vendredi 24 avril 2009
par   G. Grégori

Bernander R, Nordstrom K (1990) Chromosome replication does not trigger cell division in E. coli. Cell 60 :365-374


An essential part of the chromosome replication origin of E. coli K-12 and B/r was replaced by the plasmid pOU71. The average initiation mass of replication for pOU71 decreases with increasing temperature. The constructed strains were grown exponentially at different temperatures, and cell sizes and DNA content were measured by flow cytometry. The average DNA content increased with increasing temperature, but the cell size distribution was largely unaffected. Furthermore, cells in which DNA replication had not yet initiated (cells in the B period) became less abundant with increasing temperature. The increased DNA content could not be explained by an increase in the length of the C period. It is concluded that chromosome replication does not trigger cell division in E. coli, but that the chromosome replication and cell division cycles of E. coli run in parallel independently of each other.

Bly JE, Miller NW, Clem LW (1990) A monoclonal antibody specific for neutrophils in normal and stressed channel catfish. Dev Comp Immunol 14 :211-221


A murine monoclonal antibody, designated mAb 13C5, was found to react specifically with channel catfish neutrophils based upon its ability to identify a subpopulation of leukocytes that are phagocytic and stain positive with both Sudan Black B and nitro-blue tetrazolium. Cytofluorographic analysis with mAb 13C5 was used to assess trafficking of neutrophils in various tissues of catfish subjected to transport stress. Since no prestress neutrophil reservoir was apparent, it seems likely that stress-induced neutrophilia in catfish, as in endotherms, may result from demargination of a pool of capillary-bound neutrophils. MAb 13C5 was also used to successfully "pan" for neutrophil-enriched and depleted populations of peripheral blood leukocytes from transport stressed channel catfish. "Panning" indicated that the continued physical presence of elevated numbers of neutrophils is not responsible for the suppression of T and B cell in vitro proliferation responses to mitogens observed in stressed fish.

Bolin SR, Ridpath JF (1990) Frequency of association of noncytopathic bovine viral diarrhea virus with bovine neutrophils and mononuclear leukocytes before and after treatment with trypsin. Am J Vet Res 51 :1847-1851


Enriched populations of neutrophils and mononuclear leukocytes from 9 cattle persistently infected with noncytopathic bovine viral diarrhea virus were analyzed for frequency of association with virus, using flow cytometric procedures. Trypsinization of neutrophils decreased the frequency of viral association from 0.82% to 0.49%. Similar treatment of mononuclear leukocytes decreased the frequency of viral association from 5.53% to 4.81%. Results of immunocytochemical procedures to locate viral antigen were inconclusive for neutrophils, but viral antigen was found in the cytoplasm of mononuclear leukocytes. A distinct and highly pure population of eosinophils was identified during flow cytometric analysis of neutrophil populations from 2 of 9 cattle.

Boye E, Lobner-Olesen A (1990) Flow cytometry : illuminating microbiology. New Biol 2 :119-125


By means of flow cytometry, a technique whereby a hydrodynamically directed stream of cells is passed through a focus of exciting light, one can measure cell size and the macromolecular content of individual bacteria. The sensitivity and versatility of the flow cytometer make it a powerful tool in studies of the bacterial cell cycle, in identifying and characterizing bacterial infections, and in selecting bacteria of desired qualities. We review some of these applications of flow cytometry and conclude that this method holds great promise in many other areas of microbiology.

Boyer Kollas B, Hartle HT, Wigdahl B (1990) Analysis of major histocompatibility complex gene products in tissues isolated from the developing human nervous system. Fetal Diagn Ther 5 :124-137


We have examined the expression of the major histocompatibility complex (MHC) class I and II gene products in the developing human fetal peripheral nervous system. As determined by RNA blot hybridization analysis, MHC class I RNA was readily detectable in extracts prepared from dorsal root ganglia (DRG) obtained from aborted human fetal material. However, utilizing similar methodology, it was not possible to detect MHC class II RNA. In conjunction with these studies, expression of MHC class I and II proteins in primary human fetal DRG tissue was examined by fluorescence-activated flow cytometry and protein immunoblotting. Consistent with the detection of MHC-specific RNA, the accumulation of MHC class I-specific protein was readily detectable in human fetal DRG neural cell populations with little, if any, accumulation of MHC class II-specific protein evident. These studies suggest that MHC gene products may be expressed early in the development of the human nervous system resulting in the generation of specific immunocompetent neural cell populations.

Braverman PK, Biro FM, Brunner RL, Gilchrist MJ, Rauh JL (1990) Screening asymptomatic adolescent males for chlamydia. J Adolesc Health Care 11 :141-144


Ninety-seven asymptomatic 16-21-year-old sexually active adolescent males were evaluated for gonorrhea and chlamydia by culture, chlamydia enzyme immunoassay, and an analysis of a random urine sample for pyuria using centrifuged urine and urine cytometer. The incidence of gonorrhea was 5.3% and chlamydia by culture 12.3%. Immunoassay was superior in sensitivity and specificity (75% and 99%, respectively) to centrifuged urine (sensitivity 58%, specificity 92%) or urine cytometer (58% and 91%) in identifying asymptomatic chlamydia urethritis. Chlamydia enzyme immunoassay is an acceptable, more rapid, and less expensive alternative to culture. The absence of pyuria in asymptomatic males cannot be assumed to indicate the absence of a sexually transmitted disease.

Brickman MJ, Balber AE (1990) Trypanosoma brucei rhodesiense bloodstream forms : surface ricin-binding glycoproteins are localized exclusively in the flagellar pocket and the flagellar adhesion zone. J Protozool 37 :219-224


Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.

Evans DL, Harris DT, Staton DL, Jaso-Friedmann L (1990) Pathways of signal transduction in teleost nonspecific cytotoxic cells. Dev Comp Immunol 14 :295-304


In the present study evidence is presented that both a putative "receptor" binding monoclonal antibody (mAb) and the calcium ionophore A23187, either singly or together, increase receptor expression and lysis of IM-9 target cells by catfish NCC. NCC activity against IM-9 target cells was increased 55% after 1 h mAb treatment. Receptor expression determined by flow cytometry increased 95% following 18-h treatment. A23187 treatment of NCC produced greater than 200% increases in receptor expression. Combined treatments of NCC with 10(-4) M A23187 and 10(-7) M PMA however augmented receptor expression only 22% above that produced by A23187 alone. MAb and A23187 comodulated cytotoxicity by a 65% increase over ionophore treatment alone. MAb and 10(-10) M PMA comodulation produced only 10.6% increases in cytotoxicity compared to mAb. These data demonstrate that the moiety on NCC recognized by 5C6 may provide an activation signal for increased cytotoxicity and receptor expression. Calcium ionophore, either singly or together with mAb, provided an even stronger activation signal for increased lysis and receptor expression.

Fitzpatrick DR, Zamb TJ, Babiuk LA (1990) Expression of bovine herpesvirus type 1 glycoprotein gI in transfected bovine cells induces spontaneous cell fusion. J Gen Virol 71 ( Pt 5) :1215-1219


Bovine MDBK cells were transfected with Rous sarcoma virus-based vectors for constitutive expression of the bovine herpesvirus type 1 (BHV-1) glycoprotein, gI. Cell lines stably expressing recombinant gI were cloned and characterized. Recombinant gI was localized intracellularly, predominantly in a perinuclear region, and on the cell surface. Cells expressing gI exhibited spontaneous polykaryon formation, thus confirming the fusogenic activity described previously in gI-expressing transfected murine LMTK- cells. The recombinant form of gI synthesized in transfected MDBK cells was similar in Mr to the form expressed in BHV-1-infected MDBK cells, unlike the recombinant form of gI expressed by LMTK- cells which is deficient in N-linked glycosylation. It was concluded that cell fusion associated with the expression of BHV-1 gI in transfected mammalian cells is a reproducible phenomenon in a number of cell types and is not due to species-specific factors or expression of abnormally glycosylated gI. Cell fusion is a useful in vitro marker for gI function and may contribute to the spread of BHV-1 infections in vivo.

Flores BM, Garcia CA, Stamm WE, Torian BE (1990) Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry. J Clin Microbiol 28 :1999-2005


Monoclonal antibodies to Naegleria fowleri and Acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured N. fowleri and Acanthamoeba spp. Four monoclonal antibodies to N. fowleri were specific for N. fowleri and had no reactivity to A. polyphaga. Similarly, four monoclonal antibodies to A. polyphaga did not react with N. fowleri. Two of the four monoclonal antibodies to A. polyphaga did not react with other Acanthamoeba spp. tested, while two of the antibodies demonstrated a high degree of cross-reactivity with a putative Acanthamoeba castellanii strain by immunofluorescence microscopy ; this was confirmed by fluorescence flow cytometry for one of the antibodies. These monoclonal antibodies were used to identify Acanthamoeba trophozoites in infected brain sections of a patient who died of suspected Acanthamoeba-caused granulomatous amoebic encephalitis, demonstrating potential utility in the direct identification of N. fowleri and Acanthamoeba spp. in clinical specimens.

Foerster RJ, Wolf G (1990) Phagocytosis of opsonized fluorescent microspheres by equine polymorphonuclear leukocytes. Zentralbl Veterinarmed B 37 :481-490


Equine blood polymorphonuclear leukocytes (PMN) were isolated by buffy coat and hypotonic lysis of residual erythrocytes. A highly reproducible method is described for measuring the uptake of opsonized latex microspheres by equine PMN using flowcytometry. The use of cytochalasin D allowed for differentiation of ingested from attached particles. The kinetics of phagocytosis in vitro is shown for different experimental conditions. We developed an assay for evaluation of phagocytic capacity of PMN which allows the assessment of drugs for their influence on phagocytosis in vivo as well as in vitro.

Goldberg EH, Goble R, Jackman SH (1990) Monoclonal antibodies defining the skin-selective alloantigens, Skn. Immunogenetics 31 :393-395


Holtfreter HB, Cohen N (1990) Fixation-associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs. Cytometry 11 :676-685


We have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.

Karupiah G, Blanden RV (1990) Anti-asialo-GM1 inhibits vaccinia virus infection of murine ovaries : asialo-GM1 as an additional virus receptor ? Immunol Cell Biol 68 ( Pt 5) :343-346


The neurovirulent strain of vaccinia virus, VV-WR, or recombinants derived from VV-WR, cause highly productive infection of murine ovaries, but infection could be partially inhibited in vivo using an antiserum to asialo-GM1 (as-GM1). In vitro analysis by flow cytometry revealed that murine ovarian cells expressed a cell surface antigen identical to or cross-reactive with as-GM1. The capacity of VV-WR to infect murine ovaries appears to depend in part upon as-GM1 expression on ovarian cells.

Klebba PE, Benson SA, Bala S, Abdullah T, Reid J, Singh SP, Nikaido H (1990) Determinants of OmpF porin antigenicity and structure. J Biol Chem 265 :6800-6810


Sixty-six murine hybridomas raised to Escherichia coli B/r porin were used to identify and differentiate the epitopes of this outer membrane protein. Anti-porin monoclonal antibodies (mAb) were raised against outer membrane fragments, purified native trimeric porin (trimer), and purified sodium dodecyl sulfate-denatured monomeric porin (monomer). Immunochemical and flow cytometric methods identified five distinct cell surface-exposed determinants on OmpF. The peptide composition of porin epitopes was determined by analysis of mAb reactivity with cyanogen bromide-generated peptide fragments. Four of 43 anti-monomer mAb reacted with surface exposed sites on OmpF, defining epitopes that consist of residues within CNBr peptides d2, d3, and B. The anti-porin mAb panel was also used to evaluate changes in porin antigenic structure in strains with short ompF deletions. Flow cytometric experiments indicated that despite changes in porin permeability, little if any alteration of surface epitopes occurred in these strains. Western immunoblot analysis of the mutant porins showed loss of reactivity with numerous mAb, which was caused by changes in three spatially distinct epitopes at residues 108-111, 118-123, and 124-129. Our findings indicate that in these ompF mutants the residues responsible for altering porin permeability are not exposed on the cell surface, but are buried within the tertiary structure of the protein. One of these regions, which is apparently involved in the determination of channel permeability characteristics, is conserved among 15 of 16 different porin molecules which were screened with the anti-OmpF mAb panel.

Laffin J, Lehman JM (1990) Detection of intracellular virus and viral products. Methods Cell Biol 33 :271-284


Langer JA, Rashidbaigi A, Lai LW, Patterson D, Jones C (1990) Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein. Somat Cell Mol Genet 16 :231-240


The cellular responses to alpha and beta interferons (IFN-alpha and -beta) are mediated through the IFN-alpha/beta (type I) receptor, while the response to IFN-gamma is mediated through the IFN-gamma (type II) receptor. The receptors for IFN-alpha/beta and IFN-gamma are encoded by genes on human chromosomes 21 and 6q, respectively. The presence of chromosome 21q confers both ligand binding and responsiveness to human IFN-alpha/beta, whereas chromosome 6q confers binding of Hu-IFN-gamma, but not cellular responsiveness on somatic cell hybrids. Chromosome 6q (i.e., the Hu-IFN-gamma receptor gene) and chromosome 21q are both necessary for the cellular response of somatic cell hybrids (from fibroblasts) to Hu-IFN-gamma. It is conceivable that the factor mediating activity through the IFN-gamma receptor is, in fact, the IFN-alpha receptor, or that the two genes are distinct but part of an "interferon response" region. Here we more precisely localize on human chromosome 21 the genes for the IFN-alpha receptor and for the factor(s) mediating the action of IFN-gamma through the chromosome 6-encoded receptor. Hamster-human somatic cell hybrids containing various fragments of human chromosome 21 were used. The presence of the human IFN-alpha/beta receptor was determined by binding 32P-labeled human IFN-alpha to cells, covalently cross-linking the [32P]IFN-alpha-receptor complex, and analyzing it by SDS-polyacrylamide gel electrophoresis. The presence of the IFN-gamma receptor-related factor mediating cellular responsiveness was determined by HLA induction in hybrid cells containing the IFN-gamma receptor (chromosome 6q), a transfected copy of the human HLA-B7 gene, and various portions of chromosome 21. In all hybrids examined, the two genes cosegregate. Specifically, both genes are localized to the region of chromosome 21 containing the markers D21S58, D21S65, and GART and appear to be proximal to D21S58. The implications for IFN action are discussed.

Lebow LT, Bonavida B (1990) Purification and characterization of cytolytic and noncytolytic human natural killer cell subsets. Proc Natl Acad Sci U S A 87 :6063-6067


Natural killer (NK) cells form three functionally distinct populations of effectors : competent cytolytic effectors able to bind and kill target cells and two subsets of nonlytic effectors, one able and the other unable to bind target cells. A flow cytometric method was developed, based on size and two-color fluorescence of NK cell-target conjugates, for the characterization and sorting of highly purified subpopulations—killer cells, nonkiller binder cells, and free cells. Ultrastructural examination revealed that granule content was reduced in the killer cells and absent in most of the binder cells. Quantitative differences in the expression level of HLA class I, CD11b (C3bi receptor), and CD16 (receptor for the Fc portion of IgG) antigens could differentiate the subsets. The killer phenotype was HLAlo, CD11bvery hi, and CD16very lo ; the binder phenotype was CD11bhi and CD16lo ; and the free-cell phenotype was CD11blo and CD16hi. Cell activation was not requisite for lytic function because no difference in either expression of activation markers or cell cycle could be established among the sorted subpopulations. Although recycling function was inhibited, retention of lytic activity was enriched 4-fold in the sorted killer cell population. These results represent characterization of a successful bulk isolation of competent killer, nonkiller binder, and free cells in human NK-cell populations and should aid our understanding of NK-cell development, lineage, and function.

McSharry JJ, Costantino R, McSharry MB, Venezia RA, Lehman JM (1990) Rapid detection of herpes simplex virus in clinical samples by flow cytometry after amplification in tissue culture. J Clin Microbiol 28 :1864-1866


Murine monoclonal antibodies (MAbs) against herpes simplex virus type 1 and 2 (HSV-1 and -2, respectively) nuclear antigens were used to identify cells infected with HSV-1 or -2 by indirect immunofluorescence in conjunction with flow cytometry after virus amplification of MRC-5 cell monolayers. The results indicate that MAbs Q1, Q2, and H-640 detect HSV-1- and HSV-2-infected cells, MAb SD-1 detects HSV-2- but not HSV-1-infected cells, and MAb 58-S detects HSV-1- but not HSV-2-infected cells. MAb Q1, which detects HSV-1- as well as HSV-2-infected cells, was used to detect HSV-infected cells after inoculation and overnight (16- to 20-h) incubation of MRC-5 monolayers with clinical samples suspected of containing HSV. In comparing the efficiency of flow cytometry with cytopathic effect (CPE) in tissue culture for detecting HSV in clinical samples, HSV was detected by flow cytometry in 77% of the cases where HSV was detected by CPE in tissue culture. In many cases, flow cytometry detected HSV from 1 to 3 days before HSV was detected by CPE.

Miller JS, Quarles JM (1990) Flow cytometric identification of microorganisms by dual staining with FITC and PI. Cytometry 11 :667-675


The identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.

Moy JN, Gleich GJ, Thomas LL (1990) Noncytotoxic activation of neutrophils by eosinophil granule major basic protein. Effect on superoxide anion generation and lysosomal enzyme release. J Immunol 145 :2626-2632


Eosinophil granule major basic protein (MBP) and neutrophils have each been implicated in the inflammatory late phase events of allergic disease. Based on this association and flow cytometric evidence presented in this report for MBP binding to neutrophils, we examined the ability of MBP to activate human neutrophils. Incubation of neutrophils with 0.5 to 3.0 microM MBP at room temperature produced a concentration-dependent chemiluminescence (CL) response that peaked after 50 to 70 min. Reduced-and-alkylated MBP, eosinophil cationic protein, and eosinophil-derived neurotoxin did not induce CL. MBP-induced CL was abrogated in the absence of Ca2+ and was absent in neutrophils isolated from two individuals with chronic granulomatous disease. MBP also stimulated release of superoxide anion (O2-) and lysozyme but not beta-glucuronidase or lactate dehydrogenase. Additionally, 1.5 microM MBP in combination with FMLP or platelet-activating factor stimulated a synergistic increase in O2- release from cytochalasin B-treated neutrophils. The degree of synergism with FMLP or platelet-activating factor was inversely related (p less than 0.005) to the level of MBP-induced O2- release. These results indicate that MBP activates neutrophils in a noncytolytic fashion and provide evidence that eosinophil-neutrophil collaboration may contribute to the pathogenesis observed in allergic late phase reactions.

Nir R, Lamed R, Gueta L, Sahar E (1990) Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry. Appl Environ Microbiol 56 :2870-2875


A method is presented for encapsulating single microbial cells in small spheres suitable for analysis and sorting by flow cytometry. The entrapped cells are able to multiply and form colonies contained within their respective microspheres. The system is based on ejecting the cells suspended in a gellable liquid through an orifice vibrating at ultrasonic frequencies, thus shearing the cell-containing jet into uniform droplets. When low-melting-temperature agarose was used, the droplets could be gelled into solid spheres during flight by appropriately directed colling air streams. This gelling was accompanied by significant dehydration, resulting in a twofold decrease in bead diameter and a corresponding increase in agarose concentration. Nevertheless, the microbeads obtained were highly uniform and had diameters which could be precisely controlled in the range of 10 to 40 microns. A variety of bacterial and yeast species were entrapped in agarose beads by using this system. In all cases the cells were able to develop into microcolonies containing as many as several hundred cells. This system enables one to apply the powerful method of flow cytometry to the analysis and sorting of whole microbial colonies. Potential applications of this technology in various areas of microbiology are considered.

Obernesser MS, Socransky SS, Stashenko P (1990) Limit of resolution of flow cytometry for the detection of selected bacterial species. J Dent Res 69 :1592-1598


The enumeration of bacteria in dental plaque samples is a vital but time-consuming procedure that uses standard cultural methods. Flow cytometry has proven to be a useful tool for the analysis of eukaryotic cells. In the present investigation, the utility of this technology for the enumeration of bacteria in mixtures was explored. Rabbit antisera were produced against the putative periodontal pathogens A. actinomycetemcomitans, B. intermedius, B. gingivalis, E. corrodens, W. recta, B. forsythus, as well as the frequently isolated supragingival species S. sanguis. Cross-reactive antibodies were removed by absorption, and the specificity of each antiserum was confirmed by being tested against a panel of 235 oral microbial strains (79 genera ; 94 species) by means of ELISA. Conditions were established for the indirect immunofluorescent labeling of cells without agglutination with use of a goat anti-rabbit Ig-FITC second antibody. When an internal bead standard was used, it was found that unstained bacteria were enumerated by light-scattering parameters with poor efficiency (less than 3%). However, cells exposed to FITC either in the presence of specific or non-specific first antibody were enumerated with high efficiency (102.6 +/- 29.3%), indicating that a small amount of non-specific binding of fluorochrome facilitates bacterial detection. Clear discrimination between specifically- and non-specifically-stained bacteria was achieved with all six rabbit antisera. Mixtures of known composition were made (1) with pure cultures or (2) with a known species and supragingival plaque devoid of that species by culture. The results from both approaches with various species combinations revealed that the limit of resolution for accurate quantitation of a selected species was approximately 5%, although specific organisms could be detected qualitatively when present at approximately 1%.

Ordonez JV, Rubinstein HM, Burnett JW (1990) Flow cytometric detection of jellyfish venom induced cytotoxicity. Toxicon 28 :863-867


Crude venoms of three poisonous jellyfish produce membrane depolarization as determined by the loss of fluorescence intensity of 3,3’-dipentyloxacarbocyanine iodide loaded cells measured by flow cytometry. This method for detecting jellyfish cytotoxicity was reproducible and more sensitive than mouse lethality assays.

Pontzer CH, Russell JK, Jarpe MA, Johnson HM (1990) Site of nonrestrictive binding of SEA to class II MHC antigens. Int Arch Allergy Appl Immunol 93 :107-112


We have used the synthetic peptide approach to show that the N-terminal 45-amino acids of staphylococcal enterotoxin A (SEA), SEA(1-45), constitute an important part of its binding site on class II major histocompatibility complex (MHC) molecules. SEA(1-45) and to a lesser extent SEA(1-27) were able to displace SEA from HLA-DR on Raji cells as assessed by flow cytometry and to compete with radiolabeled SEA for interaction with HLA-DR in a direct binding assay. Specific binding of SEA to Ia on murine A-20 cells could be inhibited by the same peptides [i.e. SEA(1-45) greater than SEA(1-27)] that blocked binding to HLA-DR. Therefore, different class II MHC molecules associate with the same functional site on SEA. Further, an ELISA system was used to demonstrate that SEA(1-45) is able to directly bind to a mouse synthetic I-A beta b peptide, I-A beta b (65-85), which contains a binding site of the class II MHC molecule involved in SEA presentation to T cells. Thus, we have localized a site on SEA that is involved in selective surface association with class II MHC antigens and identified the region on the class II MHC antigen to which that site binds.

Qvist P, Aasted B, Bloch B, Meyling A, Ronsholt L, Houe H (1990) Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle. Can J Vet Res 54 :469-472


Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities.

Raponi G, Keller N, Overbeek BP, Rozenberg-Arska M, van Kessel KP, Verhoef J (1990) Enhanced phagocytosis of encapsulated Escherichia coli strains after exposure to sub-MICs of antibiotics is correlated to changes of the bacterial cell surface. Antimicrob Agents Chemother 34 :332-336


The influence of five antibiotics (netilmicin, ceftriaxone, cefepime, fleroxacin, and ciprofloxacin) on capsular polysaccharide distribution and on opsonophagocytosis by human polymorphonuclear leukocytes of unencapsulated and encapsulated Escherichia coli strains was studied. Unencapsulated E. coli strains were readily opsonized in serum and easily ingested by polymorphonuclear leukocytes, and antibiotics did not further enhance the phagocytosis rates. In contrast, encapsulated bacteria were poorly opsonized in human serum, and phagocytosis was enhanced after overnight exposure to 0.5x the MICs of the antibiotics, with the exception of cefepime. Incubation of unencapsulated as well as encapsulated bacteria in complement-inactivated serum markedly reduced the bacterial uptake by polymorphonuclear leukocytes regardless of the presence of antibiotics. Slide agglutination assays, performed either with a monoclonal antibody for capsular polysaccharide or with an antiserum raised against the stable unencapsulated mutant E. coli O7:K-, showed reduction but not lack of the capsular polysaccharide of encapsulated E. coli O7:K1, and better exposure of subcapsular epitopes, after incubation with 0.5x the MICs of antibiotics. Flow cytometric analysis of encapsulated E. coli exposed to netilmicin, ciprofloxacin, and fleroxacin revealed that the reduction in capsular material was homogeneous among the bacterial population. Treatment with cefepime and ceftriaxone induced two populations of bacteria that differed in the amount of K antigen present. These results indicate that sub-MICs of netilmicin, ceftriaxone, fleroxacin, and ciprofloxacin influenced complement-mediated opsonization, probably due to changes in the capsular polysaccharide structure.

Sedmak DD, Roberts WH, Stephens RE, Buesching WJ, Morgan LA, Davis DH, Waldman WJ (1990) Inability of cytomegalovirus infection of cultured endothelial cells to induce HLA class II antigen expression. Transplantation 49 :458-462


Cytomegalovirus infection in the renal allograft recipient has been associated with the initiation of acute rejection. The mechanism of this induction is unknown. It may be related to renal tubular epithelial and endothelial expression of HLA class II antigens or a CMV immediate-early antigen that exhibits immunologic crossreactivity with HLA DR. In this study the ability of CMV to both infect and subsequently induce class II antigen expression on cultured human umbilical-vein endothelial cells (HUVEs), in the absence of cytokines, particularly gamma interferon, was tested. Individual HUVE cell lines were first proven to express HLA class II antigens in the presence of 10, 100, and 200 units of recombinant IFN-gamma as early as 24 hr postincubation by an immunohistochemical technique and by flow cytometry. These cell lines were successfully infected with CMV strains AD169 and CMV3 as determined by the presence of early and late viral antigens and CMV DNA. The degree of infection was dose and incubation-time dependent. Infection of HUVEs with these CMV strains and a nonattenuated clinical isolate failed to induce HLA DR, DP, or DQw1 expression in the absence of IFN-gamma. These findings support the hypothesis that endothelial cells in vivo may serve as reservoirs of CMV infection. They do not support the hypothesis that CMV produces an immediate-early antigen that has immunologic cross-reactivity with HLA DR. Furthermore, there is no support for the hypothesis that CMV independently induces HLA class II antigens in the absence of IFN-gamma.

Strair RK, Towle M, Smith BR (1990) Retroviral mediated gene transfer into bone marrow progenitor cells : use of beta-galactosidase as a selectable marker. Nucleic Acids Res 18 :4759-4762


Recombinant retroviruses have been utilized as vectors for gene transfer in model systems of gene therapy. Since many of these model systems require the transplantation of genetically modified primary cells it is important to devise methods which will allow the rapid and efficient selection for transplantation of only the cells which are capable of expressing high levels of the transferred gene. This report describes the use of beta-galactosidase as such a selectable marker. Bone marrow progenitors are infected with a recombinant retrovirus encoding beta-galactosidase. Using a fluorescence assay for beta-galactosidase we demonstrate that it is possible to use cell sorting to enrich for cells which will form bone marrow colonies that express high levels of beta-galactosidase. This rapid and non-toxic selection of bone marrow cells may facilitate attempts to achieve gene therapy in a variety of model systems.

Sullam PM, Payan DG, Dazin PF, Valone FH (1990) Binding of viridans group streptococci to human platelets : a quantitative analysis. Infect Immun 58 :3802-3806


The binding of viridans group streptococci with human platelets was analyzed by two-color flow cytometry. Binding was detected within 15 s of mixing bacteria and platelets. At ratios of bacteria to platelets of 1:1, 10:1, 100:1, and 1,000:1, the percentages of bound streptococci (mean +/- standard deviation) were 93.2% +/- 5.4%, 80.0% +/- 8.6%, 39.8% +/- 11.1%, and 12.5% +/- 2.0%, respectively. Binding of labeled bacteria was reversed by adding a 500-fold excess of unlabeled streptococci. These results demonstrate that streptococcus-platelet binding is rapid, reversible, and saturable, which suggests a specific receptor-ligand interaction.

Whetter LE, Gebhard DH, MacLachlan NJ (1990) Temporal appearance of structural and nonstructural bluetongue viral proteins in infected cells, as determined by immunofluorescence staining and flow cytometry. Am J Vet Res 51 :1174-1179


The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells ; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included : (1) enumeration of the proportion of infected cells in a population ; (2) further characterization of infected cells, including estimates of their viability ; and (3) computer-assisted storage and analysis of data obtained.

Yokochi T, Ikeda H, Inoue Y, Kimura Y, Ito H, Fujii Y, Kato N (1990) Characterization of autoantigens relevant to experimental autoimmune orchitis (EAO) in mice immunized with a mixture of syngeneic testis homogenate and Klebsiella O3 lipopolysaccharide. Am J Reprod Immunol 22 :42-48


Experimental autoimmune orchitis (EAO) in mice has been induced by repeated injection of a mixture of syngeneic testis homogenate and Klebsiella O3 lipopolysaccharide (KO3 LPS) as a potent adjuvant. The antisera obtained from mice with EAO lesions defined several antigens with apparent molecular weights (MW) of 38,000 (38 kd), 86 kd, 100 kd, and greater than 200 kd by the immunoblotting method. These antigens were organ-specific and exclusively present on the acrosome of spermatozoa, suggesting that these acrosomal antigens were highly relevant to EAO. It was found that the antigen with a fairly high MW (greater than 200 kd) was expressed on spermatozoa from the epididymis. Furthermore, the acrosomal 86 kd antigen was predominantly expressed in the testis, while the 100 kd antigen was dominant in the spermatozoa from the epididymis. It was therefore suggested that the 86 kd and 100 kd antigens in the acrosome were differentially expressed on the process of maturation of spermatozoa.

Zhou C, Jong A (1990) CDC6 mRNA fluctuates periodically in the yeast cell cycle. J Biol Chem 265 :19904-19909


Using cultures synchronized by two independent procedures, alpha-factor arrest and centrifugal elutriation, we have investigated the expression of the Saccharomyces cerevisiae CDC6 gene through the cell cycle. Our results show that the CDC6 gene is periodically expressed in the yeast cell cycle. The level of CDC6 transcripts increases in late G1, reaching a peak (approximately 10-20-fold over the initial level) at about the G1/S phase boundary. The peak of CDC6 mRNA was observed to overlap or slightly precede that of the CDC8 message, and to obviously precede that of the histone H2A message by some 25 min. Unlike histone H2A mRNA, the CDC6 mRNA as well as CDC8 mRNA were not affected by hydroxyurea treatment. These results suggest that regulation of H2A mRNA is different from that of CDC6 or CDC8. We have studied the 5’-flanking regions of CDC6 and other cell cycle-regulated genes. DNA sequence analysis of the CDC6 promoter revealed two sequences, 5’-C/GACGCGNC/G-3’ and 5’-PuGNAGAAA-3’ (where Pu is a purine, and N is any nucleotide), which are repeated three times each. Similar sequence elements have also been found among several cell cycle-regulated genes, including the CDC8 gene, but are not found upstream of histone genes. The possible significance of these elements is discussed.