status.
Laguna R, Romo J, Read BA, Wahlund TM (2001) Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi. Appl Environ Microbiol 67 :3824-3831
Emiliania huxleyi is a unicellular marine alga that is considered to be the world’s major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.
Leakey A, Hirst R, La Brooy J (2001) A low molecular weight factor is a significant mediator of non-opsonic neutrophil activation by Helicobacter pylori. J Med Microbiol 50 :787-794
Helicobacter pylori is believed to trigger neutrophil activation through several factors, including the H. pylori neutrophil-activating protein (HpNAP). The aim of this study was to characterise the factors within H. pylori cell-free extracts that stimulate neutrophil activation. Neutrophil activation was found to be dose-dependent and exhibited considerable variation between different clinical isolates. Activity was attributable to more than one protein factor. A low mol. wt fraction of <3 kDa was found to contribute a large proportion of the neutrophil-stimulating activity within H. pylori cell-free extract. Additional activity was provided by a high mol. wt fraction, possibly representing HpNAP. An inhibition ELISA and neutralisation experiments failed to identify or exclude formyl methionyl leucyl phenylalanine as the active factor within the low mol. wt fraction. The importance of the putative, low mol. wt neutrophil-activating factor may have been overlooked by those studies that have used concentrated H. pylori extracts.
Lebaron P, Servais P, Agogue H, Courties C, Joux F (2001) Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems ? Appl Environ Microbiol 67 :1775-1782
The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.
Lee GC, Song BH, Lee CH (2001) Increase in the expression of human leukocyte antigen class I in human fibroblasts by soluble factors secreted from human cytomegalovirus-infected cells. Mol Cells 11 :392-398
The human cytomegalovirus (HCMV) is known to downregulate the expression of the human leukocyte antigen (HLA) class I for escape from immune surveillance. In order to understand the HCMV immune evasion mechanism, expression of HLA class I on the surface of HCMV-infected cells was investigated. A decrease in the HLA class I expression was observed at higher MOI ; whereas at a lower MOI a slight increase in the HLA class I expression was observed. When HCMV-infected and uninfected cells were separately prepared on coverslips and co-cultured, the increased HLA class I expression was observed in uninfected cells. Treatment of the uninfected cells with the culture supernatant from HCMV-infected cells resulted in an increase in the HLA class I expression. A biochemical analysis of the HCMV-infected cell culture supernatant revealed the presence of interferon (IFN) beta interleukin (IL)-1beta, and IL-6. The HLA class I-enhancing activity of the culture supernatant was mimicked by IFN beta, but not by IL1-beta or IL-6, and was partially reversed by pretreatment with an antibody to IFN beta. Therefore, it appears that the HCMV infection of human foreskin fibroblast cells induces interferon beta and other soluble factor(s) that are responsible for the up-regulation of the HLA class I expression.
Lee MS, Yen CY, Ueng SW, Shih CH, Chao CC (2001) Signal transduction pathways and apoptosis in bacteria infected chondrocytes. J Orthop Res 19 :696-702
The mechanism underlying chronic destructive arthropathy after pyogenic arthritis is not clear. This study evaluated the role of apoptosis in Staphylococcus aureus infected human articular chondrocytes and investigated the signal transduction pathways activated by bacterial infection. Chondrocytes cultured in monolayer were challenged with bacteria for 6 h and were analyzed after incubation for 2, 18, and 24 h. Chondrocytes showed morphologic and biochemical evidences of apoptosis after infection and the following incubation period. Although treatment with extensive washing and vancomycin could ameliorate the amount of apoptosis from 31% to 15% at 2 h, from 48% to 23% at 18 h, and from 58% to 33% at 24 h, the infected samples with treatment still had higher amount of apoptosis than the un-infected controls (ANOVA P < 0.001). Accompanying with the increasing amount of apoptosis, the caspase activity was upregulated in bacteria infected samples and remained high in samples with treatment (ANOVA P < 0.05). Signal transduction pathways activated by bacterial infection were assessed by co-transfection technique. After infection, the c-Jun N-terminal kinase, extracellular signal-regulated kinase, and cyclic AMP-dependent protein kinase activities were elevated by 7.6-, 7.3-, and 3.2-fold, respectively, compared to the uninfected controls. The data support the hypothesis that human chondrocytes will undergo apoptosis after infection by a single organism. Apoptosis and activated intracellular kinase activities may be related to the pathogenesis of post-infectious destructive arthropathy.
Lee YK, Soh BS, Wu JH (2001) Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis. Cytometry 43 :82-85
BACKGROUND : The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD : This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS : It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS : With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.
Letellier A, Messier S, Lessard L, Chenier S, Quessy S (2001) Host response to various treatments to reduce Salmonella infections in swine. Can J Vet Res 65 :168-172
Host response was evaluated following the administration of various treatments, such as probiotics, prebiotics, and vaccination, to reduce Salmonella in swine. Response to the treatments were studied by the evaluation of phagocytosis rates by flow cytometry, by studying the activation of whole-blood phagocytes by bioluminescence, the production of IgA against S. Typhimurium, and by histopathology. Significant differences were observed in the activation of whole-blood phagocytes in all groups of treated pigs (P = 0.0001). In SC54 vaccinated pigs, a significant reduction of Salmonella in the ileum was observed (P < 0.05) and the production of IgA against S. Typhimurium was higher in this group in comparison to uninfected control pigs (P = 0.0007). Furthermore, significant histopathological (P < 0.05) changes were observed in SC54 vaccinated pigs. Villus height and mucus and goblet cells density in the small intestine were reduced in vaccinated pigs in comparison to infected control pigs. Taken together, these findings suggest that SC54 vaccine can stimulate local immunity and reduce the presence of Salmonella in the ileum in swine. Use of SC54 vaccine should thus be considered in further field experiments.
Li WK, Dickie PM (2001) Monitoring phytoplankton, bacterioplankton, and virioplankton in a coastal inlet (Bedford Basin) by flow cytometry. Cytometry 44 :236-246
BACKGROUND : To establish the prevailing state of the ecosystem for the assessment of long-term change, the abundance of microbial plankton in Bedford Basin (Nova Scotia, Canada) is monitored weekly by flow cytometry. METHODS : Phytoplankton are detected by their chlorophyll autofluorescence. Those that contain phycoerythrin are designated as Synechococcus cyanobacteria or cryptophyte algae according to the intensity of light scatter. Bacteria and viruses are stained with DNA-binding fluorochromes and detected by green fluorescence. Distinction is made between bacterial and viral subpopulations exhibiting high and low fluorescence. RESULTS : Time series data are presented for weekly observations from 1991 to 2000. Weekly averages are computed for the complete annual cycle of temperature, salinity, river discharge, nitrate, phosphate, silicate, chlorophyll, total phytoplankton including Synechococcus and cryptophytes, total bacteria including high and low-fluorescence subpopulations, and total viruses including high and low-fluorescence subpopulations. CONCLUSIONS : The microbial biomass in the surface water of Bedford Basin is dominated by phytoplankton. The spring bloom of phytoplankton represents a maximum in algal biovolume, but not in cell number. Phytoplankton, bacteria, and viruses all attain their annual numerical maxima between the summer solstice and the autumn equinox. A vigorous microbial loop and viral shunt is envisioned to occur in the summer.
Lin TJ, Issekutz TB, Marshall JS (2001) SDF-1 induces IL-8 production and transendothelial migration of human cord blood-derived mast cells. Int Arch Allergy Immunol 124 :142-145
BACKGROUND : Mast cell numbers and expression of chemokines are known to increase in the context of angiogenesis and inflammation, but the mechanisms by which this occurs are not understood. Stromal-derived factor-1 (SDF-1) is an important chemokine in angiogenesis and cell migration. The effects of SDF-1 on human mast cells were examined. METHODS : Expression of the SDF-1 receptor CXC chemokine receptor 4 (CXCR4) on mast cells was examined by RT-PCR and flow cytometry. The ability of labeled cord blood-derived mast cells to migrate across HUVEC monolayers in response to SDF-1 was determined. The cytokine and chemokine responses of cord blood-derived mast cells to SDF-1 treatment over 24 h were examined by ELISA. RESULTS : Cord blood-derived human mast cells expressed the CXCR4 receptor for SDF-1 and migrated across HUVEC monolayers in response to this chemokine. Treatment of cord blood-derived mast cells with SDF-1 did not induce degranulation or the production of several cytokines but did induce a highly selective IL-8 response. CONCLUSION : Human mast cells can both migrate across vascular endothelium and produce the pro-angiogenic chemokine IL-8 in response to SDF-1. These responses may be important in angiogenic processes.
Lipoglavsek L, Avgustin G (2001) Application of flow cytometry for ecological monitoring of the rumen microbial ecosystem. Folia Microbiol (Praha) 46 :53-55
Flow cytometry in combination with fluorescently labeled ribosomal RNA oligonucleotide probes was used for enumeration and monitoring of ruminal bacteria. The polyanionic azo dye Trypan Blue was used for discrimination between live bacterial cells and inorganic particles and the separation was further improved by lysozyme treatment and sonication. Cy3-labeled universally conserved probe EUB338 and FITC-labeled Prevotella bryantii specific probe PBB14 were used for in situ hybridization in mixed culture experiments and in samples of crude rumen fluid. The results were analyzed by flow cytometry. The separation of P. bryantii and Butyrivibrio fibrisolvens, another ruminal bacterium, in mixed culture experiments was satisfactory and enabled monitoring of these bacteria in a test system. P. bryantii cells were detected in crude rumen fluid samples only after supplementation with pure culture cells ; this implicates a low concentration of P. bryantii cells in vivo (less than 100/nL, i.e. 10(5) per mL).
Lipski A, Friedrich U, Altendorf K (2001) Application of rRNA-targeted oligonucleotide probes in biotechnology. Appl Microbiol Biotechnol 56 :40-57
Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.
Liu L, Zou P, Guo R, Xiao J, Xu Z (2001) Bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand gene. J Tongji Med Univ 21 :215-218
The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated. FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology, PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine 2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels of mFasL protein on NIH3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3 cells membrane was investigated by the inducing apoptosis of Fas+ Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately co-cultured with Yac-1 cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants from twelve resistant clones was 8.5 x 10(5) colony-forming-unit (CFU)/ml. The NIH3T3 cells transfected by above supernatants had a higher level of mFasL (53.81 +/- 6.9%), and significantly induced the apoptosis of Fas+ Yac-1 cells (56.78 +/- 4.5%), as both were cocultured for 5 h at 1:1 ratio, whereas it is 7.08 +/- 3.4% in control group (P < 0.01). Supernatant from PLFIN-PA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.
Liu WT, Sun JR, Lin CH, Kuo RL, Kung SH (2001) An indicator cell assay for detection of human cytomegalovirus based on enhanced green fluorescent protein. J Virol Methods 96 :85-92
An indicator cell line (ML-UL54-EGFP) for the detection of human cytomegalovirus (HCMV) by a simple and direct method was developed. The stable line was constructed by introducing into mink lung cells an expression cassette that contains the enhanced green fluorescent protein (EGFP) reporter gene under the control of an HCMV-inducible promoter. The promoter was from the upstream region of the HCMV UL54 (pol) gene, an early gene promoter that is activated in the early phase of HCMV infection. Following infection with HCMV for 48 h, the stable line expressed well detectable level of the EGFP as observed under a fluorescence microscope. The sensitivity of the indicator cell assay is at least comparable with that of a plaque assay as assessed with a panel of HCMV strains. There were no detectable fluorescent cells after inoculations with several viruses other than HCMV, indicating high specificity. Analysis with flow cytometry revealed that the induced fluorescence from the infected cells was proportional to the titer of HCMV inoculated, making it possible to quantify HCMV infectious particles. In summary, the EGFP-based indicator cell line is of potential use for rapid detection and quantification of HCMV in clinical specimens.
Lloyd D, Turner NA, Khunkitti W, Hann AC, Furr JR, Russell AD (2001) Encystation in Acanthamoeba castellanii : development of biocide resistance. J Eukaryot Microbiol 48 :11-16
Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites. Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.
Ludovico P, Sansonetty F, Corte-Real M (2001) Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry. Microbiology 147 :3335-3343
In yeast the use of rhodamine 123 (Rh123) has been restricted to the evaluation of mitochondrial respiratory function including the discrimination between respiratory-competent and -deficient cells. This study describes the optimization and validation of a low-concentration Rh123 staining protocol for the flow-cytometric assessment of mitochondrial membrane potential (Delta Psi m) changes in whole yeast cells. The optimized protocol was validated by the use of compounds that specifically affect mitochondrial energetics. Epifluorescence microscopy was used to monitor Rh123 distribution within the cell. Incubation of yeast cell suspensions with Rh123 (50 nM, 10 min) gave minimal non-specific binding and cytotoxicity of the dye. The ratio (R) between the green fluorescence and forward scatter (both measured as log values) was used to measure Delta Psi m with only little dependence on cell ’volume’ and mitochondrial concentration. Cells treated with mitochondrial membrane de- or hyper-polarizing agents displayed a decrease and an increase of R values respectively, indicating that changes of the Rh123 distribution in cells indicate variations in the Delta Psi m. Live and dead cells also displayed significantly different R values. The method described here allows assessment of Delta Psi m changes in whole yeast cells in response to a given drug. Moreover, the relationship between drug effects and disorders of mitochondrial energetics might be addressed.
Lundstrom AM, Blom K, Sundaeus V, Bolin I (2001) HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains. Microb Pathog 31 :243-253
Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90% ; Hel 344, 60% ; CCUG 17875, 61% ; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath.
Makristathis A, Rokita E, Pasching E, Apfalter P, Willinger B, Rotter ML, Hirschl AM (2001) Urease prevents adherence of Helicobacter pylori to Kato III gastric epithelial cells. J Infect Dis 184 :439-445
The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated. Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB ::TnKm or P1ureA ::TnMax5) or producing the inactive apoprotein (N6ureG ::TnKm), and with urease-positive clones recovered after complementation of N6ureB ::TnKm with ureAB. Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry. With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P<.001). With clone K2(3), producing larger amounts of urease than N6, a significant reduction of bacteria load was observed, in comparison with the wild type (P<.001). N6ureG ::TnKm showed adherence characteristics similar to those of N6. The role of urease in internalization was not clear. Thus, urease significantly inhibits H. pylori adherence to Kato III cells by a mechanism largely independent of enzymatic activity.
Malacrino P, Zapparoli G, Torriani S, Dellaglio F (2001) Rapid detection of viable yeasts and bacteria in wine by flow cytometry. J Microbiol Methods 45 :127-134
The potential of using flow cytometry (FCM) in combination with fluorescent dyes for rapidly estimating counts of yeasts and malolactic bacteria in laboratory media and wines was examined. In general, there was a good correlation (regression coefficient, 0.94) between viable counts of yeasts determined by FCM and by standard plate assay. The FCM detection limit of yeasts in YPDE medium and in Pinot noir must was 10(3) cells/ml. The lowest bacterial concentration detected by FCM was 10(4) cells/ml. When yeast and malolactic bacteria populations were simultaneously analysed in wine by FCM without any previous sample treatment, difficulties were encountered in the count of bacterial cells due to their size, which is similar to natural debries present in wine. However, after the optimisation of the sample preparation, the technique appeared promising in determining the presence of such microorganisms in wine with one single measurement. Because it is rapid and easy to use, flow cytometry can be considered a useful method for microbiological quality control in wineries and for the investigation of the growth dynamics of microorganisms in wine.
Manohar M, Baumann DO, Bos NA, Cebra JJ (2001) Gut colonization of mice with actA-negative mutant of Listeria monocytogenes can stimulate a humoral mucosal immune response. Infect Immun 69 :3542-3549
We used Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study the gut mucosal immune responses following oral infection. We employed a germfree (GF) mouse model to try to accentuate the development of a humoral mucosal immune response in the gut, and we used oral colonization with one of the mutants, actA-negative (DeltaactA) L. monocytogenes, to restrict infection largely to the gut. The DeltaactA mutant was able to colonize the intestinal mucosa of formerly GF mice for long periods of time without causing disease while eliciting secretory immunoglobulin A (IgA) responses, as evidenced by gut tissue fragment culture assays. Flow cytometric analyses and immunohistochemical methods showed the development of only minimal germinal center reactions (GCR) in Peyer’s patches and more robust GCR in mesenteric lymph nodes. Pronounced increases in total (natural) IgA production occurred in gut tissues by day 7 and were maintained for up to 90 days. Levels of specific IgA were modest in gut tissues on day 14, increased until day 76, and stabilized at day 90. We also observed a significant rise in serum IgA and IgG1 levels following oral infection by listeriae. Upon colonization, the organisms mainly infected the intestines and intestinal lumen, and we only sporadically observed few colony-forming bacteria in the liver and spleen. We observed a marked rise in IgA-secreting cells, including listeria-specific IgA antibody-secreting cells, in the lamina propria of the small intestine by enzyme-linked immunospot assays. To ascertain whether some of the IgA was specific for listeriae, we performed Western blot analysis to test the reactivity of IgA from fragment cultures to antigens in sonicates of L. monocytogenes. We detected IgA binding to antigenic proteins with molecular masses of 96, 60, 40, and 14 kDa in the Listeria sonicates.
Marie D, Partensky F, Vaulot D, Brussaard C (2001) Enumeration of phytoplankton, bacteria, and viruses in marine samples. Curr Protoc Cytom Chapter 11 :Unit 11 11
For many years, a small but dedicated group of scientists have been using flow cytometry for the evaluation of marine microorganisms. One of these scientists now provides us with a detailed series of protocols in this area, spelling out the variations in method and instrument operation that are crucial to the successful extraction of quality flow data from marine organisms. In addition, the use of a number of less frequently employed fluorescent probes gives some insight into alternative staining procedures. As our collection of microbiologically oriented techniques increases, this knowledge database becomes invaluable.
Marie D, Simon N, Guillou L, Partensky F, Vaulot D (2001) DNA/RNA analysis of phytoplankton by flow cytometry. Curr Protoc Cytom Chapter 11 :Unit 11 12
The past 10 years or so have seen the combination of molecular and biochemical techniques within the confines of cytometry. The use of flow cytometry in microbiology is finally coming of age. This unit carefully defines the criteria for evaluation of DNA and RNA in phytoplankton. Of course not everyone works with phytoplankton, but the methods outlined are very appropriately representative for other organisms. In addition, the unit discusses the methods for evaluating cell cycle and discriminating specific taxa using fluorescent oligonucleotide probes targeted to 18S rRNA.
Maruyama J, Nakajima H, Kitamoto K (2001) Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS. Biosci Biotechnol Biochem 65 :1504-1510
Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidutans histone H2B protein tagged by EGFP (H2B ::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B ::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B ::EGFP were put through FACS (fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.
Masse M (2001) Universal leukoreduction of cellular and plasma components : process control and performance of the leukoreduction process. Transfus Clin Biol 8 :297-302
Many countries in Europe and over the world are currently or will be concerned in the near future, by the implementation of universal leukoreduction (ULR) for red blood cells (RBC), platelets (PT) and now also for plasma. Recommended by several advisory committees, this decision to implement ULR must be considered as a recognition of the benefit of early leukocyte removal, and also as a precautionary measure to increase blood safety. The leukodepletion technology for RBC, PT and plasma has become increasingly more elaborated and integrated in the collection or in the component preparation process. To reach this aim and to assure that the end-products meet local specifications (1 or 5 x 10(6) residual leukocytes), a process control and validation program for leukoreduction has been described in the specific guidelines. Tested on a wide scale by a group of centers, flow cytometry is emerging as reference method for residual leukocyte enumeration. Validation protocols (linearity, precision, accuracy) have been defined in numerous national or international studies (PSL and BEST Working Party). The sensitivity of the method is greatly improved by concentration of the sample, with a detection limit equivalent to 10 cells/mL for RBC or PT, and 0.5 cells/mL for plasma. Furthermore, monitoring of the performance of the leukoreduction process includes a quality control program based on a general statistical model with a parametric or non parametric approach, sampling plan, ongoing control, process capability assessment, confidence limit, detection of failure, and estimation of the non conforming units rate.
McGinnes LW, Sergel T, Chen H, Hamo L, Schwertz S, Li D, Morrison TG (2001) Mutational analysis of the membrane proximal heptad repeat of the newcastle disease virus fusion protein. Virology 289 :343-352
Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, that have been implicated in the fusion activity of the protein. Peptides from these two domains form a six-stranded, coiled-coil with the HR1 sequences forming a central trimer and three molecules of the HR2 helix located within the grooves in the central trimer (Baker et al., 1999, Mol. Cell 3, 309 ; Zhao et al. 2000, Proc. Natl. Acad. Sci. USA 97, 14172). Nonconservative mutations were made in the HR2 domain of the Newcastle disease virus fusion protein in residues that are likely to form contacts with the HR1 core trimer. These residues form the hydrophobic face of the helix and adjacent residues ("a" and "g" positions in the HR2 helical wheel structure). Mutant proteins were characterized for effects on synthesis, steady-state levels, proteolytic cleavage, and surface expression as well as fusion activity as measured by syncytia formation, content mixing, and lipid mixing. While all mutant proteins were transport competent and proteolytically cleaved, these mutations did variously affect fusion activity of the protein. Nonconservative mutations in the "g" position had no effect on fusion. In contrast, single changes in the middle "a" position of HR2 inhibited lipid mixing, content mixing, and syncytia formation. A single mutation in the more carboxyl-terminal "a" position had minimal effects on lipid mixing but did inhibit content mixing and syncytia formation. These results are consistent with the idea that the HR2 domain is involved in posttranslational interactions with HR1 that mediate the close approach of membranes. These results also suggest that the HR2 domain, particularly the carboxyl-terminal region, plays an additional role in fusion, a role related to content mixing and syncytia formation.
Merchant M, Longnecker R (2001) LMP2A survival and developmental signals are transmitted through Btk-dependent and Btk-independent pathways. Virology 291 :46-54
The latent membrane protein 2A (LMP2A) of Epstein—Barr virus (EBV) has been implicated in controlling viral latency due to the ability of LMP2A to block B cell antigen receptor (BCR) signaling in vitro and to alter B cell development and enhance B cell survival in vivo. These LMP2A functions require interactions with the protein tyrosine kinases Syk and Lyn. However, a role for the Bruton’s tyrosine kinase (Btk) has not been investigated for these LMP2A functions. To investigate whether Btk is important for LMP2A developmental and survival signals in vivo, LMP2A transgenic animals were mated to Btk deficient (Btk(-/-)) mice. Unlike LMP2A(+), Btk(+/+) transgenic littermate controls, LMP2A(+), Btk(-/-) animals do not generate immunoglobulin (Ig) receptorless B cells in the periphery and instead produce Ig(+) B cells similar to those in the Btk(-/-) mice. Interestingly, however, LMP2A(+), Btk(-/-) animals produce B cells at a vastly reduced level compared to Btk(-/-) littermates, indicating that LMP2A affects B cell development in the absence of Btk. In the RAG-1(-/-), Btk(-/-) double knockout background, LMP2A is still capable of enhancing the survival of Ig-receptorless B cells. Use of Btk phosphopeptide-specific antibodies reveals that Btk is constitutively phosphorylated in LMP2A-expressing cell lines. These data indicate that LMP2A initiates both Btk-dependent and Btk-independent pathways, resulting in altered B cell development and enhanced B cell survival.
Moe GR, Zuno-Mitchell P, Lee SS, Lucas AH, Granoff DM (2001) Functional activity of anti-Neisserial surface protein A monoclonal antibodies against strains of Neisseria meningitidis serogroup B. Infect Immun 69 :3762-3771
Neisserial surface protein A (NspA) is currently being investigated with humans as a candidate vaccine for the prevention of meningococcal disease. Although NspA is highly conserved, the ability of anti-NspA antibodies to bind to or elicit complement-mediated bactericidal activity against diverse Neisseria meningitidis serogroup B strains is controversial. To evaluate strain differences in NspA surface accessibility and susceptibility to bactericidal activity, we prepared murine immunoglobulin G2a anti-NspA monoclonal antibodies (MAbs) and evaluated their functional activity against 10 genetically diverse N. meningitidis serogroup B strains. By colony Western blot, all 10 strains expressed NspA as detected by one or more MAbs. By flow cytometry, two MAbs were found to bind to the bacterial surface of 6 of the 10 strains. In addition, two strains showed variable NspA surface accessibility for the MAbs despite being uniformly positive for NspA expression by colony Western blotting. Only 4 of the 10 strains were susceptible to anti-NspA complement-mediated bacteriolysis. Passively administered MAb protected infant rats from developing bacteremia after challenge with N. meningitidis serogroup B strain 8047 (surface binding positive, susceptible to anti-NspA bacteriolysis), was poorly protective against strain BZ232 (surface binding variable, resistant to bacteriolysis), and did not protect against strain M986 (surface binding negative, resistant to bacteriolysis). Finally, NspA does not appear to be critical for causing bacteremia, as an NspA knockout from strain 8047 was highly virulent in infant rats. Taken together, these findings suggest that an NspA-based vaccine will need to incorporate additional antigens to elicit broad protection against N. meningitidis serogroup B.
Nasirudeen AM, Singh M, Yap EH, Tan KS (2001) Blastocystis hominis : evidence for caspase-3-like activity in cells undergoing programmed cell death. Parasitol Res 87 :559-565
We have shown previously that the human intestinal protozoan, Blastocystis hominis, undergoes apoptosis-like programmed cell death (PCD) when exposed to a cytotoxic monoclonal antibody (mAb), 1D5. In the present study, ELISA and immunoblot assays employing chicken anti-CPP32 antibody suggest that caspase-3-like antigens exist in B. hominis. Using colorimetric and flow cytometric assays for caspase-3 activity, we also observed an increase in activity between 1 h and 6 h after exposure to mAb 1D5, with greatest activity at 6 h. These findings suggest that caspase-3-like proteases play an important role in B. hominis undergoing PCD, similar to the phenomenon in higher eukaryotic organisms.
Nasirudeen AM, Tan KS, Singh M, Yap EH (2001) Programmed cell death in a human intestinal parasite, Blastocystis hominis. Parasitology 123 :235-246
Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis ; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.
Nasreen N, Mohammed KA, Hardwick J, Van Horn RD, Sanders KL, Doerschuk CM, Hott JW, Antony VB (2001) Polar production of interleukin-8 by mesothelial cells promotes the transmesothelial migration of neutrophils : role of intercellular adhesion molecule-1. J Infect Dis 183 :1638-1645
Migration of polymorphonuclear neutrophils (PMNL) from the vascular compartment into the pleural space occurs rapidly during the development of parapneumonic effusions. This study investigated the polarized secretion of interleukin (IL)-8 in activated pleural mesothelial cells (PMC) and the migration of PMNL across resting, activated PMC monolayers. Results show that PMC produce IL-8 in a polar manner. When PMC were stimulated with Staphylococcus aureus or IL-1beta at the basal or at the apical surface, significantly (P< .05) more IL-8 was released toward the apical surface. This polarized production of IL-8 was confirmed by in situ hybridization. PMNL migration was higher from the basilar to apical than from the apical to basilar surface of PMC. Neutralizing antibodies against IL-8 and intercellular adhesion molecule (ICAM)-1 significantly (P< .001) blocked PMNL migration across activated monolayers. Thus, during pleural inflammation, PMC regulate the influx of PMNL into the pleural space by polar production of IL-8 and expression of ICAM-1.
Nickerson K, Sisk TJ, Inohara N, Yee CS, Kennell J, Cho MC, Yannie PJ, 2nd, Nunez G, Chang CH (2001) Dendritic cell-specific MHC class II transactivator contains a caspase recruitment domain that confers potent transactivation activity. J Biol Chem 276 :19089-19093
The MHC class II transactivator (CIITA) is a critical transcription factor that regulates genes involved in antigen presentation function. At least three functional forms of CIITA gene products are transcribed from three different promoters. The CIITA gene expressed in dendritic cells (DC-CIITA) has a unique first exon encoding an extended N-terminal region of CIITA. Here, we show that the N terminus of DC-CIITA has high homology to a caspase recruitment domain (CARD) found in components of apoptosis and nuclear factor-kappaB signaling pathways. However, DC-CIITA does not regulate cell death, nor does it induce nuclear factor-kappaB activity. Instead, DC-CIITA is transcriptionally a more potent activator of the MHC class II gene than the form expressed in B cells. A single amino acid substitution in the CARD of DC-CIITA, predicted to disrupt CARD-CARD interactions, diminished the transactivation potential of DC-CIITA. These results indicate that the CARD in the context of CIITA serves as a regulatory domain for transcriptional activity and may function to selectively enhance MHC class II gene expression in dendritic cells.
Nisapakultorn K, Ross KF, Herzberg MC (2001) Calprotectin expression inhibits bacterial binding to mucosal epithelial cells. Infect Immun 69 :3692-3696
Squamous mucosal epithelial cells constitutively express calprotectin in the cytoplasm. To study how this antimicrobial protein complex confers epithelial resistance to invading bacteria, an epithelial cell line was stably transfected to express the calprotectin complex. Cells expressing calprotectin resist invasion by Listeria monocytogenes and Salmonella enterica serovar Typhimurium. Calprotectin expression was accompanied by altered actin organization, increased alpha3 integrin expression, and spreading cell morphology. In this study, we assessed whether calprotectin expression affects bacterial binding and uptake. Threefold-fewer Listeria organisms bound to the surfaces of calprotectin-expressing cells, and 10-fold fewer were localized intracellularly by immunofluorescence. Similarly, fewer Salmonella organisms bound to cells expressing calprotectin. Calprotectin-expressing and sham-transfected cells showed similar levels of expression of surface E-cadherin and intracellular adhesion molecule 1 (ICAM-1) by flow cytometry. Calprotectin-expressing transfectants expressed calprotectin on the cell surface as well as in the cytosol. In conclusion, two bacterial pathogens showed reduced binding to calprotectin-expressing epithelial cells. Calprotectin-expressing cells appeared to have internalized disproportionately fewer Listeria organisms, suggesting that reduced binding and translocation supplemented direct antimicrobial effects in calprotectin-expressing cells.
Oberyszyn AS, Robertson FM (2001) Novel rapid method for visualization of extent and location of aerosol contamination during high-speed sorting of potentially biohazardous samples. Cytometry 43 :217-222
BACKGROUND : Containment of potentially biohazardous aerosols that result from high-speed sorting of human cells has been an increasingly important problem in analytical cytometry. The current method for assessing the efficiency of aerosol containment involves detection of aerosols containing sorted T4 bacteriophage on lawns of T4-susceptible Escherichia coli on plates that are placed in and around the sort area. Although this method is sensitive, it is time consuming and involves maintenance and handling of bacteria and sorting of bacteriophage that may themselves serve as sources of contamination for sorted viable human cells. METHODS : Glo Germ (5-microm melamine copolymer resin beads), which are fluorescent under black light illumination, were sorted on a Beckman-Coulter Elite ESP sorter in order to visualize deposition of aerosols under normal and mock failure modes. RESULTS : Glo Germ was successfully used under both normal sorting conditions, as well as mock failure mode, to visualize aerosol formation. CONCLUSIONS : We have developed a method to examine aerosol containment using modified Glo Germ, a product used for teaching aseptic technique in hospitals, industry, restaurants, and schools. Use of this technique represents a rapid, inexpensive, qualitative analysis of the extent and location of aerosol contamination from cell sorters.
Oh SC, Nam SY, Kwon HC, Kim CM, Seo JS, Seong RH, Jang YJ, Chung YH, Chung HY (2001) Generation of fusion genes carrying drug resistance, green fluorescent protein, and herpes simplex virus thymidine kinase genes in a single cistron. Mol Cells 11 :192-197
We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required.
Orme IM (2001) Immunology and vaccinology of tuberculosis : can lessons from the mouse be applied to the cow ? Tuberculosis (Edinb) 81 :109-113
The nature of both innate and acquired immunity in the lungs are both still poorly understood, and how these two sets of mechanisms intersect with each other may have considerable bearing on the overall expression of host resistance. While the central role of CD4 T cells in the acquired phase is well established, cells bearing this marker may also contribute to innate immunity in the guise of both NK-positive and negative innate populations capable of secreting gamma interferon. In contrast, expansion of an antigen-specific memory T cell population is the purpose of current vaccine strategies, and several interesting and promising types of new candidates are briefly discussed.
Oumouna M, Jaso-Friedmann L, Evans DL (2001) Flow cytometry-based assay for determination of teleost cytotoxic cell lysis of target cells. Cytometry 45 :259-266
BACKGROUND : The nonradiometric assays previously developed to detect cellular cytotoxic activity have been hindered by many difficulties. Among the problems are the requirement for expensive commercial kits and the use of techniques that produce high background noise and decreased sensitivity. In addition, these assays did not account for bidirectional apoptosis (activation-induced cell death [AICD]). Most attempts to derive cytometry-based cytotoxicity assays have been unsuccessful because individual effectors and targets could not be identified (i.e., "separated") using gating techniques. METHODS : In the present study, teleost nonspecific cytotoxic (NCC) and mammalian target cells were each sufficiently different in size to identify them by flow cytometry (FCM). Using appropriate gating and discriminator techniques, these two cell populations were differentiated based on scatter properties and propidium iodide (PI) binding. Total capacity for PI binding was obtained by permeabilization of the targets with ice-cold acetone. Spontaneous PI binding was relatively low. This technique detected cytotoxicity at effector-to-target ratios (E:T) of 1:1 and after only 30 min cocultivation. RESULTS : Tilapia NCC from peripheral blood kill human transformed target cells by necrosis and apoptosis as identified by PI binding. Maximum killing of HL-60 targets (approximately 100%) occurred by 180 min cocultivation. For the same time, the killing of IM-9 did not exceed 60%. Almost 90% of IM-9 targets are lysed following 14 h of cocultivation. The maximum killing of both HL-60 and IM-9 targets was observed at a 25:1 E:T ratio after 14 h. Comparisons of the chromium(>51) release assay with flow detection of cytotoxicity revealed that FCM detected 55% lysis of the target cells compared with 2% cytotoxicity by chromium release, after a cocultivation time of 240 min. DISCUSSION : FCM detection of (teleost) NCC lysis of target cells using PI uptake is more sensitive than standard chromium release assays. This level of sensitivity was observed because NCC and targets were sufficiently different in size such that they could be resolved by scatter plots. Using FCM, cytotoxicity was detected earlier and at lower E:T ratios than previously reported for chromium release assays. Although tilapia were reported previously to be not capable of lysing IM-9 targets by chromium release detection, the more sensitive method of FCM detected cytotoxicity using PI uptake. HL-60 lysis by tilapia NCC exhibited saturable kinetics but occurred at different times post-cocultivation.
Papi A, Papadopoulos NG, Stanciu LA, Degitz K, Holgate ST, Johnston SL (2001) Effect of desloratadine and loratadine on rhinovirus-induced intercellular adhesion molecule 1 upregulation and promoter activation in respiratory epithelial cells. J Allergy Clin Immunol 108 :221-228
BACKGROUND : Rhinoviruses have been recently associated with the majority of asthma exacerbations for which current therapy is inadequate. Intercellular adhesion molecule 1 (ICAM-1) has a central role in airway inflammation in asthma, and it is the receptor for 90% of rhinoviruses. Rhinovirus infection of airway epithelium induces ICAM-1. Desloratadine and loratadine are compounds belonging to the new class of H(1)-receptor blockers. Anti-inflammatory properties of antihistamines have been recently documented, although the underlying molecular mechanisms are not completely defined. OBJECTIVE : We have investigated the effects of desloratadine and loratadine on rhinovirus-induced ICAM-1 expression, mRNA upregulation, and promoter activation. METHODS : Cultured primary bronchial or transformed (A549) respiratory epithelial cells were pretreated with desloratadine and loratadine for 16 hours and infected with rhinovirus type 16 for 8 hours. ICAM-1 surface expression was evaluated with flow cytometry, and ICAM-1 mRNA was evaluated with specific RT-PCR. In A549 cells promoter activation was evaluated with a chloramphenicol acetyltransferase assay, and binding activity of nuclear factor kappa B in nuclear extracts was evaluated with an electrophoretic mobility shift assay. RESULTS : Desloratadine and loratadine (0.1-10 micromol/L) inhibited rhinovirus-induced ICAM-1 upregulation in both primary bronchial or transformed (A549) respiratory epithelial cells. In A549 cells the 2 compounds showed a dose-dependent inhibition with similar efficacy (inhibitory concentration of 50%, 1 micromol/L). Desloratadine and loratadine also inhibited ICAM-1 mRNA induction caused by rhinovirus infection in a dose-dependent manner, and they completely inhibited rhinovirus-induced ICAM-1 promoter activation. Desloratadine also inhibited rhinovirus-induced nuclear factor kappa B activation. Desloratadine and loratadine had no direct effect on rhinovirus infectivity and replication in cultured epithelial cells. CONCLUSION : These effects are unlikely to be mediated by H(1)-receptor antagonism and suggest a novel mechanism of action that may be important for the therapeutic control of virus-induced asthma exacerbations.
Park JW, Choi YJ, Suh SI, Baek WK, Suh MH, Jin IN, Min DS, Woo JH, Chang JS, Passaniti A, Lee YH, Kwon TK (2001) Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity. Carcinogenesis 22 :1633-1639
Resveratrol has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. In the present study, we determined the effect of high intracellular levels of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-gamma1 degradation and cytochrome c release during resveratrol-induced apoptosis. For this, we used U937/vector and U937/Bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with U937/vector, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 60 or 100 microM resveratrol for 24 h produced morphological features of apoptosis and DNA fragmentation in U937/vector cells, respectively. This was associated with caspase-3 activation and PLC-gamma1 degradation. In contrast, resveratrol-induced caspase-3 activation and PLC-gamma1 degradation and apoptosis were significantly inhibited in U937/Bcl-2 cells. Bcl-2 overexpressing cells exhibited less cytochrome c release and sustained expression levels of the IAP proteins during resveratrol-induced apoptosis. In addition, these findings indicate that Bcl-2 inhibits resveratrol-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase-3 that is involved in the execution of apoptosis.
Peng X, Cebra JJ, Adler MW, Meissler JJ, Jr., Cowan A, Feng P, Eisenstein TK (2001) Morphine inhibits mucosal antibody responses and TGF-beta mRNA in gut-associated lymphoid tissue following oral cholera toxin in mice. J Immunol 167 :3677-3681
In this study, we investigated the effect of morphine on the mucosal immune system using fragment cultures of ileal segments, Peyer’s patches (PPs), and mesenteric lymph nodes. Mice were implanted s.c. with a morphine slow release pellet. Control groups received a naltrexone slow release pellet, a placebo pellet, or both a morphine and a naltrexone pellet. After 48 h, mice were orally immunized with cholera toxin (CT) and were boosted orally 1 wk later. Animals were sacrificed 1 wk after the booster immunization, and PPs, mesenteric lymph nodes, and ileal segments were cultured in 24-well plates for 12 days. Morphine resulted in a highly significant inhibition of CT-specific IgA and IgG production in fragment culture supernatants of all three tissues compared with placebo. Naltrexone blocked the reduction in Ab levels induced by morphine, indicating that the effect is opioid receptor mediated. Morphine did not significantly alter total IgA levels in any of the tissue culture supernatants. Morphine also inhibited CT-specific IgA and IgG levels in serum. By flow cytometry, morphine did not alter the lymphoid cell composition in PPs compared with placebo. The effect of morphine on TGF-beta, IL-5, and IL-6 mRNA expression in PPs and ileal segments was determined following oral immunization with CT. Morphine significantly decreased TGF-beta mRNA compared with that in the placebo group, and naltrexone blocked this effect. These results indicate that morphine inhibits Ag-specific IgA responses in gut-associated lymphoid tissue at least partially through the inhibition of TGF-beta, a putative IgA switch factor, in the gastrointestinal tract.
Perez-Prieto S, Garcia-Rosado E, Rodriguez S, Castro D, Borrego JJ (2001) Antigenic properties and experimental transmission to several fish species of a marine birnavirus isolated from sole (Solea senegalensis). Vet Microbiol 82 :11-25
A cross-neutralization test was used to study the antigenic relationship of an aquabirnavirus isolated from sole (Solea senegalensis), named solevirus, and several infectious pancreatic necrosis virus (IPNV) strains. Solevirus was antigenically similar to IPNV strain Sp. Transmission of the solevirus to other fish species has been determined by inoculation to freshwater and marine fish species (two salmonids and gilt-head seabream). A higher pathogenicity was obtained for the marine fish species, although solevirus caused an asymptomatic infection in all species tested, as demonstrated by the detection of viral RNA and of viral antigens in fish leucocytes, respectively, using polymerase chain reaction (PCR) and flow cytometry (FC).
Pernthaler A, Pernthaler J, Eilers H, Amann R (2001) Growth patterns of two marine isolates : adaptations to substrate patchiness ? Appl Environ Microbiol 67 :4077-4083
During bottle incubations of heterotrophic marine picoplankton, some bacterial groups are conspicuously favored. In an earlier investigation bacteria of the genus Pseudoalteromonas rapidly multiplied in substrate-amended North Sea water, whereas the densities of Oceanospirillum changed little (H. Eilers, J. Pernthaler, and R. Amann, Appl. Environ. Microbiol. 66:4634-4640, 2000). We therefore studied the growth patterns of two isolates affiliating with Pseudoalteromonas and Oceanospirillum in batch culture. Upon substrate resupply, Oceanospirillum lagged threefold longer than Pseudoalteromonas but reached more than fivefold-higher final cell density and biomass. A second, mobile morphotype was present in the starved Oceanospirillum populations with distinctly greater cell size, DNA and protein content, and 16S rRNA concentration. Contrasting cellular ribosome concentrations during stationary phase suggested basic differences in the growth responses of the two strains to a patchy environment. Therefore, we exposed the strains to different modes of substrate addition. During cocultivation on a single batch of substrates, the final cell densities of Oceanospirillum were reduced three times as much as those Pseudoalteromonas, compared to growth yields in pure cultures. In contrast, the gradual addition of substrates to stationary-phase cocultures was clearly disadvantageous for the Pseudoalteromonas population. Different growth responses to substrate gradients could thus be another facet affecting the competition between marine bacteria and may help to explain community shifts observed during enrichments.
Pina-Vaz C, Sansonetty F, Rodrigues AG, Costa-de-Oliveira S, Martinez-de-Oliveira J, Fonseca AF (2001) Susceptibility to fluconazole of Candida clinical isolates determined by FUN-1 staining with flow cytometry and epifluorescence microscopy. J Med Microbiol 50 :375-382
The susceptibility of clinical Candida isolates to fluconazole was assayed by flow cytometry (FCM) and epifluorescence microscopy (EFM), with FUN-1 staining. In all, 25 clinical isolates of Candida spp. (12 sensitive, 3 dose-dependently sensitive and 10 resistant to fluconazole according to the NCCLS M27-A protocol) were treated with increasing concentrations of fluconazole during 1 or 2 h staining with FUN-1 for 30 min and analysed, respectively, by FCM at 575 nm (FL2) and by EFM. Fluconazole-susceptible strains showed an increased accumulation of FUN-1 in comparison with controls as determined by FCM and a reduced metabolic processing of the probe, confirmed by EFM. Conversely, resistant strains showed decreased FUN-1 staining and were able to process the probe. The fluconazole minimal inhibitory concentrations (MICs) determined by FCM or EFM after FUN-1 staining compared very well with the corresponding values determined by the M27-A protocol, indicating that FUN-1 staining can be used as an alternative to the conventional method. MIC values of resistant strains, with the exception of C. krusei, were lower when treatment with fluconazole followed pre-incubation with 0.1 mM sodium azide, a concentration known to inhibit the activity of efflux pumps. These results show that FUN-1 staining can be used as an alternative and rapid method for the assessment of susceptibility of Candida clinical isolates to fluconazole. Furthermore, the results suggest that resistance of Candida cells to fluconazole, with the exception of C. krusei strains, is likely to be due to the activity of efflux pumps.
Pina-Vaz C, Sansonetty F, Rodrigues AG, Costa-Oliveira S, Tavares C, Martinez-de-Oliveira J (2001) Cytometric approach for a rapid evaluation of susceptibility of Candida strains to antifungals. Clin Microbiol Infect 7 :609-618
OBJECTIVE : To achieve a fast and reliable determination of the susceptibility of Candida strains to amphotericin B (Am B), fluconazole (Flu) and 5-fluorocytosine (5-FC), using cytometric methods as an alternative to the classical dilution method. METHODS : Twenty-three clinical isolates of Candida with different susceptibility patterns were treated for 1 h with two concentrations each of Am B (2 and 8 mg/L), Flu (8 and 64 mg/L) and 5-FC (4 and 32 mg/L), followed by staining with three different fluorochromes, under conditions previously defined through an optimisation study. These were 1 mg/L propidium iodide (PI)/10(6) cells for 30 min at 30 degrees C (a marker that only penetrates cells with severe lesions of the membrane) ; 0.5 microM FUN-1/10(6) cells for 30 min at 30 degrees C (a fluorescent probe which after entering the yeast cell is converted, by metabolically active yeasts, from a diffuse cytosolic pool with a yellow-green fluorescence into red cylindrical intravacuolar structures) and 0.25 microM of JC-1/10(6) cells for 15 min at 37 degrees C (a monomer that changes reversibly from green to red the J-aggregates, with the increased membrane potential). About 50 000 yeast cells were analysed by flow cytometry (FCM), at FL3 (red, 620 nm) for PI and FL2 (yellow-green, 575 nm) for FUN-1 and the ratio of FL3 to FL1 was determined (red, 620 nm/green, 525 nm) for JC-1 ; 200 cells of each suspension were also analysed by epifluorescence microscopy (EPM). Viability studies were performed in parallel to count the number of colony forming units. RESULTS : Susceptible (S) strains exposed to Am B and stained with JC-1 showed a dose-dependent decrease in the mitochondrial potential, i.e. a decreased ratio between red/green fluorescence by FCM and a decrease in J-aggregates by EPM. Neither FUN-1 nor PI was useful in the study of Am B activity. Susceptibility to Flu and 5-FC could be detected with FUN-1 staining : metabolic changes were detected by an increase in yellow-green intensity of fluorescence by FCM or a decrease of cylindrical intravacuolar structure formation by EPM, although no decrease in total viability was registered. Staining with JC-1 could predict resistance to both drugs, but did not allow distinction between sensitive dose-dependent strains (S-DD) or intermediate (I) resistance to Flu or 5-FC, respectively, from S strains. PI did not stain Candida cells treated with Flu or 5-FC under our experimental conditions. CONCLUSION : Susceptibility patterns of Candida strains to Am B can be determined by using JC-1, and to Flu and 5-FC by using FUN-1. PI was not a useful probe with which to study the effect of such antifungals under the conditions described here.
Pouwels PH, Vriesema A, Martinez B, Tielen FJ, Seegers JF, Leer RJ, Jore J, Smit E (2001) Lactobacilli as vehicles for targeting antigens to mucosal tissues by surface exposition of foreign antigens. Methods Enzymol 336 :369-389
Raybourne RB (2001) Flow cytometric detection of pathogenic E. coli in food. Curr Protoc Cytom Chapter 11 :Unit 11 16
E. coli O157:H7 is one of the more important food pathogens, andrapid, quantitative methods to evaluate foods for the presence of this pathogen are needed. This unit provides exactly that : a very much simplified flow cytometric assay for detection of E. coli O157:H7 in a well established vehicle of infection, ground beef. The method uses commercially available FITC-conjugated specific antibody to this bacterial serotype. Sample preparation and bacterial enrichment procedures are described. Direct and indirect approaches for quantification of the number of bacteria are given. A key feature of the assay is the reduction in time compared with plate-counting methods ; the tradeoff is a slight reduction in sensitivity. Particularly useful is the simultaneous inclusion of a spiked sample to ensure a positive control. In addition, the unit provides hints on sorting the organisms if desired.
Regeniter A, Haenni V, Risch L, Kochli HP, Colombo JP, Frei R, Huber AR (2001) Urine analysis performed by flow cytometry : reference range determination and comparison to morphological findings, dipstick chemistry and bacterial culture results—a multicenter study. Clin Nephrol 55 :384-392
AIM : To validate whether quantitative flow-cytometric analysis of particulate matter in urine would allow for accurate and rapid enumeration of red blood cells (RBC), leukocytes (WBC), squamous epithelial cells (EC), casts, and bacteria, a Sysmex UF-100 analyzer was tested in a multicenter study. MATERIAL AND METHODS : At first, reference values were established and found to be < 14 for RBC, < 16 for WBC, < 9 for EC, < 2 for casts and < 173 for bacteria, respectively (counts per microl ; 97.5 percentile). Due to the wide use of dipstick and microscopic sediment analysis in routine urine diagnostics, comparative studies on 950 random urine samples were performed. Bacterial counting combined with WBC enumeration was further compared in 266 routine urinary microbiologic cultures. RESULTS : Good correlations were found comparing UF-100 results of RBC (r = 0.89), WBC (r = 0.94), and EC (r = 0.74) with Fuchs-Rosenthal Chamber (FRC) counts. However, some misclassification of casts (r = 0.32) could be observed. Correlations of UF-100 with dipstick and sediment testing was significant (p < 0.001), but the scatter of the latter two methods is too wide to consider them as quantitative methods. Promising results further revealed that the analyzer has a good negative predictive value (NPV) for microbiologically negative cultures, especially for cultures with bacterial counts of 10(5)/l (NPV = 95%). CONCLUSION : The analyzer is capable of providing rapid and reliable urine analysis of cellular particles avoiding the known imprecision of dipstick and sediment methodology. Thus, when used in an algorithm, combined with dipstick or quantitative urine chemistry analysis (for hemoglobin, esterase, protein, glucose, etc.), this analyzer might serve as a rapid and accurate screening tool in routine urine analysis, thereby reducing manual reviewing rate as well as the number of missed samples, compared to screening with dipstick alone.
Resina-Pelfort O, Comas-Riu J, Vives-Rego J (2001) Effects of deflected droplet electrostatic cell sorting on the viability and exoproteolytic activity of bacterial cultures and marine bacterioplankton. Syst Appl Microbiol 24 :31-36
The cell-sorting capability of flow cytometers makes it possible to isolate specific populations of cells with pre-defined cytometric characteristics. A better knowledge of the biological effects of the sorting process is necessary for the future cell sorting applications. In this paper we report the effects of flow cytometric sorting on bacterial viability and exoproteolytic activity (EPA) of bacterial cultures and marine bacterioplankton. Sorting bacterial cultures and bacterioplankton samples reduce viability as assessed by plate counts and produce variations in the exoproteolytic activity. These effects indicate that deflected electrostatic sorting may significantly alter the biological properties of the sorted bacteria.
Riffard S, Douglass S, Brooks T, Springthorpe S, Filion LG, Sattar SA (2001) Occurrence of Legionella in groundwater : an ecological study. Water Sci Technol 43 :99-102
The natural habitat of Legionella is the water environment. Little is known about their presence in groundwater in spite of the fact that many millions around the globe regularly rely on groundwaters. This pilot study was aimed at evaluating the occurrence of Legionella in groundwater samples (water and biofilms) collected from various sites. Water and biofilm samples from selected groundwater sources were examined for Legionella using culture media (selective and non-selective) and a semi-nested PCR assay. Innovative approaches such as immunomagnetic separation (IMS) in combination with cultivation and flow cytometry were also evaluated. The findings available thus far show that (a) Legionella could be readily recovered from groundwater samples by cultivation even though their numbers showed considerable variations, (b) surprisingly, the PCR methodology was not yet as sensitive as cultivation and (c) flow cytometry was not directly applicable on natural samples because of debris and the high number of heterotrophic associated microflora from which some members were likely to cross-react with the monoclonal antibody used for separation procedures (IMS).
Robaczewska M, Guerret S, Remy JS, Chemin I, Offensperger WB, Chevallier M, Behr JP, Podhajska AJ, Blum HE, Trepo C, Cova L (2001) Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver. Gene Ther 8 :874-881
Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.
Rodriguez ME, Van der Pol WL, Van de Winkel JG (2001) Flow cytometry-based phagocytosis assay for sensitive detection of opsonic activity of pneumococcal capsular polysaccharide antibodies in human sera. J Immunol Methods 252 :33-44
The development of efficient vaccines against Streptococcus pneumoniae is of major importance for public health. The efficacy of pneumococcal vaccination and induced protection are thought to be reflected by the opsonic antibody titers in sera from vaccines. We describe a novel two-color flow cytometry technique for quantification of antibody-mediated pneumococcal phagocytosis. Serum-opsonised fluorescein-isothiocyanate (FITC)-labelled S. pneumoniae were allowed to attach to neutrophils, split into two aliquots and further incubated either at 4 degrees C (to avoid phagocytosis) or 37 degrees C (to allow phagocytosis). Cell-surface residual opsonic IgG was detected by phycoerythrin (PE)-conjugated anti-human IgG in both samples. The fraction of FITC-labelled bacteria phagocytosed via antibody (F(i)) could be estimated from FITC and PE labels, and reflected the opsonic activity of sera. The technique displayed high sensitivity for the detection of opsonic antibodies, as shown by experiments using pre- and post-immune sera, which documented significantly increased phagocytosis after vaccination, and the observed increase in phagocytosis rates at higher antibody levels. The intrinsic variation of the assay was low, and could be further reduced by the use of effector cells from donors with similar IgG receptor (FcgammaR) allotypes. The method described in this study should be generally applicable to test vaccine efficacy, to evaluate the interaction of bacteria and phagocytes, and to discriminate between antibody-mediated and antibody-independent interactions between bacteria and phagocytes.
Rodriguez Saint-Jean S, Borrego JJ, Perez-Prieto SI (2001) Comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fish. J Virol Methods 97 :23-31
In the present study, six diagnostic methods for the detection of infectious pancreatic necrosis virus (IPNV) (indirect immunofluorescence, flow cytometry, immunoperoxidase, immunodot blot, immunostaphylococcus-protein A, and RT-PCR) have been comparatively evaluated using the seroneutralization as the reference assay, and 83 Spanish isolates and 3 reference strains. The most reliable methods were flow cytometry and RT-PCR which could detect virus at titers of 1x10(2) and 1x10(3) TCID50/ml, respectively. At a multiplicity of infection of 50, both assays allowed the earliest detection of IPNV at 4 h post-inoculation. Indirect immunofluorescence and immunoperoxidase assays required at least 6 h post-inoculation to detect viral antigens. The immunodot blot assay possesses low sensitivity and the immunostaphylococcus-protein A test cannot be applied for routine examination of IPNV. Positive reactions were obtained in 100% of the samples tested by seroneutralization and RT-PCR, 90.4% by the flow cytometry, 80.7% by the indirect immunofluorescence assay, 67.5% by the immunoperoxidase, 62.6% by the immunodot blot, and only 27.7% by immunostaphylococcus-protein A test. Therefore, RT-PCR and flow cytometry were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish.
Schoier J, Ollinger K, Kvarnstrom M, Soderlund G, Kihlstrom E (2001) Chlamydia trachomatis-induced apoptosis occurs in uninfected McCoy cells late in the developmental cycle and is regulated by the intracellular redox state. Microb Pathog 31 :173-184
Infections with the obligate intracellular bacterium Chlamydia trachomatis are characterized by avoidance of fusion between chlamydia-containing endosomes and lysosomes, bacterial persistence and development of post-infectious sequelae. In this report we show that C. trachomatis induces apoptosis in McCoy and HeLa cells. Apoptosis was monitored by three different techniques ; enzyme-linked immunoassay (EIA) of fragmented nucleosomes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and flow cytometry of propidium iodide-stained cells. Apoptosis occurred in uninfected cells, was induced late in the chlamydial developmental cycle, beyond 24 h post-infection and was dependent on bacterial protein synthesis. Apoptosis was not significantly increased in infected, inclusion-containing cells. Treatment of cells with the antioxidants ascorbic acid (10 microM) and alpha-tocopherol (10 microM) reduced the degree of apoptosis. These results suggest that host cells infected with C. trachomatis generate proapoptotic stimuli that induce apoptosis in uninfected, neighbouring cells and that the redox state of the cell is a regulator in chlamydia-induced apoptosis.
Schuberth HJ, Krueger C, Zerbe H, Bleckmann E, Leibold W (2001) Characterization of leukocytotoxic and superantigen-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis. Vet Microbiol 82 :187-199
Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays.Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described.The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41% ; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2% ; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.
Schulein R, Seubert A, Gille C, Lanz C, Hansmann Y, Piemont Y, Dehio C (2001) Invasion and persistent intracellular colonization of erythrocytes. A unique parasitic strategy of the emerging pathogen Bartonella. J Exp Med 193 :1077-1086
The expanding genus Bartonella includes zoonotic and human-specific pathogens that can cause a wide range of clinical manifestations. A productive infection allowing bacterial transmission by blood-sucking arthropods is marked by an intraerythrocytic bacteremia that occurs exclusively in specific human or animal reservoir hosts. Incidental human infection by animal-adapted bartonellae can cause disease without evidence for erythrocyte parasitism. A better understanding of the intraerythrocytic lifestyle of bartonellae may permit the design of strategies to control the reservoir and transmittable stages of these emerging pathogens. We have dissected the process of Bartonella erythrocyte parasitism in experimentally infected animals using a novel approach for tracking blood infections based on flow cytometric quantification of green fluorescent protein-expressing bacteria during their interaction with in vivo-biotinylated erythrocytes. Bacteremia onset occurs several days after inoculation by a synchronous wave of bacterial invasion into mature erythrocytes. Intracellular bacteria replicate until reaching a stagnant number, which is sustained for the remaining life span of the infected erythrocyte. The initial wave of erythrocyte infection is followed by reinfection waves occurring at intervals of several days. Our findings unravel a unique bacterial persistence strategy adapted to a nonhemolytic intracellular colonization of erythrocytes that preserves the pathogen for efficient transmission by blood-sucking arthropods.
Sergel TA, McGinnes LW, Morrison TG (2001) Mutations in the fusion peptide and adjacent heptad repeat inhibit folding or activity of the Newcastle disease virus fusion protein. J Virol 75 :7934-7943
Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the fusion activity of the protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999 ; X. Zhao, M. Singh, V. N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172-14177, 2000). We have extended our previous mutational analysis of the HR1 domain of the Newcastle disease virus fusion protein, focusing on the role of the amino acids forming the hydrophobic core of the trimer, amino acids in the "a" and "d" positions of the helix from amino acids 123 to 182. Both conservative and nonconservative point mutations were characterized for their effects on synthesis, stability, proteolytic cleavage, and surface expression. Mutant proteins expressed on the cell surface were characterized for fusion activity by measuring syncytium formation, content mixing, and lipid mixing. We found that all mutations in the "a" position interfered with proteolytic cleavage and surface expression of the protein, implicating the HR1 domain in the folding of the F protein. However, mutation of five of seven "d" position residues had little or no effect on surface expression but, with one exception at residue 175, did interfere to various extents with the fusion activity of the protein. One of these "d" mutations, at position 154, interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most "d" position residues do affect fusion activity argues that a stable HR1 trimer is required for formation of the six-stranded coiled coil and, therefore, optimal fusion activity. That most of the "d" position mutations do not block folding suggests that formation of the core trimer may not be required for folding of the prefusion form of the protein. We also found that mutations within the fusion peptide, at residue 128, can interfere with folding of the protein, implicating this region in folding of the molecule. No characterized mutation enhanced fusion.
Sergueev K, Yu D, Austin S, Court D (2001) Cell toxicity caused by products of the p(L) operon of bacteriophage lambda. Gene 272 :227-235
Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.
Setiyono A, Yamaguchi T, Ogawa M, Fukushi H, Hirai K (2001) Isolation of monoclonal antibodies that inhibit the binding of infectious bursal disease virus to LSCC-BK3 cells. J Vet Med Sci 63 :215-218
Three hybridoma cell lines producing monoclonal antibodies (MAbs) against LSCC-BK3 cells which are susceptible to infectious bursal disease virus (IBDV) infection were produced and characterized. The MAbs, designated T7, Q11 and Q13, inhibited the attachment of IBDV to LSCC-BK3 cells. Furthermore, these MAbs bound to LSCC-BK3 but not to nonpermissive cells in flow cytometry. MAb T7 detected a 110-kDa membrane protein of LSCC-BK3 cells, whereas Q11 and Q13 reacted with membrane proteins of molecular weights 58-, 85-, 90- and 110-kDa. These observations imply that the 110-kDa protein recognized by all the MAbs is associated with IBDV binding. The MAbs established in this study are useful for studying the interaction between IBDV and its target cell.
Shapiro HM (2001) Microbiology. Clin Lab Med 21 :897-909, x-xi
Although flow cytometry can detect microorganisms rapidly in low concentrations in some clinical samples, it has not proved cost-effective for clinical use in this application. Adaptation of fluorescence multiplexing methodology recently developed for flow cytometric bead immunoassays, and the introduction of hybrid cytometers based on microfluidic technology, however, may make it possible to place cost-effective cytometric apparatus in clinical microbiology laboratories in the near future.
Shen Z, Chen M, Cai W, Shen J, Chen J, Hong C, Zeng Y (2001) [The effects of sodium butyrate on proliferation, differentiation and apoptosis in immortalized esophageal epithelial cells]. Zhonghua Bing Li Xue Za Zhi 30 :121-124
OBJECTIVE : To study the effects of sodium butyrate on proliferation, differentiation and apoptosis of immortalized esophagus epithelial cells. METHODS : SHEE, an immortalized human fetal esophageal epithelial cell line induced by HPV18 E6E7, was cultivated in culture flasks and 24-well plates. Two experiment groups of cultured cells were treated with 1 and 5 mmol/L of sodium butyrate respectively for 4 days, and one group of untreated cells set aside as control. The numbers of cloned cells were calculated. The ultra-structure of SHEE cells was examined by transmission electron microscopy (TEM). The cell cycle and number of apoptotic cells were measured by flow cytometry, Ki-67 and cytokeratin of cells were detected by immunohistochemistry method and F-actin of cells labeled with phalloidin was examined by laser confocal scanning microscopy. RESULTS : Colony formations showed a significant decrease in the 2 experiment groups after 4 days of culture (P < 0.01). In the 1 mmol/L group, the cells at S phase were diminished and arrested at G(0)/G(1) phase. Compared with control group, Ki-67 positive cells were found decreased, while F-actin and cytokeratin were increased. Apoptotic cells in 5 mmol/L group were increased markedly. CONCLUSIONS : Sodium butyrate may induce SHEE cells growth arrest, differentiation and apoptosis. The effects depend on sodium butyrate concentration and time of exposure. Whether it can be used in combination with other anticancer drugs should be further studied.
Shepel M, Boyd J, Luider J, Gibb AP (2001) Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets. J Med Microbiol 50 :1030-1038
Yersinia enterocolitica is a bacterium capable of growth at 4 degrees C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry, enterocolitica did not cause platelet aggregation or activation, not even when grown at 22 degrees C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37 degrees C but not with pYV- strains nor with strains grown at 22 degrees C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22 degrees C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37 degrees C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.
Sincock SA, Robinson JP (2001) Flow cytometric analysis of microorganisms. Methods Cell Biol 64 :511-537
Sisk TJ, Roys S, Chang CH (2001) Self-association of CIITA and its transactivation potential. Mol Cell Biol 21 :4919-4928
The major histocompatibility complex (MHC) class II transactivator (CIITA) regulates the expression of genes involved in the immune response, including MHC class II genes and the interleukin-4 gene. Interactions between CIITA and sequence-specific, DNA-binding proteins are required for CIITA to function as an activator of MHC class II genes. CIITA also interacts with the coactivators CBP (also called p300), and this interaction leads to synergistic activation of MHC class II promoters. Here, we report that CIITA forms complexes with itself and that a central region, including the GTP-binding domain is sufficient for self-association. Additionally, this central region interacts with the C-terminal leucine-rich repeat as well as the N-terminal acidic domain. LXXLL motifs residing in the GTP-binding domain are essential for self-association. Finally, distinct differences exist among various CIITA mutant proteins with regard to activation function, subcellular localization, and association with wild-type protein and dominant-negative potential.
Smith PK, Wang SZ, Dowling KD, Forsyth KD (2001) Leucocyte populations in respiratory syncytial virus-induced bronchiolitis. J Paediatr Child Health 37 :146-151
OBJECTIVES : To enumerate the cellular composition of the airways in infants with acute bronchiolitis. METHODOLOGY : Cells were obtained by airway lavage from the upper and lower airway and the peripheral blood of infants with respiratory syncytial virus (RSV)+ bronchiolitis, RSV- bronchiolitis and age-matched controls. RESULTS : Neutrophils are the predominant cells present in the upper and lower airway. Neutrophils are present at a higher number/unit volume in the airway than in the peripheral blood. CONCLUSIONS : Neutrophils, being the dominant cellular infiltrate into the airway, are likely to contribute to the pathophysiology of bronchiolitis. Therapies targeted at limiting neutrophil influx or neutrophil-mediated damage in the airway may have a therapeutic role.
Stecchini ML, Del Torre M, Donda S, Maltini E, Pacor S (2001) Influence of agar content on the growth parameters of Bacillus cereus. Int J Food Microbiol 64 :81-88
Factors governing Bacillus cereus colony growth on agar media as modified by the agar content (1-7%, w/v) were studied. Agar had a significant effect on the radial growth rate which diminished as the agar content increased. Cell density in colonies (colony density) was found to decrease during the incubation time, with lower values occurring in the presence of 1% agar. Size and DNA content of the cells grown on 1 and 7% agar were similar. An increasing proportion of cell population growing on 7% agar produced spores during the 24-h incubation period. It was shown that ’water condition’ on the agar surface could be associated with colony density, with 7% agar media presenting the thinnest nominal thickness of the liquid film. On the other hand, the partial drying phenomena of the agar media which occur during preparation and incubation, could not account for the observed differences in colony growth.
Stopa PJ, Mastromanolis SA (2001) The use of blue-excitable nucleic-acid dyes for the detection of bacteria in well water using a simple field fluorometer and a flow cytometer. J Microbiol Methods 45 :143-153
The blue-excitable nucleic acid dyes Coriphosphine O (CPO), YOYO-1, and YOPRO-1 were evaluated to rapidly detect the presence of bacteria in well water samples using a simple field fluorometer (Turner Designs, Sunnyvale, CA, Model 10-AU-005) and a tabletop flow cytometer (Coulter Epics XL). The dyes were first titrated on the Turner Designs Model 10-AU field fluorometer with log-fold dilutions of Esherichia coli, since this organism is the indicator organism for water contamination. A detection limit of 10(4) Colony Forming Units per ml (CFU/ml) was established for YOPRO-1 and 10(5) CFU/ml for YOYO-1. The detection limit with CPO was determined to be 10(7) CFU/ml due to the high background fluorescence of the dye. The dyes were also evaluated with ragweed pollen to gauge the effect of a biological interferent. Ten well-water samples were subsequently analyzed using the technique. The results showed that only YOYO-1 correctly detected all the samples that were positive according to the reference laboratory. YOPRO-1 correctly detected only one of four positive samples. Analysis with the CPO dye was inconclusive due to high background fluorescence. The samples were then subjected to analysis on the flow cytometer. Results obtained with YOYO-1 compared well to those obtained on the fluorometer and by the reference techniques. YOPRO-1 performed better on the flow cytometer than with the simple fluorometer, correctly detecting three of four positive samples. Although the CPO results showed a very slight increase of green fluorescence with positive samples, they were largely indistinguishable from negative samples. This study suggests YOYO-1 could be useful with either a simple fluorometer or with a tabletop flow cytometer in screening water samples for the presence of bacterial contamination.
Taylor SL, Jaso-Friedmann L, Allison AB, Eldar A, Evans DL (2001) Streptococcus iniae inhibition of apoptosis of nonspecific cytotoxic cells : a mechanism of activation of innate immunity in teleosts. Dis Aquat Organ 46 :15-21
Nonspecific cytotoxic cells (NCC) may provide innate anti-bacterial resistance against Streptococcus iniae infections in tilapia. The mechanism of immunity would be elaboration and release of various cytokines, augmentation of inflammation and amplification of increased antigen processing. To investigate bacterial regulation of NCC function, 2 different processes of cellular pathology were examined : apoptosis and necrosis. Different isolates of S. iniae from diseased teleosts, a dolphin and a human were tested. All isolates were examined for their ability to produce apoptosis and/or necrosis on freshly purified tilapia NCC and on a tilapia continuous cell line (i.e. TMB-8 cells). Two different isolates (9033 and 173) inhibited the outer membrane expression of phosphatidylserine (PS) by NCC, an early sign of apoptosis. This occurred at 4 h post-treatment and lasted throughout the 24 h treatment period. All other isolates either did not differ from control levels or produced a small increase in PS expression by NCC. The early reduction in PS expression occurred concomitantly with increased necrosis associated with nonspecific DNA fragmentation. Two-color flow cytometry (Annexin-V vs propidium iodide staining) demonstrated the specificity of Annexin-V binding. Experiments were also done to determine the effects of S. iniae on TMB-8 cells. Treated TMB-8 cells did not produce appreciable Annexin-V binding. Compared to the ATCC strain, 9033 produced high levels of necrosis-associated DNA fragmentation of TMB-8 cells at 4 and 8 h post-treatment. These data indicated that different isolates of S. iniae may regulate NCC anti-bacterial resistance by causing reduced levels of programmed cell death (PCD), increased necrosis and associated enhancement of inflammatory responses. Understanding the relevance of these bacterial effects on NCC may be an important consideration in the evaluation of isolates used in vaccine/ bacterin production.
Tobiason DM, Seifert HS (2001) Inverse relationship between pilus-mediated gonococcal adherence and surface expression of the pilus receptor, CD46. Microbiology 147 :2333-2340
Pilus-mediated adherence to mucosal epithelial cells is a critical step for Neisseria gonorrhoeae to establish an infection in the human host. CD46, the defined receptor for the gonococcal pilus, is a complement-regulatory protein that is expressed on all human nucleated cells. It was observed that a piliated, Opa(-) variant of gonococcal strain FA1090 adhered with different efficiencies to the human epithelial cell lines tested (Chang, ME180, HEC-1B and PC-3). Surprisingly, these differences in adherence levels did not correlate with levels of CD46 expressed by these cell lines. In fact, there was an inverse relationship between total surface-exposed CD46 and gonococcal adherence. Four major isoforms of CD46 are produced due to alternative RNA splicing of a surface-exposed region and the cytoplasmic tail. The relative isoform surface expression of each cell line was determined, and each was found to express different ratios of the four CD46 isoforms. No correlation could be derived between CD46 isoform surface expression and pilus-mediated gonococcal adherence, indicating that CD46 does not act as a classic receptor for gonococcal pili.
Tosello-Trampont AC, Brugnera E, Ravichandran KS (2001) Evidence for a conserved role for CRKII and Rac in engulfment of apoptotic cells. J Biol Chem 276 :13797-13802
Apoptosis or programmed cell death occurs in multicellular organisms throughout life. The removal of apoptotic cells by phagocytes prevents secondary necrosis and inflammation and also plays a key role in tissue remodeling and regulating immune responses. The molecular mechanisms that regulate the engulfment of apoptotic cells are just beginning to be elucidated. Recent genetic studies in the nematode Caenorhabditis elegans have implicated at least six genes in the removal of apoptotic cell corpses. The gene products of ced-2, ced-5, and ced-10 are thought to be part of a pathway that regulates the reorganization of the cytoskeleton during engulfment. The adapter proteins CrkII and Dock180 and the small GTPase Rac represent the mammalian orthologues of the ced-2, ced-5 and ced-10 gene products, respectively. It is not known whether CrkII, Dock180, or Rac proteins have any role during engulfment in mammalian cells. Here we show, using stable cell lines and transient transfections, that overexpression of wild-type CrkII or an activated form of Rac1 enhances engulfment. Mutants of CrkII failed to mediate this increased engulfment. The higher CrkII-mediated uptake was inhibited by coexpression of a dominant negative form of Rac1 but not by a dominant a negative Rho protein ; this suggested that Rac functions downstream of CrkII in this process, which is consistent with genetic studies in the worm that place ced-10 (rac) downstream of ced-2 (crk) in cell corpse removal. Taken together, these data suggest that CED-2/CrkII and CED-10/Rac are part of an evolutionarily conserved pathway in engulfment of apoptotic cells.
Triantafilou M, Wilson KM, Triantafilou K (2001) Identification of Echovirus 1 and coxsackievirus A9 receptor molecules via a novel flow cytometric quantification method. Cytometry 43 :279-289
BACKGROUND : Virus-receptor binding is an essential step in every virus infectious process. Many viruses employ more than one receptor molecule or even receptor complexes for attachment. In this study, we investigate the binding of Echovirus 1 (Echo1) and Coxsackievirus A9 (CAV-9) on cell surface molecules. CAV-9 has been reported to utilize integrin alpha v beta(3) in binding to cells, whereas Echo1 has been known to utilize integrin alpha 2 beta(1). METHODS AND RESULTS We directly test whether the presence of these molecules alone was sufficient for virus binding. We devised a novel flow cytometric binding assay that enables us to quantify virus particles bound on host cells and to further determine the extent to which viruses utilize specific receptors. CONCLUSIONS : By quantifying virus particles and possible receptor molecules, we found that Echo1 utilizes mainly integrin alpha 2 beta(1). CAV-9 utilizes integrin alpha v beta(3) to a much lesser extent (40%), indicating that CAV-9 also utilizes other receptor(s).
Valsamakis A, Kaneshima H, Griffin DE (2001) Strains of measles vaccine differ in their ability to replicate in an damage human thymus. J Infect Dis 183 :498-502
A number of strains of live attenuated measles virus are in use worldwide as measles vaccines. All effectively induce protective immunity, but many differences in immunogenicity and, potentially, in safety are seen between strains in young infants. However, no system for assessing biologic differences between vaccine strains has been available. The SCID-hu thymus model system was used to compare AIK-c, Edmonston-Zagreb (EZ), and Moraten-3 common vaccine strains derived from the Edmonston prototype virus-for replication and pathologic changes in human thymus. EZ replicated best and induced substantial thymocyte death ; AIK-c replicated poorly but induced moderate thymocyte death ; Moraten replicated moderately well but did not affect thymic architecture. Thus, live attenuated measles vaccines differ in replicative capacity and pathogenicity in vivo. These differences may account for strain-dependent variations in immunization.
Viviani MA, Esposto MC, Cogliati M, Montagna MT, Wickes BL (2001) Isolation of a Cryptococcus neoformans serotype A MATa strain from the Italian environment. Med Mycol 39 :383-386
Cryptococcus neoformans is a heterothallic basidiomycete which possesses a bipolar mating system based on two mating type alleles, MATa and MATalpha. In the type variety, C. neoformans var. neoformans, both mating types have been found among strains of one serotype, serotype D, whereas only MATalpha was identified after extensive survey of serotype A strains. Serotype A MA Ta appeared to be extinct or to exist only in a vestigial, non-functional form. We report the isolation of a C. n. var. neoformans serotype A MATa strain from the Italian environment. The strain was serotyped by slide agglutination test, genotyped by polymerase chain reaction (PCR) fingerprinting using the (GACA)4 primer, and its haploid state was determined by flow cytometry. The mating type was identified by PCR amplification of the pheromone a gene. In addition, the amplification of the four STE20 alleles, specific for the mating type of serotypes A and D, showed that the strain contains only the MATa locus. By crossing experiments the strain was found to be fertile. The interest in the finding of this fertile isolate is related to the possibility to construct a congenic pair of serotype A MATa/MATalpha strains to be used in genetic and pathogenesis studies.
Vogt B, Roscher S, Abel B, Hildinger M, Lamarre A, Baum C, von Laer D (2001) Lack of superinfection interference in retroviral vector producer cells. Hum Gene Ther 12 :359-365
Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retroviral vectors have become more feasible for clinical gene transfer protocols since stable tetracycline (tet)-regulated packaging cell lines have become available. Here, we analyzed superinfection interference in VSV-G-pseudotyped and classic amphotropic packaging cell lines. No superinfection interference was observed in VSV-G-pseudotyped packaging cell lines. Thus, integrated retroviral vector genomes accumulated during culture. Similar results were obtained with the amphotropic packaging cells, but to a lesser degree. In addition, VSV-G packaging cells were susceptible to infection with vector particles devoid of envelope proteins, which are produced by these cells in high titers when VSV-G expression is suppressed by tetracycline. For both packaging systems, superinfection could be blocked by azidothymidine (AZT). With regard to safety, this study suggests that in clinical protocols amphotropic producer clones should be tested for superinfection interference and VSV-G packaging cells should always be cultured in the presence of AZT.
Walberg M, Steen HB (2001) Flow cytometric monitoring of bacterial susceptibility to antibiotics. Methods Cell Biol 64 :553-566
Wang J, Wang D, Feng Z, Wang RJ, Lu Y, Shen J, Xu Y (2001) [Regulatory effect of Bcl-2 on the cortical neurons of primary cultured mice with HSV-1 infection]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 15 :228-229
OBJECTIVE : To study the effect of Bcl-2 on the cortical neurons of primary cultured mice with HSV-1 infection. METHODS : Analysis of the expression of Bcl-2 on the cortical neurons of primary cultured fetal mice in vitro infected with HSV-1 for 11 hours or exposed to sorbitol for 5 hours was made by flow cytometry and Western blotting. RESULTS : The Bcl-2 of the cultured neurons undergoing HSV-1 infection expressed upregulating compared with the mock untreated neurons. The Bcl-2 protein of HSV-1 infected and exposed to sorbitol neurons expressed upregulating compared with the control group. The Bcl-2 protein of the primary cultured 3 day cells expressed higher than that of the primary cultured 7 day cells. The higher neurovirulence, the higher expression of Bcl-2. CONCLUSIONS : Bcl-2 might protect the primary cultured cortical neurons of fetal mice from apoptosis where infected with HSV-1, and thereby the lifespan of host cells may be prolonged.
Wang SJ, Yao K, Xie FD, Ji XH (2001) Effects of Tripterygium wilfordii saponins and interleukin-10 on dendritic cells from human peripheral blood. Acta Pharmacol Sin 22 :721-724
AIM : To study the effects of Tripterygium wilfordii (TII) and IL-10 on human leukocyte antigen-DR (HLA-DR) and CD80 expressions and IL-12 p40 subunit production and transcription of dendritic cells (DC) in human peripheral blood from healthy volunteers in vitro. METHODS : DC were generated by culturing plastic-adherent peripheral blood mononuclear cells with GM-colony stimulating factor (GM-CSF), IL-4, and TNFalpha. The expressions of HLA-DR and CD80 were examined by flow cytometry after the cells were stained with immunofluorescence antibodies. Enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction analysis were used to detect IL-12 p40 protein level and mRNA transcription, respectively. RESULTS : TII 5 - 20 mg/L and IL-10 50 - 200 microg/L greatly down-regulated the membrane expressions of HLA-DR and CD80 on DC in a concentration-dependent manner. IL-12 p40 production and mRNA transcription were also inhibited in DC both by TII and by IL-10. CONCLUSION : TII and IL-10 exert immunosuppressive role via inhibiting membrane expressions of HLA-DR and CD80 and synthesis of IL-12 p40 subunit in DC.
Warner S, Hartley CA, Stevenson RA, Ficorilli N, Varrasso A, Studdert MJ, Crabb BS (2001) Evidence that Equine rhinitis A virus VP1 is a target of neutralizing antibodies and participates directly in receptor binding. J Virol 75 :9274-9281
Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathione S-transferase (GST) fusion protein (GST-VP1). GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV. An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro. Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated GST-VP1 in receptor binding. Importantly, anti-GST-VP1 antibodies inhibited the binding of ERAV virions to Vero cells, suggesting that these antibodies exert their neutralizing effect by blocking viral attachment. Thus ERAV VP1, like its counterpart in FMDV, appears to be both a target of protective antibodies and involved directly in receptor binding. This study reveals the potential of recombinant VP1 molecules to serve as vaccines and diagnostic reagents for the control of ERAV infections.
Wasylnka JA, Simmer MI, Moore MM (2001) Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species. Microbiology 147 :869-877
ASPERGILLUS : fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition : the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.
Weisman R, Choder M (2001) The fission yeast TOR homolog, tor1+, is required for the response to starvation and other stresses via a conserved serine. J Biol Chem 276 :7027-7032
Targets of rapamycin (TORs) are conserved phosphatidylinositol kinase-related kinases that are involved in the coordination between nutritional or mitogenic signals and cell growth. Here we report the initial characterization of two Schizosaccharomyces pombe TOR homologs, tor1(+) and tor2(+). tor2(+) is an essential gene, whereas tor1(+) is required only under starvation and other stress conditions. Specifically, Deltator1 cells fail to enter stationary phase or undergo sexual development and are sensitive to cold, osmotic stress, and oxidative stress. In complex with the prolyl isomerase FKBP12, the drug rapamycin binds a conserved domain in TORs, FRB, thus inhibiting some of the functions of TORs. Mutations at a conserved serine within the FRB domain of Saccharomyces cerevisiae TOR proteins led to rapamycin resistance but did not otherwise affect the functions of the proteins. The S. pombe tor1(+) exhibits different features ; substitution of the conserved serine residue, Ser(1834), with arginine compromises its functions and has no effect on the inhibition that rapamycin exerts on sexual development in S. pombe.
West NJ, Schonhuber WA, Fuller NJ, Amann RI, Rippka R, Post AF, Scanlan DJ (2001) Closely related Prochlorococcus genotypes show remarkably different depth distributions in two oceanic regions as revealed by in situ hybridization using 16S rRNA-targeted oligonucleotides. Microbiology 147 :1731-1744
An in situ hybridization method was applied to the identification of marine cyanobacteria assignable to the genus Prochlorococcus using horseradish-peroxidase-labelled 16S rRNA-targeted oligonucleotide probes in combination with tyramide signal amplification (TSA). With this method very bright signals were obtained, in contrast to hybridizations with oligonucleotides monolabelled with fluorochromes, which failed to give positive signals. Genotype-specific oligonucleotides for high light (HL)- and low light (LL)-adapted members of this genus were identified by 16S rRNA sequence analyses and their specificities confirmed in whole-cell hybridizations with cultured strains of Prochlorococcus marinus Chisholm et al., 1992, Prochlorococcus sp. and Synechococcus sp. In situ hybridization of these genotype-specific probes to field samples from stratified water bodies collected in the North Atlantic Ocean and the Red Sea allowed a rapid assessment of the abundance and spatial distribution of HL- and LL-adapted Prochlorococcus. In both oceanic regions the LL-adapted Prochlorococcus populations were localized in deeper water whereas the HL-adapted Prochlorococcus populations were not only distinct in each region but also exhibited strikingly different depth distributions, HLI being confined to shallow water in the North Atlantic, in contrast to HLII, which was present throughout the water column in the Red Sea.
Wouters PC, Bos AP, Ueckert J (2001) Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields. Appl Environ Microbiol 67 :3092-3101
Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products.
Zaman Z, Roggeman S, Verhaegen J (2001) Unsatisfactory performance of flow cytometer UF-100 and urine strips in predicting outcome of urine cultures. J Clin Microbiol 39 :4169-4171
UF-100 flow cytometer and urine strip results were cross-interpreted to predict culture outcomes. The best negative predictive value was obtained with bacteria at > or =1,000/microl, white blood cells at > or =20/microl, or leukocyte esterase positivity. Nine of 24 false negatives were clinically significant. Thus, UF-100 and urine strip results do not accurately predict the outcome of cultures.
Zerbe H, Ossadnik C, Leibold W, Schuberth HJ (2001) Influence of Escherichia coli and Arcanobacterium pyogenes isolated from bovine puerperal uteri on phenotypic and functional properties of neutrophils. Vet Microbiol 79 :351-365
When cows develop endometritis after birth, Escherichia coli and Arcanobacterium pyogenes are usually the most prominent bacteria present in bovine uterine lochial secretions. A. pyogenes alone is rarely found in the course of a disturbed puerperium. This was confirmed in this study, since average and high-grade uterine contaminations were always associated with the presence of both bacteria. The contamination grade was positively correlated with uterine polymorphonuclear granulocyte (PMN) numbers and negatively correlated with blood PMN numbers. Whether E. coli and A. pyogenes affect the phenotype and function of bovine PMN in a similar or differential way was subject to in vitro studies. PMN were tested in the presence of washed bacterial fragments or culture supernatants taken as a source for soluble and/or secreted bacterial products. Fragments and soluble products differed only quantitatively in their effects on PMN. Usually, long-time exposure (24h) of PMN to fragments induced the strongest effects. Accelerated death of granulocytes was only moderately induced by both E. coli and A. pyogenes products. Both E. coli and A. pyogenes products induced the enhanced expression of a membrane molecule detected by mAb IL-A110 and of CD11b. Expression of other surface structures remained largely unchanged (MHC class I, CD11c). Functional parameters of PMN (phagocytosis ; generation of reactive oxygen species, ROS ; antibody-independent cellular cytotoxicity, AICC) generally declined after pre-incubation for 24h with products of E. coli or A. pyogenes. Interestingly, soluble products of A. pyogenes stimulated the phagocytosis of PMN. However, co-incubation with E. coli products abrogated this stimulatory effect. The results supply evidence for similar modes of action of the gram-negative E. coli and the gram-positive A. pyogenes on bovine PMN. Alterations in PMN function and phenotype are mainly triggered by direct contact between bacterial fragments and PMN. Inhibition experiments with polymyxin B demonstrated that E. coli-mediated effects were not solely due to the action of lipopolysaccharide. The dominant functional depression of neutrophils by E. coli products strengthens the suggestion that the earlier appearance of E. coli in the uterus may support the co-infection of this organ by A. pyogenes at later times.
Zhang YJ, Wang XP, Deng JH, Salinas RA, Oishi N, Gao SJ (2001) Suppression of oncogenic viral interferon regulatory factor (vIRF) of Kaposi’s sarcoma-associated herpesvirus by ribozyme-mediated cleavage. Cancer Gene Ther 8 :285-293
Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8) has been etiologically associated with several malignancies including Kaposi’s sarcoma and primary effusion lymphoma. Oncogenic viral interferon regulatory factor (vIRF) encoded by KSHV ORF-K9 is a homologue of cellular interferon regulatory factor (IRF), and has been demonstrated to inhibit type I/II interferon signal transduction and transform NIH3T3 cells through the interactions with IRF-1, IRF-3, and CBP/p300 proteins. To counteract vIRF’s pathogenic role, we have developed five ribozymes targeting ORF-K9 mRNA to suppress vIRF expression. The vIRF RNA substrates were cleaved up to 80% in a substrate-specific manner in transcript cleavage assays in vitro. In a transient transfection assay, two of the ribozymes efficiently suppressed the expression of vIRF protein measured by dual-color immunofluorescence assay that simultaneously detects the expression of both vIRF protein and ribozyme. Flow cytometry analysis showed that these ribozymes reduced vIRF expression up to 76%. A mutant ribozyme had no cleavage activity in vitro, but exhibited antisense effect in vivo. These results suggest that the ribozymes may provide a new approach for functional knockout of vIRF gene, and are potential candidates of antiviral therapy for KSHV-related malignancies.
Zhao LJ, Liu HQ, Cao J, Feng GS, Qi ZT (2001) Activation of Intracellular MAPK/ERK Initiated by Hepatitis C Virus Envelope Protein E2 in HepG2 Cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 33 :691-695
CD81, widely expressed on the surface of various human cells including hepatocytes, is a protein involved in intracellular signal transduction pathways. Recent studies suggested that human CD81 could specifically interact with hepatitis C virus (HCV) envelope protein E2. Therefore, CD81 has been identified as a putative cellular receptor for HCV. The HCV E2-CD81 interaction was considered a molecular mechanism contributing to HCV infection and pathogenicity. MAPK/ERK is characteristically associated with cell proliferation and hypertrophy. To investigate the effect of HCV on MAPK/ERK, human HepG2 cells were used in this study. CD81 expression on HepG2 cell surface was determined by flow cytometry with method of immunofluorescence. The cells were cultured in DMEM medium without fetal calf serum for 7 h, and then treated with HCV E2 protein at different time courses. Activation of MAPK/ERK in the cells was measured by Western blot, immunohistochemical and immunofluorescent analyses. Phosphorylation of MAPK/ERK was related to the concentration of HCV E2 proteins and to the time length of stimulation. MAPK/ERK in HepG2 cells was activated by HCV E2 protein, suggesting that HCV E2-CD81 interaction might be involved in intracellular signal transduction and might play an active role in HCV pathogenicity.
Zhu Z, Lee CS, Tejeda KM, Giannobile WV (2001) Gene transfer and expression of platelet-derived growth factors modulate periodontal cellular activity. J Dent Res 80 :892-897
Platelet-derived growth factor (PDGF) is a potent stimulator of wound healing. PDGF gene therapy may promote greater periodontal regeneration than local protein application, due to sustained growth factor delivery to the target tissue. This investigation tested the ability of recombinant adenoviruses (rAds) encoding PDGF-A or PDGF-1308 (a PDGF-A dominant-negative mutant that disrupts endogenous PDGF bioactivity) to affect cells derived from the periodontium. Osteoblasts, periodontal ligament fibroblasts, and gingival fibroblasts were transduced with rAds, and gene expression, DNA synthesis, and cell proliferation were evaluated. The results revealed strong message for the PDGF-A gene for 7 days following gene delivery. Ad2/PDGF-A enhanced the mitogenic and proliferative response in all cell types, while Ad2/PDGF-1308 potently inhibited mitogenesis and proliferation. In conclusion, Ad2/PDGF can effectively transduce cells derived from the periodontium and promote biological activity equivalent to PDGF-AA. These studies support the potential use of gene therapy for sustained PDGF release in periodontal tissues.
Zubkov MV, Fuchs BM, Burkill PH, Amann R (2001) Comparison of cellular and biomass specific activities of dominant bacterioplankton groups in stratified waters of the Celtic Sea. Appl Environ Microbiol 67 :5210-5218
A flow-sorting technique was developed to determine unperturbed metabolic activities of phylogenetically characterized bacterioplankton groups with incorporation rates of [(35)S]methionine tracer. According to fluorescence in situ hybridization with rRNA targeted oligonucleotide probes, a clade of alpha-proteobacteria, related to Roseobacter spp., and a Cytophaga-Flavobacterium cluster dominated the different groups. Cytometric characterization revealed both these groups to have high DNA (HNA) content, while the alpha-proteobacteria exhibited high light scatter (hs) and the Cytophaga-Flavobacterium cluster exhibited low light scatter (ls). A third abundant group with low DNA (LNA) content contained cells from a SAR86 cluster of gamma-proteobacteria. Cellular specific activities of the HNA-hs group were 4- and 1.7-fold higher than the activities in the HNA-ls and LNA groups, respectively. However, the higher cellular protein synthesis by the HNA-hs could simply be explained by their maintenance of a larger cellular protein biomass. Similar biomass specific activities of the different groups strongly support the main assumption that underlies the determination of bacterial production : different bacteria in a complex community incorporate amino acids at a rate proportional to their protein synthesis. The fact that the highest growth-specific rates were determined for the smallest cells of the LNA group can explain the dominance of this group in nutrient-limited waters. The metabolic activities of the three groups accounted for almost the total bacterioplankton activity, indicating their key biogeochemical role in the planktonic ecosystem of the Celtic Sea.
zur Hausen A, van Grieken NC, Meijer GA, Hermsen MA, Bloemena E, Meuwissen SG, Baak JP, Meijer CJ, Kuipers EJ, van den Brule AJ (2001) Distinct chromosomal aberrations in Epstein-Barr virus-carrying gastric carcinomas tested by comparative genomic hybridization. Gastroenterology 121 :612-618
BACKGROUND & AIMS : Approximately 10% of gastric adenocarcinomas carry the human pathogenic Epstein-Barr virus (EBV). The role of EBV in the pathogenesis of these carcinomas remains to be established. METHODS : To obtain a comprehensive overview of chromosomal aberrations in EBV-carrying and EBV-negative gastric carcinomas we performed comparative genomic hybridization (CGH) on 44 gastric carcinomas, 10 EBV-positive, and 34 EBV-negative. Additionally, DNA flow cytometry was done. RESULTS : Loss of chromosome 4p (P < 0.001) and of 11p (P < 0.02) was exclusively restricted to EBV-carrying gastric carcinomas. In addition, loss of 18q (P < 0.02) was significantly more frequent in EBV-carrying gastric carcinomas. The latter involves loci, which have already been linked to gastric carcinogenesis such as the DCC and SMAD4 gene. In contrast, the losses on chromosome 4 and 11 do not yet harbor a gene related to gastric carcinogenesis. No significant correlation was found between DNA-ploidy and the EBV-status. A number of chromosomal aberrations were found at comparable frequencies in both groups, i.e., losses of chromosome 17, 12q, and loss of 1p. Interestingly, gains of 13q (10/34) and 3q (5/34) and loss of 1q (5/34) were solely observed in EBV-negative gastric carcinomas. CONCLUSIONS : These data indicate that EBV-carrying and EBV-negative gastric carcinomas have different pathogenetic pathways in which EBV might play a crucial role.