vendredi 24 avril 2009
par   G. Grégori

Akerlund K, Bjork L, Fehniger T, Pohl G, Andersson J, Andersson U (1996) Sendai virus-induced IFN-alpha production analysed by immunocytochemistry and computerized image analysis. Scand J Immunol 44 :345-353


IFN-alpha production in Sendai virus-stimulated human buffy coat cultures could readily be demonstrated in individual cells at a protein level by the use of a novel immuno-enzymatic staining procedure. A distinctive rounded, juxtanuclear staining pattern was generated in producer cells by the accumulation of the intracellularly synthesized IFN-alpha in the Golgi stacks. The technology is based upon acquiring a video image of stained monolayers of cells, viewed in a microscope by a colour camera, which then transfers binary images directly into a computer-controlled operating system. The characteristic appearance of the immunocytochemical staining enabled a computerized image-analysis system to measure IFN-alpha producing cells based on defined criteria set for morphology, intensity, colour and size. The automated system could accurately and reproducibly register a range of 0.1-7.0% of the total cell population as IFN-alpha producing cells during the kinetic studies of the response. Congruent results were obtained with manual microscopy and image analysis concerning the assessment of the incidence of IFN-alpha producing cells in the total cell populations. All IFN-alpha producing cells expressed surface HLA-DR molecules and 95% of these cells belonged to the myelomonocytic lineage. The image analysis system provided, in contrast to conventional microscopy, an opportunity to assess and document differences of signal intensity and cell size of individual IFN-alpha producing as well as non-producing cells.

Belyavskyi M, Westerman M, DiMichele L, Wilson VG (1996) Perturbation of the host cell cycle and DNA replication by the bovine papillomavirus replication protein E1. Virology 219 :206-219


A stable cell line expressing the bovine papillomavirus E1 protein (C2E1) was compared with an E1 minus control line (CNEO) to study the effects of E1 protein on host cell growth. C2E1 and CNEO cells were synchronized either at mitosis or at the G1/S boundary by the cell cycle inhibitors nocodazole and mimosine, respectively. After release from the drug-induced cell cycle block, the progression through the succeeding stages of the cell cycle was temporally monitored using flow cytometry. In addition, incorporation of bromodeoxyuridine (BrdUrd) was used to determine precisely the time of initiation of DNA synthesis in C2E1 and CNEO cells after release from drug-induced cell cycle arrest. Expression of E1 protein decreased the duration of G1 phase and increased S and G2 phase durations without affecting the overall cell doubling time. In conjunction with the increase in G2 phase duration, histone H1 kinase activity was prolonged during the G2 to M phase transition in C2E1 cells, which suggested that E1 protein may affect the mechanisms which ensure proper timing of kinase inactivation. During the G1 to S phase transition in C2E1 cells, the timing of appearance and abundance of cyclin D1 were altered compared to CNEO cells, while cyclin E levels were unaffected. Consequently, E1 protein may affect G1 phase duration through a cyclin D1-dependent pathway. Finally, a subpopulation of cells with a greater than G2 DNA content (>G2 DNA), and which was still capable of incorporating BrdUrd, was shown to exist only in the E1-expressing cell line. These combined results demonstrate that the viral replication protein E1 has the potential to influence the host cell environment significantly, which may contribute to pathogenesis and viral persistence.

Bernard S, Olivier M, Bernard F, Berthon P, Doucet F, Lantier F (1996) Flow cytometric analysis of the reactivity of workshop monoclonal antibodies on sheep lymph node cells, after infection with Salmonella abortusovis. Vet Immunol Immunopathol 52 :403-409


Of the 302 monoclonal antibodies included in the Third Workshop on Ruminant Leukocyte Antigens, 167 have been tested for their reactivity on uninfected and Salmonella-infected sheep lymph node leukocytes using FACS analysis. Only 47 of them showed specificities which could be related to those of some control monoclonal antibodies, such as the anti-CD5, anti-Ig light chain and anti-MHC Class II monoclonal antibodies.

Christensson M, Bremme K, Shanwell A, Westgren M, Christensson B (1996) Flow cytometric quantitation of serum anti-D in pregnancy. Transfusion 36 :500-505


BACKGROUND : The major cause of fetal hemolytic disease is maternal immunization to D in D-incompatible pregnancies. To prevent complications, D-incompatible pregnancies are monitored for the level of maternal anti-D. At present, the monitoring of anti-D levels is performed by the indirect antiglobulin test complemented by quantitation by the technique used in an automated antibody detection and quantitation instrument. STUDY DESIGN AND METHODS : Flow cytometry was used to quantitatively determine the level of anti-D in serum and to analyze the IgG subclass distribution and the presence of IgM anti-D in these samples. The results were compared to the indirect antiglobulin test titer and to the results obtained by the technique used in an automated antibody detection and quantitation instrument. RESULTS : Flow cytometry allowed sensitive and accurate determinations of anti-D levels with low interassay and intra-assay variability, both for serum samples and standard curves. CONCLUSION : Flow cytometry is a simple, rapid, and reliable method for determining the serum levels of D antibodies and their Ig subclass distribution. It is therefore well suited for the monitoring of women during D-incompatible pregnancies.

Clyne M, Drumm B (1996) The urease enzyme of Helicobacter pylori does not function as an adhesin. Infect Immun 64 :2817-2820


Helicobacter pylori urease is essential for colonization of the gastric mucosa irrespective of whether the stomach is acidic or hypochlorhydric. It has therefore been speculated that the enzyme functions as an adhesin. The aim of this study was to compare the adherence of H. pylori N6 with the adherence of an isogenic urease-negative mutant, strain N6(ureB ::TnKm), to gastric cells. Strain N6 originated from a patient with gastritis. Strain N6(ureB ::TnKm) is specifically modified in the gene which encodes the large subunit of urease, UreB, and hence does not form a UreA-UreB enzyme complex. We have used flow cytometry to assess the adherence of H. pylori to the cells. We have also used phase-contrast microscopy to assess the adherence of the organism to Kato III cells. In the absence of urea both strains bound to Kato III cells and to primary gastric cells. Binding of both strains to the cells occurred rapidly. The presence of urea in the incubation medium decreased the binding of strain N6 to the cells. This was due to a rise in the pH of the incubation medium, which caused loss of viability of the organism. Urea had no effect on the adherence of strain N6(ureB ::TnKm). We conclude that the urease of H. pylori does not play a role in the adherence of the organism to gastric cells.

Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173 :33-38


We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.

Cuff CF, Zhao W, Nukui T, Schafer R, Barnett JB (1996) 3,4-Dichloropropionanilide-induced atrophy of the thymus : mechanisms of toxicity and recovery. Fundam Appl Toxicol 33 :83-90


The herbicide 3,4-dichloropropionanilide (propanil) has several well-documented neurotoxic and immunotoxic effects on mice. We report here a detailed characterization of the effects of propanil exposure on the thymus. We found that at doses of 100-200 mg/kg, propanil induces significant thymic atrophy between 2 and 7 days postexposure. This atrophy is characterized by a decrease in thymus/body ratio and a decrease in cellularity. Flow cytometric analyses of thymuses from propanil- and vehicle-treated mice indicate that the CD4(+) CD8(+) population of immature cells, is most significantly decreased in propanil-exposed mice. We performed cell cycle analysis of thymocyte populations using two-color surface staining and the DNA binding dye 7-aminoactinomycin D to determine whether thymic atrophy was associated with changes in the percentages of cells in the S, G2, and M phases of the cell cycle. We found a high percentage of proliferating CD4(+)CD8(+) thymocytes 4 days after exposure. Thus, recovery of the thymus occurs following increases in thymocyte proliferation, most notably the immature CD4(+) CD8(+) thymocytes. We tested the hypothesis that glucocorticoids play a role in the observed atrophy by examining thymuses in adrenalectomized, propanil-treated mice. No atrophy was observed in those animals. These results suggest that propanil has an immunotoxic effect on the thymus that appears to be mediated, in part, by endogenous glucocorticoids.

D’Apice L, Fenizia D, Capparelli R, Scala F, Iannelli D (1996) Detection of antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry. Res Vet Sci 60 :179-181


An assay has been developed to detect antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry. The method was the protein A-deficient strain Wood 46 of S aureus incubated with milk samples and fluorescein-labelled rabbit anti-water buffalo antiserum. The assay can detect antibodies when the pathogen is not detectable by bacterial tests and can determine the antibody titre directly on undiluted samples.

Davey HM, Kell DB (1996) Flow cytometry and cell sorting of heterogeneous microbial populations : the importance of single-cell analyses. Microbiol Rev 60 :641-696


The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is a property of an individual cell, with macroscopic measurements of culture variables such as ATP content, respiratory activity, and so on, must inevitably fail. It is therefore necessary to make physiological measurements on individual cells. Flow cytometry is such a technique, which allows one to analyze cells rapidly and individually and permits the quantitative analysis of microbial heterogeneity. It therefore offers many advantages over conventional measurements for both routine and more exploratory analyses of microbial properties. While the technique has been widely applied to the study of mammalian cells, is use in microbiology has until recently been much more limited, largely because of the smaller size of microbes and the consequently smaller optical signals obtainable from them. Since these technical barriers no longer hold, flow cytometry with appropriate stains has been used for the rapid discrimination and identification of microbial cells, for the rapid assessment of viability and of the heterogeneous distributions of a wealth of other more detailed physiological properties, for the analysis of antimicrobial drug-cell interactions, and for the isolation of high-yielding strains of biotechnological interest. Flow cytometric analyses provide an abundance of multivariate data, and special methods have been devised to exploit these. Ongoing advances mean that modern flow cytometers may now be used by nonspecialists to effect a renaissance in our understanding of microbial heterogeneity.

Duan Q, Zhao Z, Huang C, Zhang S (1996) [Analysis of surface antigen molecule expression on serovar stains of Neisseria gonorrhoeae by flow cytometry]. Wei Sheng Wu Xue Bao 36 :379-384


In the present experiment, flow cytometry was employed for analysing expression characteristics of the antigen molecules distingushed by 10 monoclonal antibodies against Neisseria gonorrhoeae lipooligosaccharides. Stability of antigen expression and amount of epitope on the surface of N. gonorrhoeae were quantitative determined. Reactivity of the monoclonal antibodies with serovar strains of N. gonorrhoeae were evaluated.

Eliasson A, Bernander R, Nordstrom K (1996) Random initiation of replication of plasmids P1 and F (oriS) when integrated into the Escherichia coli chromosome. Mol Microbiol 20 :1025-1032


We have constructed intP1 and intFs strains of Escherichia coli in which the basic replicons of either plasmid P1 or plasmid F (oriS) were integrated into an inactivated oriC, such that chromosome replication is controlled by the integrated plasmid replicon. In this study, we have further analysed these strains, and density-shift experiments revealed that chromosome replication occurred randomly during the cell cycle. Flow-cytometry analyses of exponentially growing populations supported this conclusion, and also showed that the DNA/mass ratio of the strains decreased with increasing growth rate. Flow cytometry of exponentially growing cultures treated with rifampicin demonstrated that initiation of replication was uncoordinated in cells containing multiple replication origins.

Gordon S, Houldsworth S, Duncan K, Roberts IS, Andrew PW (1996) Rapid measurement of antimycobacterial drug activity. Res Microbiol 147 :79-86


Gudmundsson GH, Agerberth B, Odeberg J, Bergman T, Olsson B, Salcedo R (1996) The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur J Biochem 238 :325-332


The peptide FA-LL-37, previously termed FALL-39, was originally predicted from on ORF of a cDNA clone isolated from a human bone marrow library. This peptide was synthesized and found to have antibacterial activity. We have now characterized and sequenced the complete gene for FA-LL-37, termed FALL39. It is a compact gene of 1963 bp with four exons. Exons 1-3 code for a signal sequence and the cathelin region. Exon 4 contains the information for the mature antibacterial peptide. Our results indicate that FALL39 is the only member of the cathelin gene family present in the human genome. Potential binding sites for acute-phase-response factors are identified in the promoter and in intron 2. A possible role for the cytokine interleukin-6 in the regulation of FALL 39 is discussed. Anti-(FA-LL-37) IgG located the peptide in granulocytes and we isolated the mature peptide from these cells after degranulation. Structural analysis determined the mature peptide to be LL-37. To obtain LL-37 for antibacterial assays, synthetic FA-LL-37 was degraded with dipeptidyl-peptidase I. This analysis showed that mature LL-37 is a potent antibacterial peptide.

Heidner HW, Knott TA, Johnston RE (1996) Differential processing of sindbis virus glycoprotein PE2 in cultured vertebrate and arthropod cells. J Virol 70 :2069-2073


A step in the maturation of Sindbis virus glycoproteins is the cleavage of the precursor glycoprotein PE2 into E3 and E2 by furin or a furin-like host cell protease. The results presented here suggest that PE2 cleavage is an obligatory event for Sindbis virus maturation in C6/36 cells and demonstrate that certain mutants display a cell-specific PE2 cleavage phenotype. We previously have described Sindbis virus variants which fail to cleave PE2 because of incorporation of a signal for N-linked glycosylation immediately adjacent to the PE2 cleavage site but are viable in BHK-21 cells by virtue of an additional mutation at E2 216 or E2 191 (TRSB-NE2G216 and TRSB-NE2T191, respectively) (H. W. Heidner, K. L. McKnight, N. L. Davis, and R. E. Johnston, J. Virol. 68:2683-2692, 1994). Other viable PE2 cleavage-defective mutants were constructed by substituting the parental residue at E2 position 1 (Arg), with Leu or Val (TRSB-E2L1 and TRSB-E2V1, respectively) (H.W. Heidner and R. E. Johnston, J. Virol. 68:8064-8070, 1994). When grown in BHK-21 cells, all four of these viruses replicated normally and incorporated PE2 in place of E2 in released virions. However, growth of TRSB-NE2G216 and TRSB-NE2T191 was severely restricted in cultured arthropod cells (C6/36 cells). Analysis of infected C6/36 cells by flow cytometry demonstrated that the restricted growth of TRSB-NE2G216 and TRSB-NE2T191 was not due to an impaired ability to initiate infection. In addition, TRSB-NE2G216 and TRSB-NE2T191 remained growth restricted in C6/36 cells following introduction of in vitro transcriptions by electroporation. In contrast, the PE2 cleavage defect of TRSB-E2L1 and TRSB-E2V1 was cell type specific. In C6/36 cells, the majority of PE2 was converted to E2, and these viruses replicated normally in C6/36 cells. These results demonstrated a consistent link between expression of a PE2 cleavage defect and restricted growth in C6/36 cells and suggest that cleavage of PE2 is required for maturation of Sindbis virus late in infection of C6/36 cells.

Hock B (1996) Advances in immunochemical detection of microorganisms. Ann Biol Clin (Paris) 54 :243-252


Immunology and microbiology have been linked together since their infancies. The discovery more than 100 years ago that antibodies (Abs) constitute one of the pillars of the body’s defense against bacteria and viruses led immediately to the development of serological tests for diagnosis and identification of microorganisms. More recent approaches are based on immunoassay technology, which does not require cross-linking of antigens by Abs. High sensitivity results from the use of labels, such as fluorescent dyes or enzymes, for the detection of antigen binding by Abs. As an example, the quantification of members of the Enterobacteriaceae in drinking water using enzyme immunoassays (Elisa) is presented, which is now available as a DIN standard. Related techniques such as dipstick or dot blot tests originated from the necessity of shortening the analysis time. Immunofluorescence flow cytometry represents the most sophisticated development of this technology to date. A major technological leap is expected from immunosensors, miniaturized measuring devices that selectively detect their targets and provide concentration-dependent signals. When Abs as part of the receptor unit bind their ligands, there is a variation in optical properties, electric charge, mass, or heat, which can be detected directly, ie without tracers, by a variety of transducers. Since sensitivity is directly related to the affinity of the ligand binding, high sensitivity excludes reversibility. Immunochemical methodology is still limited by the availability of selective and sensitive Abs. Future progress will be significantly accelerated by the application of recombinant techniques for Ab production. The main emphasis is directed toward the generation of recombinant Ab libraries, as they are already available for the generation of anti-HIV Ab fragments. It is not surprising therefore that immunochemical methodology, together with PCR-based techniques, belongs to the most promising branch of modern diagnosis.

Hudson KM, Denko NC, Schwab E, Oswald E, Weiss A, Lieberman MA (1996) Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins. Blood 88 :3465-3473


Cytotoxic necrotizing factor (CNF) toxins, isolated from certain Escherichia coli strains known to cause intestinal and extra intestinal infections, induce reorganization of the actin cytoskeleton and generate hyperploidy in adherent cell lines. We have examined the effect of CNF toxin on one of the few cell types that naturally increase nuclear DNA content, megakaryocytes. Our studies show that only hematopoietic cells capable of differentiating along the megakaryocyte lineage responded to the CNF2 toxin by becoming polyploid and by reorganizing actin. The K562, HEL, and CHRF-288-11 cell lines can be induced with phorbol ester to differentiate along the megakaryocyte lineage, and these cells also respond to the toxin with increased DNA content and actin cytoskeletal rearrangements. Interestingly, treatment of the K562 and HEL cell lines with CNF2 does not result in an increase in production of the megakaryocytic marker glycoprotein IIIa, unlike phorbol ester treatment. Conversely, two T-cell leukemic cell lines, CEM and Molt4, and the promyelocytic HL-60 cell line, which do not differentiate along the megakaryocyte lineage in response to phorbol myristate acetate, do not respond to CNF2, by increased expression of gpIIIa, increased nuclear DNA content, or actin reorganization. A potential target of these toxins, RhoA, is expressed by both megakaryocytic and nonmegakaryocytic cell lines, as shown by reverse transcription-polymerase chain reaction and Western blot. Although it is clear that the CNF toxins can affect a wide variety of adherent nonhematopoietic cell lines, we propose that the response to CNF, in terms of reorganizing actin structure and increase in DNA content in hematologic suspension cells, correlates with the capability of these target cells to differentiate along the megakaryocytic lineage.

Hughes EE, Matthews-Greer JM, Gilleland HE, Jr. (1996) Analysis by flow cytometry of surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Can J Microbiol 42 :859-862


Antisera were produced in mice immunized with 18 synthetic peptide conjugates representing various regions throughout the length of the outer membrane protein F molecule of Pseudomonas aeruginosa and analysed by flow cytometry to identify those antisera capable of binding to the surface of whole cells of P. aeruginosa. Antibodies to peptides 9, 18, 10, and 4 were significantly cell-surface reactive. The maximum median percentage of antibody-binding cells in this assay was 36.6%. Over six different determinations, peptide 9 antisera binding to the cells ranged from 16.9 to 57.0% of the cell population. We propose that the surface accessibility of protein F epitopes varies during the cell cycle.

Ishibashi K, Yamaguchi O, Shiraiwa Y, Ogihara M, Shigeta S (1996) Combination therapy of Pseudomonas aeruginosa pyelonephritis in neutropenic mice with human antilipopolysaccharide monoclonal antibody and cefsulodin. J Urol 155 :2094-2097


PURPOSE : These studies were designed to determine the combined inhibitory effect of a human monoclonal antibody (MAb) and cefsulodin on Pseudomonas aeruginosa renal infection in a neutropenic condition. MATERIALS AND METHODS : Protection against the infection of mice was estimated by survival rate and bacterial numbers in the kidney and blood. Opsonophagocytic assay by human polymorphonuclear neutrophils (PMNs) and fluorescence activated cell sorter (FACS) analysis were also examined. RESULTS : Treatment of infected mice with MAb combined with a suboptimal dose of cefsulodin prevented the mice from developing pyelonephritis and bacteremia and resulted in a significantly higher survival rate than treatment with either MAb or cefsulodin alone (p < 0.01). When bacteria were preexposed to cefsulodin, a significant enhancement in opsonophagocytic killing with MAb was observed. Fluorescence activated cell sorter analysis suggested that the bacteria incubated with 1/4 minimal inhibitory concentration (MIC) of cefsulodin showed greater binding of MAb to bacteria than the control. CONCLUSION : The combination therapy with human antilipopolysaccharide MAb and cefsulodin is useful for P. aeruginosa pyelonephritis in neutropenic hosts.

Ji Y, McLandsborough L, Kondagunta A, Cleary PP (1996) C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect Immun 64 :503-510


Group A streptococcal C5a peptidase (SCPA) specifically cleaves the human serum chemotaxin C5a at the polymorphonuclear leukocyte (PMNL) binding site. This study tested the proposal that SCPA contributes to virulence by retarding the influx of inflammatory cells and clearance of streptococci during the first few hours after infection. To investigate the specific contribution of SCPA to the virulence of group A streptococci, scpA insertion and deletion mutants were created by directed plasmid insertion into scpA and gene replacement. The precise locations of insertion and deletion mutations were confirmed by PCR and DNA sequence analysis. The impact of mutation on virulence was investigated with a mouse air sac model of inflammation. Experiments evaluated clearance of streptococci from the air sac within 4 h after infection. SCPA- streptococci were cleared more efficiently than wild-type bacteria. Localization of streptococci in lymph nodes and spleens of infected mice revealed a significant difference between mutant and wild-type streptococci. PMNLs and other granulocytes that infiltrated the air sac were quantitated by single-color flow cytometry. The total cellular infiltrate was greater and PMNLs dominated the granulocytic infiltrates of air sacs inoculated with SCPA- mutant bacteria. The data obtained are consistent with the possibility that SCPA- streptococci are initially cleared from the site of infection primarily by PMNLs. Moreover, mutant and wild-type streptococci followed different paths of dissemination. SCPA- bacteria were transported to lymph nodes, whereas wild-type streptococci avoided transport to the lymph nodes and rapidly spread to the spleen.

Kogerman P, Sy MS, Culp LA (1996) CD44 protein levels and its biological activity are regulated in Balb/c 3T3 fibroblasts by serum factors and by transformation with the ras but not with the sis oncogene. J Cell Physiol 169 :341-349


CD44s (standard isoform) levels and hyaluronan-binding activity were investigated in Balb/c 3T3 cells and their derivatives transformed with ras or sis oncogenes as a function of serum concentration in the medium. 3T3 cells contained low levels of CD44 and did not bind hyaluronan when grown in medium containing 0.5 or 10% serum. In 5% serum, however, the cells had much higher levels of CD44 and were able to bind hyaluronan. CD44 levels also increased in 3T3 cells restimulated with either 5 or 10% serum after prior maintenance in low serum. In cells restimulated with 5% serum, high levels of CD44 were sustained for at least 72 hr. In cells restimulated with 10% serum, however, the increase in CD44 levels reverted by 48 hr. Transformation of 3T3 cells with ras (but not with sis) oncogene rendered CD44 levels insensitive to serum modulation : ras-transformed cells contained high levels of CD44 and bound hyaluronan at all serum concentrations and at all time points tested. Sis-transformed cells behaved like 3T3 cells in these modulatory changes. Platelet-derived growth factor (PDGF), when supplementing 0.5% serum, mimicked the effects of serum on the levels and hyaluronan-binding capacity of CD44 in 3T3 cells and the CD44-upregulating activity of serum was neutralized by incubation with anti-PDGF antibodies. These data demonstrate that serum factors, specifically PDGF, mediate regulation of CD44 levels in BAlb/c 3T3 cells and that transformation of 3T3 cells by ras renders CD44 expression insensitive to the modulating effects of serum in vitro. These results correlate with the metastatic capacity of these cells in vivo.

Kristoffersen EK, Matre R (1996) Surface annexin II on placental membranes of the fetomaternal interface. Am J Reprod Immunol 36 :141-149


PROBLEM : The phospholipidbinding membrane protein annexin II has been demonstrated to possess FcR activity for IgG and has been localized to the outer part of the syncytiotrophoblast cell layer. The question has arisen whether annexin II is exposed on the surface of syncytiotrophoblast cells thus enabling it to take part in the transport of IgG across the maternal barrier. METHOD : Syncytiotrophoblast microvillous plasma membranes were analyzed by flow cytometry for annexin II as well as established surface molecules. Fresh, fixed placental tissue was preincubated with antibodies to annexin II or known trophoblast surface molecules, and analyzed by confocal laser scanning microscopy. RESULTS : Annexin II and its subunit p11 were expressed on the surface of the syncytiotrophoblast microvillous plasma membranes as were other established surface proteins (CD46, CD59, placental alkaline phosphatase), using both flow cytometry and confocal microscopy. Annexin was not detected on the surface of viable cultured trophoblast cells. CONCLUSION : Annexin II is exposed on the surface of syncytiotrophoblast cells as a heterotetramer together with its light chain p11. It is exposed to maternal blood and may be instrumental in IgG transport across the placental barrier by binding.

Laakel M, Bouchard M, Lagace J (1996) Measurement of mouse anti-phospholipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in an ELISA. J Immunol Methods 190 :267-273


Conventional solid-phase immunoassays measuring interactions between anti-phospholipid antibodies and phospholipids are generally characterized by problems of reproducibility and high levels of non-specific binding. Here we describe two immunoassays based on the use of phospholipids in the form of solid-phase microspheres to measure the presence of anti-phospholipid antibodies in sera. Following the production of antibodies in mice against liposomes containing lipid A, we show that flow cytofluorometric analysis provides a reproducible and sensitive way to detect anti-phospholipid antibodies. We also present a sensitive, rapid and reproducible enzyme-linked immunosorbent assay (ELISA) using Alcian blue pretreated microtitre plates and solid-phase microspheres as coating antigen. This ELISA permitted the detection of antibodies to 1/1000 dilution, while untreated plates gave negative results. Such modified ELISA procedures may be applicable to other types of molecule exhibiting solid-phase binding problems e.g. synthetic peptides (J. Immunol. Methods 175 (1994) 131-135).

Lam KM, Kabbur MB, Eiserich JP (1996) Newcastle disease virus-induced functional impairments and biochemical changes in chicken heterophils. Vet Immunol Immunopathol 53 :313-327


The GB strain of Newcastle disease virus (NDV) is used to infect chicken heterophils in vitro. Heterophils have a decreased ability to phagocytize bacteria 3 h after infection, and those that did engulfed fewer bacteria relative to non-infected heterophils. Infected heterophils have a decreased H2O2 production as shown by flow cytometry, but an increased nitric oxide production, suggesting that NDV can stimulate heterophils to produce and/or utilize nitrogen intermediates but not oxygen intermediates. DNA extracted from the infected heterophils shows a marked fragmentation, suggesting that NDV infection may cause heterophils to undergo apoptosis.

Lybarger L, Dempsey D, Franek KJ, Chervenak R (1996) Rapid generation and flow cytometric analysis of stable GFP-expressing cells. Cytometry 25 :211-220


Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably expression GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1lo CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

MacCormac LP, Grundy JE (1996) Human cytomegalovirus induces an Fc gamma receptor (Fc gammaR) in endothelial cells and fibroblasts that is distinct from the human cellular Fc gammaRs. J Infect Dis 174 :1151-1161


The expression of a trypsin-sensitive receptor for the Fc portion of IgG (Fc gammaR) was demonstrated by flow cytometry on the surface of human umbilical vein endothelial cells and fibroblasts infected with human cytomegalovirus (CMV). Double-labeling experiments showed strong expression of the CMV Fc gammaR in a perinuclear region of infected cells but not in bystander uninfected cells. The CMV Fc gammaR did not react with a panel of murine monoclonal antibodies directed against the known human IgG Fc receptors, Fc gammaRI, Fc gammaRII, and Fc gammaRIII. The cytoplasmic form but not the cell surface form of CMV Fc gammaR bound murine IgG3 moderately and murine IgG1 more weakly, while both forms bound rabbit IgG almost as strongly as human IgG. The function of CMV Fc gammaR is unclear, but it may allow CMV to evade host antibody responses. However, the binding of immune complexes to infected endothelium might also contribute to immunopathology.

Martinez I, Dornburg R (1996) Mutational analysis of the envelope protein of spleen necrosis virus. J Virol 70 :6036-6043


Spleen necrosis virus (SNV) is an amphotropic type C retrovirus originally isolated from a duck. The envelope protein is related to that of type D retroviruses, and SNV appears to use the same receptor as do simian retroviruses. However, little is known about envelope-receptor interactions of SNV. We constructed a series of envelope mutants to characterize the SU peptide of SNV. Point mutations were introduced throughout SU in regions that are conserved among all retroviruses belonging to the same receptor interference group. The biological and biochemical properties of these mutants were analyzed. All mutants were transported efficiently to the cell surface. Almost all mutations in the amino-terminal one-third caused a conformational change of the envelope and a significant drop in infectivity and abolished the ability to confer superinfection interference. Similar observations were made with only two of seven mutants with mutations in the middle of SU. Four mutations in this region had little or no effect on biological activity. One mutant envelope protein (Asp to Arg at position 192) was processed normally but showed little infectivity and had no ability to confer superinfection interference. A detailed mutational analysis suggested that this amino acid forms a hydrogen bond to its cellular receptor. Mutations within the carboxy-terminal part of SU had very little or no effect on biological function. Aberrantly processed envelope proteins were proteolytically cleaved at a new point upstream of and differing in sequence from the conserved retroviral SU/TM cleavage site. Surprisingly, these mutants still retained some infectivity (0.01 to 1% of that of the wild type). Our data indicate that the envelope of SNV behaves in a manner very different from that of the envelopes of other studied retroviruses.

Moeck GS, Tawa P, Xiang H, Ismail AA, Turnbull JL, Coulton JW (1996) Ligand-induced conformational change in the ferrichrome-iron receptor of Escherichia coli K-12. Mol Microbiol 22 :459-471


Ferrichrome-iron is actively transported across the outer membrane of Escherichia coli by the TonB-dependent receptor FhuA. To obtain FhuA in a form suitable for secondary-structure analyses, a hexahistidine tag was inserted into a surface-located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand-binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of beta-sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti-FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N-terminus and which is exposed to the periplasm. These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA. It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.

Morris JG, Jr., Sztein MB, Rice EW, Nataro JP, Losonsky GA, Panigrahi P, Tacket CO, Johnson JA (1996) Vibrio cholerae O1 can assume a chlorine-resistant rugose survival form that is virulent for humans. J Infect Dis 174 :1364-1368


Vibrio cholerae can shift to a "rugose" colonial morphology associated with expression of an amorphous exopolysaccharide that promotes cell aggregation. Flow cytometric studies indicated that up to 3% of particles in rugose cultures represented aggregates of >5 bacterial cells. Rugose variants of our test strains displayed resistance to killing by chlorine, with viable cells persisting for >30 min in 2 mg/L free chlorine ; strains also showed resistance to killing by complement-mediated serum bactericidal activity. Six volunteers fed 10(6) cfu of a rugose variant of V. cholerae O1 El Tor Inaba N16961 developed symptoms typical of cholera, with a mean diarrheal stool volume of 2.2 L (range, 1.4-4.3). Isolates recovered from the stool of infected volunteers retained the rugose phenotype. The data suggest that rugose strains cause human disease. The role of these strains in the epidemiology of cholera remains to be determined.

Nystrom T, Larsson C, Gustafsson L (1996) Bacterial defense against aging : role of the Escherichia coli ArcA regulator in gene expression, readjusted energy flux and survival during stasis. Embo J 15 :3219-3228


Using two-dimensional gel electrophoresis and N-terminal amino acid sequencing analysis, we demonstrate that a mutant of the global regulatory protein ArcA fails to decrease the synthesis of the TCA cycle enzymes malate dehydrogenase, isocitrate dehydrogenase, lipoamide dehydrogenase E3 and succinate dehydrogenase in response to stasis, while the increased production of the glycolysis enzymes phosphoglycerate mutase and pyruvate kinase is unaffected. Microcalorimetric and respiratory measurements show that the continued production of TCA cycle enzymes in the (delta)arcA mutant is manifested as an elevated rate of respiration and total metabolic activity during starvation. The (delta)arcA mutant is severely impaired in surviving prolonged periods of exogenous carbon starvation, a phenotype that can be alleviated by overproducing the superoxide dismutase SodA. In addition, flow cytometry demonstrates that starving (delta)arcA mutant cells, in contrast to wild-type cells, fail to perform reductive division, remain large and contain multiple chromosomal copies. We suggest that the ArcA-dependent reduced production of electron donors and the decreased level and activity of the aerobic respiratory apparatus during growth arrest is an integral part of a defense system aimed at avoiding the damaging effects of oxygen radicals and controlling the rate of utilization of endogenous reserves.

Ogawa T, Uchida H (1996) Differential induction of IL-1 beta and IL-6 production by the nontoxic lipid A from Porphyromonas gingivalis in comparison with synthetic Escherichia coli lipid A in human peripheral blood mononuclear cells. FEMS Immunol Med Microbiol 14 :1-13


Porphyromonas gingivalis 381 lipid A possesses 1-phospho beta(1-6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2’-positions, respectively. P. gingivalis lipid A indicated lower activities in inducing interleukin-1 beta (IL-1 beta) mRNA expression, pro-IL-1 beta protein synthesis and IL-1 beta production than those of synthetic Escherichia coli lipid A (compound 506) in human peripheral blood mononuclear cells (PBMC). The induction of IL-6 mRNA and IL-6 synthesis by P. gingivalis lipid A were comparable to those of compound 506. Herbimycin A, H-7 and H-8, inhibitors of tyrosine kinase, protein kinase C and cyclic nucleotide-dependent protein kinase, inhibited P. gingivalis lipid A- and compound 506-induced IL-1 beta and IL-6 synthesis. W-7, an inhibitor of calmodulin (CaM) kinase, inhibited only P. gingivalis lipid A-induced IL-1 beta production. The result suggests that the CaM kinase-dependent cascade is involved in the down-regulation of IL-1 beta production by P. gingivalis lipid A. P. gingivalis lipid A and compound 506 also functioned in the induction of tyrosine and serine/threonine phosphorylation of several proteins in PBMC. P. gingivalis lipid A inhibited specific binding of fluorescein-labelled E. coli LPS to the PBMC. The nontoxic lipid A of P. gingivalis, having a chemical structure different from toxic compound 506, appears to induce the up- and down-regulation of the differential cytokine-producing activities following the activation of various intracellular enzymes including the CaM kinase through the common receptor sites of LPS.

Ozanne V, Ortalo-Magne A, Vercellone A, Fournie JJ, Daffe M (1996) Cytometric detection of mycobacterial surface antigens : exposure of mannosyl epitopes and of the arabinan segment of arabinomannans. J Bacteriol 178 :7254-7259


The physical arrangement of cell envelope components leads to the exposure of selected structural motifs which in turn may influence host-parasite interactions. To gain insight into the exposed epitopes, the present study describes a flow cytometric method designed to probe defined molecules on dispersed mycobacteria. The hydrophobic fluorophore N-hexadecanoyl aminofluorescein inserted in the mycobacterial cell envelope permitted focusing of fluorescence-activated cell sorter analysis on cells that were further labeled with defined monoclonal antibodies and fluorochrome-coupled streptavidin. The use of antibodies directed against the lipooligosaccharide of Mycobacterium tuberculosis demonstrated the specific detection of the antigen on the cell surface of a Canetti-like strain of M. tuberculosis, and not on those of mycobacterial strains that were devoid of the glycolipid. Thus, the method was applied to investigate the relative amounts of surface-exposed mannosylated compounds and D-arabinan-containing substances of different strains of the tubercle bacillus and a strain of the rapidly growing nonpathogenic species Mycobacterium smegmatis. Both M. tuberculosis and M. smegmatis are endowed with mannosyl and arabinan epitopes on their surfaces, although there are many differences in terms of exposed mannosyl epitopes between the various strains of the tubercle bacillus examined. These differences are correlated with the amounts of terminal mannosyl residues that cap the surface-exposed arabinomannans (A. Ortalo-Magne, A. B. Andersen, and M. Daffe, Microbiology 142:927-935, 1996) but not with the degrees of virulence of the strains. This novel approach could provide new insights into the distribution of defined surface-exposed antigens and thereby into the architecture of the cell envelopes.

Pierard GE, Arrese JE, De Doncker P, Pierard-Franchimont C (1996) Present and potential diagnostic techniques in onychomycosis. J Am Acad Dermatol 34 :273-277


The problem of onychomycosis has been frequently addressed during recent years. To make the diagnosis of onychomycosis dermatologists have relied on clinical presentation, culture, and microscopy. These approaches are hampered by false-negative and false-positive results that have confused treatment outcomes. Two new diagnostic techniques, immunohistochemistry and flow cytometry, provide an effective means of identifying different dermatophytes, yeasts, and nondermatophytic molds. Immunohistochemistry employs antibodies to certain fungi to enable positive identification in situ, whereas flow cytometry differentiates fungi on the basis of molecular differences. These techniques provide new evidence that nondermatophytic molds and yeasts can actively invade nail tissue and that mixed infections occur. These findings could have important implications for the treatment of onychomycosis.

Podbielski A, Schnitzler N, Beyhs P, Boyle MD (1996) M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes. Mol Microbiol 19 :429-441


The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsonophagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.

Popov S, Hubbard JG, Ward ES (1996) A novel and efficient route for the isolation of antibodies that recognise T cell receptor V alpha(s). Mol Immunol 33 :493-502


Studies of the T cell repertoire have been hindered by the lack of antibodies that recognise V region families, particularly for V alpha regions. In this report, single chain Fv (scFv) fragments have been isolated that recognise both recombinant V alpha(s) and native V alpha(s) on the surface of T cells. Mice have been immunised with purified soluble T cell receptors (TCRs) and antibody heavy and light chain variable domain (VH and VL, respectively) genes isolated from splenocytes using the polymerase chain reaction (PCR). The VH and VL genes have been assembled as scFv gene libraries and a bacteriophage display system used to isolate scFvs that recognise a soluble V alpha. Five scFvs have been purified and characterised in detail using enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Three of these five scFvs recognise native V alpha(s) on the surface of T cell hybridomas. This method therefore offers a rapid route to the generation of scFvs that recognise native TCRs and can readily be extended to the production of anti-human TCR antibodies for use in therapy and diagnosis.

Porter J, Deere D, Pickup R, Edwards C (1996) Fluorescent probes and flow cytometry : new insights into environmental bacteriology. Cytometry 23 :91-96


Recent trends in flow cytometry have established new techniques in bacteriology. Advances in fluorescent dye technology complement these improvements, offering probes for a variety of cellular functions. Bacterial ecology requires the application of new techniques to help answer questions unanswerable by traditional methods alone. Here we review some aspects of how coupling the two technologies has enabled researchers to directly study individual bacterial cells, and revealed the complexity and heterogeneity present in both laboratory cultures and in environmental samples. Results are discussed with respect to viability analysis, stress induced changes, specific cell detection and cell sorting.

Qi YM, Peng SW, Hengst K, Evander M, Park DS, Zhou J, Frazer IH (1996) Epithelial cells display separate receptors for papillomavirus VLPs and for soluble L1 capsid protein. Virology 216 :35-45


We examined the distribution of putative receptors for papillomavirus (PV) capsid proteins on various cell types, using either Hexahis HPV6b L1 fusion protein or synthetic HPV6b virus-like particles (VLPs). Specific, saturable binding of VLPs to CV-1 cells was demonstrated using 35S-labeled VLPs, with an average receptor number of 1 x 10(4)/cell and a binding affinity constant (Ka) of 4 x 10(7) M. VLP binding was quantitated by flow cytometry using a monoclonal antibody to the L1 capsid protein. Intense staining of epithelial and mesenchymal cells was observed. Some immature bone marrow-derived cells bound VLPs weakly, while the majority of B lymphoma cells demonstrated no binding. Binding to 12 of 16 VLP receptor positive cell lines was abolished by trypsin pretreatment of cells. Removal of cellular sialic acid or O-linked oligosaccharides separately did not affect VLP binding, which was enhanced about 25% when cells were pretreated with both neuraminidase and O-glycosidase. Culture of cells with sufficient tunicamycin to inhibit Concanavalin A binding did not diminish the binding of VLPs. Denatured L1 protein, either from VLPs or expressed from Escherichia coli as a Hexahis fusion protein, bound to a trypsin-resistant structure on a range of cell types and did not block the binding of VLPs to cells. Dual-fluorescence assay with a Burkitt lymphoma line BL72 demonstrated that Hexahis L1 protein and VLPs bind to separate cell surface molecules on BL72 cells. We conclude that the first binding of PV virus to cells is via a widely distributed membrane protein receptor(s) and that subsequent processing of particles may involve other non-trypsin-sensitive structure(s) also displayed on the cell membrane.

Rollwagen FM, Li YY, Pacheco ND, Nielsen TB (1996) Systemic bacteraemia following haemorrhagic shock in mice : alleviation with oral Interleukin 6. Cytokine 8 :121-129


A murine model of haemorrhagic shock was used to investigate bacterial translocation from the gut and subsequent systemic immunoreduction. Anaesthetized mice were bled from the femoral artery, and held at a mean arterial blood pressure of 35 mm Hg for one hour then resuscitated with shed blood and two-fold volume lactated Ringer’s solution. Upon awakening, they were given cytokines or control media orally. Bacteriological cultures of livers, spleens and mesenteric lymph nodes from haemorrhaged mice given cytokine had significantly fewer bacteria/gm of tissue than those given media. Recombinant IL-6 mimicked the effects seen with crude cytokines. Reduction of proliferation among spleen cells from haemorrhaged mice was observed and could be partially returned to normal by cytokine feeding. Mixing experiments in which cells from haemorrhaged mice were added to those of normal mice in an MLR showed no suppressor activity. Flow cytometry analysis revealed a reduction in CD 3+ cells at 16 hours post-haemorrhage in mice fed control media or cytokines, suggesting that reduced proliferative capacity may be due to loss of function rather than active suppression. Histological examination of the intestines of haemorrhaged mice fed cytokines or media revealed restoration of intestinal mucosal integrity by cytokine administration. These results suggest that oral administration of IL-6 may be an important treatment for the prevention of systemic sepsis following haemorrhage.

Roumen FJ, Boon ME, van Velzen D, Dieben TO, Coelingh Bennink HJ (1996) The cervico-vaginal epithelium during 20 cycles’ use of a combined contraceptive vaginal ring. Hum Reprod 11 :2443-2448


The aim of the study was to evaluate the influence of a combined contraceptive vaginal ring (CCVR) made of Silastic on the cervico-vaginal epithelium during 20 cycles of use. A total of 76 volunteers used the CCVR releasing 0.120 mg etonogestrel and 0.015 mg ethinyloestradiol daily. Cytological samples were taken of the vaginal epithelium, the ectocervix and the endocervix before the start, at 4 and 12 months, and at the end of the study. Cytology, hormonal profiles, human papilloma virus (HPV) status, DNA-flow cytometry, bacterial flora, and morphometry was performed on these samples. Colposcopy and histopathology of biopsy specimens were performed at the end. No cytological changes of the squamous epithelium or the columnar epithelium were found. HPV was detected in three samples of three different women. At least two of them reverted to HPV negative during the rest of the study period. Aneuploidy was diagnosed in 11 women before the study. Seven of them changed to diploid during the study. No changes from diploid to aneuploid were seen. Aneuploidy was not seen in any of the HPV positive samples. Although bacterial flora showed considerable variation during the study, no significant influence of the CCVR could be established. Morphometrical analysis showed an increasing nucleus:cytoplasm ratio of the squamous cells during the study. Mild dysplasia was detected in one woman at the end of the study. It was concluded that no unfavourable cytological or bacteriological changes of the cervico-vaginal epithelium were demonstrated during 20 cycles of CCVR use. The vaginal epithelium became more progestogenic during the study.

Russell H, O’Toole DT, Bardsley K, Davis WC, Ellis JA (1996) Comparative effects of bluetongue virus infection of ovine and bovine endothelial cells. Vet Pathol 33 :319-331


Bluetongue virus (BTV) infection results in disparate clinical syndromes among ruminant species. An in vitro model system of BTV/target cell interaction was developed using umbilical vein endothelial cells (EC)from fetal lambs and calves. These cells had microscopic, ultrastructural, and immunocytochemical features typical of EC. BTV infection in these cells was examined using virus binding assays, plaque assays, a whole-cell enzyme-linked immunosorbent assay, flow cytometry, electron microscopy, and a bioassay for interferon activity. EC from both species supported cytopathic BTV infections. Ovine EC bound more BTV initially and produced more virus over time, whereas bovine EC underwent more rapid lysis subsequent to infection. An ultrastructural comparison of BTV-infected ovine and bovine EC, grown as differentiated capillary-like cords on a laminin-rich matrix or as monolayers, revealed no significant interspecies differences in viral morphogenesis between 1 minute and 24 hours after infection. The intracellular distribution of BTV nonstructural protein 1, which localized to virus inclusion bodies and tubules, was identical for ovine and bovine endothelial cells. Ovine and bovine EC produced a soluble mediator of interferon activity in response to BTV infection ; however, ovine EC produced higher levels of interferon activity at lower levels of infection. These findings indicate differences in BTV-EC interaction that may contribute to the pathogenesis of the severe inflammatory disease that is characteristic of clinical bluetongue disease in sheep.

Schmitt MJ, Klavehn P, Wang J, Schonig I, Tipper DJ (1996) Cell cycle studies on the mode of action of yeast K28 killer toxin. Microbiology 142 ( Pt 9) :2655-2662


The virally encoded K28 killer toxin of Saccharomyces cerevisiae kills sensitive cells by a receptor-mediated process. DNA synthesis is rapidly inhibited, cell viability is lost more slowly and cells eventually arrest, apparently in the S phase of the cell cycle with a medium-sized bud, a single nucleus in the mother cell and a pre-replicated (1n) DNA content. Cytoplasmic microtubules appear normal, and no spindle is detectable. Arrest of a sensitive haploid yeast strain by alpha-factor at START gave complete protection for at least 4 h against a toxin concentration that killed non-arrested cells at the rate of one log each 2.5 h. Cells released from alpha-factor arrest were killed by toxin at a similar rate ; arrest occurred with medium-sized buds within the same cell cycle. Cells arrested by hydroxyurea, with unreplicated DNA, or by the spindle poison methylbenzimidazol-2yl-carbamate, with unseparated chromosomes, both arrest at the checkpoint at the G2/M boundary ; these arrested cells were not protected against toxin, losing about one log of viability every 4 h. Following release from the cell cycle block, a majority of these toxin-exposed cells progressed through the cell cycle and arrested in the following S-phase, again with medium-sized buds. Killing by K28 toxin apparently requires entry into the nuclear division and bud cycles, but can result from inhibition of either early or late events in these cycles. Morphogenesis in moribund cells is uniformly blocked in early S-phase with an immature bud. Toxin action causes either independent blockage of both DNA synthesis and the budding cycle, or inhibits some unknown step required for both events.

Spiller OB, Morgan BP, Tufaro F, Devine DV (1996) Altered expression of host-encoded complement regulators on human cytomegalovirus-infected cells. Eur J Immunol 26 :1532-1538


Human cytomegalovirus (HCMV)-infected cells persist in the presence of anti-HCMV antibody, suggesting that HCMV has evolved mechanisms to evade host immune defenses. Insofar as no virus-encoded complement inhibitors have been identified for HCMV, we hypothesized that HCMV infection may alter the expression of host-encoded cell surface complement inhibitors. Herein, we report that cell surface expression of two complement regulator proteins, CD55 and CD46, which are members of the regulators of complement activation (RCA) gene cluster, increased up to eightfold following infection of fibroblasts or glioblastoma cells with HCMV, but not after infection with HSV-1 or adenovirus. However, the cell surface expression of a third complement regulator, CD59, which is not a member of the RCA gene cluster, was not altered during HCMV infection. Functional studies using purified complement components demonstrated that up-regulation of CD55 suppressed the activity of cell-associated C3 convertases on HCMV-infected cells. Furthermore, increased CD55 expression protected infected cells from complement-mediated lysis, an effect which directly correlated with the length of HCMV infection. Increased expression of host-encoded complement regulator proteins may provide protection of HCMV-infected cells from the host immune response in vivo, through increasing the resistance of infected cells to complement-mediated lysis and decreasing the deposition of C3-derived products on the cell surface.

Suga S, Tsurudome M, Ohgimoto S, Tabata N, Watanabe N, Nishio M, Kawano M, Komada H, Sakurai M, Ito Y (1996) Intracellular localization of antigens recognized by anti-vimentin monoclonal antibodies (mAbs) : cross-reactivities of anti-vimentin mAbs with other cellular components. Eur J Cell Biol 70 :84-91


Monoclonal antibodies were raised against immunoaffinity-purified fusion regulatory protein (FRP)-1 complex from membrane fraction of HeLa cells. Immunoblotting and immunoprecipitation studies showed all ten antibodies reacted with a 55 kDa band of cell lysate and purified vimentin. Interestingly, one of the antibodies (mAb57) cross-reacted with purified tropomyosin and myosin. Further analyses using vimentin chemically cleaved by 2-nitro-5-thio-cyanobenzoic acid, and lambda gt 11 cDNA which encoded a partial sequence of vimentin indicated that six mAbs recognized epitopes between amino acids 1 and 313 and the other four mAbs recognized epitopes in the area between residues 314 and 326. Indirect immunofluorescence microscopy using 3% formalin-fixed, 0.1% Triton X-100 treated HeLa cells revealed that seven antibodies stained various intracellular components other than vimentin, while three antibodies stained vimentin filaments alone. Furthermore, flow cytometric analysis showed one of the antibodies (mAb25) clearly stained the surface of unfixed HeLa cells. All immunofluorescent findings were the same when HeLa, baby hamster kidney (BHK) and murine L229 cells were examined. These results indicate that we could obtain unique anti-vimentin mAbs which show cross-reactivities with previously undescribed cell surface and intracellular molecules including tropomyosin and myosin. Taken together, there are two possibilities that explain our findings : (1) The unknown molecules may have structural similarity to vimentin. (2) Our anti-vimentin mAbs can react specifically with structurally distinct epitopes present on both unknown molecules and vimentin. In either case, our cross-reactive mAbs, which recognized undescribed epitopes on vimentin, maybe provide useful tools for studying intermediate filaments and related cellular components.

Sullam PM, Bayer AS, Foss WM, Cheung AL (1996) Diminished platelet binding in vitro by Staphylococcus aureus is associated with reduced virulence in a rabbit model of infective endocarditis. Infect Immun 64 :4915-4921


The direct binding of platelets by bacteria is a postulated central mechanism in the pathogenesis of endocarditis. To address the role of binding more definitively, we employed Tn551 insertional mutagenesis of Staphylococcus aureus parental strain ISP479 to generate an isogenic variant (strain PS12) that bound platelets minimally. As compared with the binding of ISP479, the binding of PS12 to platelet monolayers was reduced by 67.2%. Similarly, the binding of PS12 to platelets in suspension was reduced by 71.3%, as measured by flow cytometry. The low-binding phenotype was transducible into both ISP479 and S. aureus Newman. Southern blotting indicated that a single copy of Tn551 was inserted within the chromosomes of PS12 and the transductants. When tested in a rabbit model, animals inoculated with PS12 were significantly less likely to develop endocarditis and had lower densities of organisms (CFU per gram) within vegetations and a decreased incidence of renal abscess formation, as compared with animals inoculated with the parental strain. The diminished virulence of PS12 was not attributable to a reduction in the initial attachment of organisms to the damaged endocardium, since 30 min after inoculation, PS12-infected animals had microbial densities on the valve surface comparable to those seen with the parental strain. These results indicate that the direct binding of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Staphylococcus-platelet binding appears to be critical for pathogenetic events occurring after the initial colonization of the valve surface, such as vegetation formation and septic embolization.

Superti F, Ammendolia MG, Tinari A, Bucci B, Giammarioli AM, Rainaldi G, Rivabene R, Donelli G (1996) Induction of apoptosis in HT-29 cells infected with SA-11 rotavirus. J Med Virol 50 :325-334


Rotavirus infection is associated both in vivo and in vitro with a series of subcellular pathological alterations leading to cell lysis. It has been suggested that these modifications can play a key role in the pathogenesis of rotavirus-associated diarrheal disease. We describe the effects of SA-11 rotavirus infection in HT-29 cells, a human enterocyte-like cell line. Cytological analyses suggested that the viral-induced cytopathic process, including chromatin clumping, can be referred to as apoptosis, the cell death pathway alternative to necrosis. A time course of the process was performed to investigate whether rotavirus-associated cell death showed specific injury signs. HT-29-infected cells were analyzed by scanning and transmission electron microscopy and features of apoptosis such as blebbing of the plasma membrane, peripheral condensation of chromatin, and fragmentation of the nucleus were observed. Specific changes occurring in cell-substrate adhesion and in some organelles relevant for viral maturation, i.e., rough endoplasmic reticulum, were detected. These findings indicate a role for apoptosis in the rotavirus infection process and its related cytopathology, and also suggested that specific histological alterations such as derangement of enterocytes are associated with the pathogenesis of rotavirus-induced diarrheal disease and could be a direct consequence of viral-triggered apoptosis.

Tsao YP, Li SF, Kuo SW, Liu JC, Chen SL (1996) Reversal of the temperature-shift-induced growth restriction of a temperature-sensitive simian virus 40 T-antigen-transformed human fibroblast cell line by treatment with retinoic acid. Biochem J 317 ( Pt 3) :707-711


We previously established a human fibroblast cell line, HFL 6-2, that contains a temperature sensitive simian virus 40 (SV40) T antigen, permitting cell growth at 35 degrees C but restricting growth at 39 degrees C. p21 (Waf1/Cip1) was significantly induced by temperature shifts in HFL 6-2 cells. Here we show that all-trans-retinoic acid (RA) treatment prevented the growth restriction of HFL 6-2 cells at 39 degrees C. In the presence of RA, HFL 6-2 cells proliferated into sizeable colonies even at 39 degrees C. [3H]Thymidine incorporation and flow cytometry analysis revealed that cells exposed to RA maintained DNA synthesis at 39 degrees C. Prevention of growth restriction by RA was correlated with a lack of induction of p21 at the transcription level. These observations suggest that RA may prevent the senescence process by repressing p21 gene expression, and perturb the growth regulation of somatic cells.

van der Waaij LA, Limburg PC, Mesander G, van der Waaij D (1996) In vivo IgA coating of anaerobic bacteria in human faeces. Gut 38 :348-354


The bacterial flora in the human colon, although extremely diverse, has a relatively stable composition and non-infectious anaerobic bacteria are dominant. The flora forms a pool of numerous different antigens separated from mucosal immunocompetent cells by just a single layer of epithelial cells. Despite this thin barrier, however, the colonic mucosa is physiologically only mildly inflamed. This study looked at the mucosal humoral immune response against faecal anaerobes. By flow cytometric analysis the in vivo immunoglobulin coating of anaerobic bacteria in faecal samples of 22 healthy human volunteers was determined. In a previous study flow cytometric analysis of faecal bacteria has been found to be a very sensitive method to detect immunoglobulins on faecal bacteria. This technique showed that in vivo many bacteria are coated with IgA (24-74%) and less with IgG and IgM. The presence of many bacteria coated with IgA implies that IgA coating does not result in permanent removal of the species from the colon. The absence of immunoglobulin coating suggests that there is immunological unresponsiveness for anaerobic bacterial antigens. It is concluded that both immunological unresponsiveness and preferential coating with IgA are responsible for the relative absence of colonic mucosal inflammation.

Wang Y, Casadevall A (1996) Susceptibility of melanized and nonmelanized Cryptococcus neoformans to the melanin-binding compounds trifluoperazine and chloroquine. Antimicrob Agents Chemother 40 :541-545


Cryptococcus neoformans is an opportunistic fungal pathogen which becomes heavily melanized in the presence of phenolic substrates such as L-dopa. Various drugs are known to bind to melanin with high affinity, including the antipsychotic agent trifluoperazine and the antimalarial agent chloroquine. We hypothesized that drugs which bind melanin may have different toxicities for melanized and nonmelanized C. neoformans cells. The effects of trifluoperazine and chloroquine or C. neoformans were determined by measuring cell viability after exposure to these drugs. Cell viability was measured by CFU determination and flow cytometry with propidium iodide staining. Melanized cells were more susceptible than nonmelanized cells to the fungicidal effects of trifluoperazine. Chloroquine had no fungicidal effect on either melanized or nonmelanized C. neoformans under the conditions studied. Flow cytometry of trifluoperazine-treated C. neoformans cells stained with the mitochondrial stain dihydrorhodamine 123 revealed fluorescence changes consistent with mitochondrial damage. Our results indicate that melanized and nonmelanized C. neoformans cells can differ in susceptibility to certain drugs and suggest that strategies which target melanin may be productive for antifungal-drug discovery.

Zeze A, Hosny M, Gianinazzi-Pearson V, Dulieu H (1996) Characterization of a highly repeated DNA sequence (SC1) from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in planta. Appl Environ Microbiol 62 :2443-2448


A highly repeated DNA sequence from the genome of an arbuscular mycorrhizal fungus has been isolated and characterized. This 1,202-bp sequence (SC1) represents about 0.24% of the Scutellospora castanea genome, estimated to be 1 pg by flow cytometry. The sequence was shown to be a Scutellospora-specific probe in Southern blots and dot blot hybridizations. After complete sequencing of SC1, PCR primers were generated and used to amplify a 907-bp fragment from spores of S. castanea or from colonized Allium porrum roots. No amplification products were obtained with DNA from either spores or mycorrhizal root of other species of arbuscular mycorrhizal fungi. These primers were sufficiently specific for unequivocal detection of S. castanea in planta.