Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.

Recent studies suggest that purified villous cytotrophoblasts are largely contaminated by mononucleated syncytial fragments and therefore unsuitable for studies of trophoblast differentiation. We assessed highly purified (>99.99 per cent) populations of villous trophoblasts for fragment contamination using the syncytial markers placental alkaline phosphatase (PLAP, by immunohistochemistry) and exteriorized phosphatidyl serine (ePS, by flow cytometric analysis). The preparations contained from 4-46 per cent syncytial fragments. However, we find that PLAP negative cells preferentially adhere to tissue culture surfaces and that all preparations were <2 per cent PLAP positive after routine plating and washing procedures. A second purification procedure eliminated dead (propidium iodide permeable) cells and separated viable syncytial fragments (ePS-positive) from viable cytotrophoblasts (ePS-negative) by two colour fluorescence activated cell sorting (FACS). Viable ePS-positive cells were ultrastructurally apoptotic, adhered poorly in culture and those that adhered rapidly underwent apoptosis. Viable ePS-negative cells contained large heterochromic nuclei and cytoplasmic structures, adhered strongly in culture and remained viable. The latter population (putative true villous CT) differentiated into syncytialized cells when cultured with EGF. We conclude that villous CT can be routinely purified, are viable in culture and can undergo syncytial fusion without extensive preformed syncytium.

Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles.

An effective in vitro model of the placental villous syncytium cultured on semi-permeable substrata is essential for studies of infectious pathogen transmission from mother to fetus. Current models using amniotic membranes or thinner artificial membranes show significant leakage, suggesting disruption of tight junctions or the presence of gaps between syncytial units. Such disruption and discontinuity of trophoblast cultures are probably the result of high stromal cell contamination, poor viability and lack of proliferation in culture. We have successfully cultured confluent layers of tight-junctioned syncytium on semi-permeable insert membranes using highly viable purified cytotrophoblasts and an alternating multiple seeding and differentiation technique. Using criteria including transepithelial diffusion of high and low molecular weight substances, electrical resistance and directional secretion of the matrix metalloproteinase, MMP-9, we demonstrate that these cultures form effective and functional physical barriers that can be maintained for up to 1 month.

Evidence that herpes simplex virus VP16 is required for viral egress downstream of the initial envelopment event.

During infection with herpes simplex virus type 1 (HSV-1), VP16 serves multiple functions, including transcriptional activation of viral immediate early genes and downregulation of the virion host shutoff protein vhs. Furthermore, VP16 has been shown to be involved in some aspect of virus assembly and/or maturation. Experiments with a VP16 null virus, 8MA, suggested that VP16 plays a direct role in virion assembly, since removal of VP16 from the HSV-1 genome results in reduced levels of encapsidated DNA and a failure to produce extracellular enveloped particles. However, VP16 null mutants display a severe translational arrest due to unrestrained vhs activity, thus complicating interpretation of these data. We examine here the role of VP16 in virion assembly and egress in the context of a vhs null background, using the virus 8MA/DeltaSma (VP16(-) vhs(-)). Comparison of 8MA and 8MA/DeltaSma with respect to viral DNA accumulation and encapsidation and accumulation of the major capsid protein, VP5, revealed that the 8MA lethal phenotype is only partially due to uncontrolled vhs activity, indicating that VP16 is required in HSV-1 virion formation. Electron microscopy confirmed these results and further showed that VP16 is required for HSV-1 egress beyond the perinuclear space. In addition, we describe the isolation and characterization of an 8MA derivative capable of propagation on Vero cells, due to second site mutations in the vhs and UL53 (gK) genes. Taken together, these results show that VP16 is required for viral egress downstream of the initial envelopment step and further underscore the importance of VP16 in controlling vhs activity within an infected cell.

Antigenic mimicry between Helicobacter pylori and gastric mucosa: failure to implicate heat-shock protein Hsp60 using immunoelectron microscopy.

BACKGROUND: Helicobacter pylori infection induces autoantibodies that cross-react with human gastric mucosa from infected individuals. Candidates for the antigens responsible for molecular mimicry causing autoreactivity include the heat-shock protein HspB (Hsp60, sometimes called Hsp54) or Lewis x and Lewis y carbohydrate antigens. OBJECTIVE: Our goal was to investigate the involvement of HspB (Hsp60) in autoreactivity between H. pylori and gastric biopsy tissue. MATERIALS AND METHODS: Immunoelectron microscopy was used to study cross-reactivity among biopsy tissues from a patient with gastritis, gastric ulcer, and duodenal ulcer and his own serum as well as reactivity with serum raised against HspB from H. pylori and monoclonal antibodies against Lewis antigens. RESULTS: The patient serum reacted with gastric mucosa, and the antibodies involved were predominantly IgG. Antibody raised to H. pylori HspB (Hsp60) reacted only with H. pylori cells but not with gastric mucosal tissue. In contrast, monoclonal antibodies specific for Lewis x and Lewis y antigens reacted with both H. pylori and human gastric epithelial tissue. CONCLUSIONS: Hsp60 (Hsp54) is unlikely to be involved in autoreactivity seen in individuals infected with H. pylori. In contrast, we could not rule out the role of Lewis x and Lewis y carbohydrate antigens, expressed as a component of H. pylori lipopolysaccharides, in molecular mimicry and autoantibody production.

Molecular genetic basis for the variable expression of Lewis Y antigen in Helicobacter pylori: analysis of the alpha (1,2) fucosyltransferase gene.

Helicobacter pylori lipopolysaccharides (LPS) express human oncofetal antigens Lewis X and Lewis Y. The synthesis of Lewis Y involves the actions of alpha (1,3) and alpha (1,2) fucosyltransferases (FucTs). Here, we report the molecular cloning and characterization of genes encoding H. pylori alpha (1,2) FucT (Hp fucT2) from various H. pylori strains. We constructed Hp fucT2 knock-out mutants and demonstrated the loss of Lewis Y production in these mutants by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The Hp fucT2 gene contains a hypermutable sequence [poly (C) and TAA repeats], which provides a possibility of frequent shifting into and out of coding frame by a polymerase slippage mechanism. Thus, the Hp fucT2 gene displays two major genotypes, consisting of either a single full-length open reading frame (ORF; as in the strain UA802) or truncated ORFs (as in the strain 26695). In vitro expression of Hp fucT2 genes demonstrated that both types of the gene have the potential to produce the full-length protein. The production of the full-length protein by the 26695 fucT2 gene could be attributed to translational-1 frameshifting, as a perfect translation frameshift cassette resembling that of the Escherichia coli dnaX gene is present. Examination of the strain UA1174 revealed that its fucT2 gene has a frameshifted ORF at the DNA level, which cannot be compensated by translation frameshifting, accounting for its Lewis Y off phenotype. In another strain, UA1218, the fucT2 gene is apparently turned off because of the loss of its promoter. Based on these data, we proposed a model for the variable expression of Lewis Y by H. pylori, in which regulation at the level of replication slippage (mutation), transcription and translation of the fucT2 gene may all be involved.

Lack of correlation between Lewis antigen expression by Helicobacter pylori and gastric epithelial cells in infected patients.

BACKGROUND & AIMS: Lewis antigens are expressed by both human gastric epithelial tissue and Helicobacter pylori. We examined Lewis antigens expressed by gastric epithelium and by H. pylori isolated from the corresponding biopsy tissue. METHODS: H. pylori Lewis expression was determined by enzyme immunoassays, and immunoelectron microscopy was used to confirm the Lewis antigens on some H. pylori cells and in some biopsy specimens. Histopathology using identical monoclonal antibodies specific for Lewis A, B, X, and Y antigens was used to detect these antigens in 24 gastric biopsy specimens. RESULTS: We identified Lewis Y in 100%, Lewis X and Lewis B in 95.8%, and Lewis A in 87.5% of biopsy specimens. In H. pylori, 87.5% expressed Lewis Y, 79.2% Lewis X, and 4.2% (one strain) Lewis B. No Lewis A was detected. Antibody specific for Lewis X labeled the bacteria and associated adhesion pedestal. The cagA gene was present in 92% of strains. CONCLUSIONS: There was no direct relationship between Lewis antigen expression by H. pylori and gastric epithelial cells in infected patients. Expression of Lewis X and Lewis Y by H. pylori suggests the possibility of their requirement for establishment and/or maintenance of infection. An immunoelectron micrograph of H. pylori interaction with the gastric epithelial adhesion pedestal suggests a tentative role for Lewis X in the adhesion process.

In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium.

Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.

Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster.

The IncHI2 plasmid R478 specifies resistance to potassium tellurite (Te(r)), to some bacteriophages (Phi), and to pore-forming colicins (PacB). The genes encoding the three phenotypes are linked, and an 8.4-kb fragment of R478 DNA encoding them cannot be subcloned unless cocloned with a second section of the plasmid. Subclone pKFW4A contains a 5.9-kb BamHI-EcoRI fragment which caused some toxicity when present in Escherichia coli cells. Bacterial cells containing freshly transformed pKFW4A, examined by light microscopy and electron microscopy, had a filamentous morphology consistent with a block in septation. Insertion of transposon Tn1000 into terZ, -A, -B, and -C genes of pKFW4A resulted in the loss of the filamentation phenotype. Deletion of several regions of the clone confirmed that these latter components are involved in the filamentation phenotype. The region specifying protection from toxicity caused by the larger 8.4-kb fragment (encompassing this cluster and the entire 5.9-kb section of pKFW4A) was sequenced and analyzed by T7 polymerase expression and Tn1000 mutagenesis. Three open reading frames, terW, terY, and terX, were identified in a 2.6-kb region. Two polypeptides with approximate molecular masses of 18 and 28 kDa were expressed in CSRDE3 cells and were consistent with TerW (17.1 kDa; 155 amino acids [aa]) and TerY (26.9 kDa; 248 aa), whereas a protein of 213 aa deduced from terX was not observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The terX gene product shows strong identity with the previously identified TerE, TerD, and TerZ polypeptides, and there is a conserved motif of 13 residues, GDN(R/L)TG(E/A)GDGDDE, within this group of polypeptides. Complementation analysis indicated that terW, located approximately 6.0 kb upstream of terZ, brings about protection of cells from toxic effects of components of the Te(r), Phi, and PacB cluster.

Functional domains of chlamydial histone H1-like protein.

Chlamydial trachomatis is one of the few prokaryotic organisms known to contain proteins that bear amino acid similarity to eukaryotic histone H1. It is also appreciated that chlamydial histone-like proteins, designated Hc1 and Hc2, can bind DNA and are presumably involved in the condensation of infectious elementary bodies. However, there is no information on either the orientation of Hc1 and Hc2 or the mechanism of their DNA-protein and protein-protein interactions. Whereas the C-terminal domain of Hc1 between amino acids 63 and 125 shows best alignment with sea-urchin histone H1, and N-terminus between amino acids 1 and 62 is highly conserved among various chlamydial species, suggesting a bifunctional role for this unique protein. In order to delineate the regions responsible for the Hc1 characteristics, we have expressed these two fragments independently in Escherichia coli and studied the binding of double-stranded DNA to either whole Hc1 protein or its two termini. Our results support the role of the carboxyl portion in DNA-protein interaction, a function similar to its eukaryotic counterpart. Although this interaction initiates DNA condensation in the absence of the N-terminal domain, it is not sufficient to produce complete compaction. Intra- or inter-molecular protein-protein interactions may be necessary to achieve such an effect.

Helicobacter pylori expresses a complex surface carbohydrate, Lewis X.

Monoclonal antibodies (MAbs) specific for Lewis X (Lex) reacted with whole cells of Helicobacter pylori NCTC11637, UA799, UA802, UA825, UA861, UA1182, and UA1206 in immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) experiments. These MAbs have documented specificity to Lex, whereas MAbs for Lea and Leb were negative in both immunoelectron microscopy and ELISA. H. pylori coccoid forms also reacted with the MAbs, whereas the flagellum lacking the sheath showed no reactivity. The Lex structures were associated with membrane fractions in the ELISA experiments, and silver-stained sodium dodecyl sulfate-polyacrylamide gels confirmed the presence of lipopolysaccharides which reacted with the MAbs in immunoblots. Serum from an H. pylori-infected individual contained immunoglobulins which blocked the binding of the Lex MAbs, indicating that part of the host immune response to H. pylori is to the Lex structure. The ability of this gastric pathogen to mimic an oncofetal antigen (self) could explain the down regulation of anti-H. pylori T-cell response seen in H. pylori-infected individuals.

The biosynthesis of Lewis X in Helicobacter pylori.

The biosynthesis of the Lewis X determinant (Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta-) in three strains of Helicobacter pylori has been investigated. Strains UA 861, UA 802 and UA 1182 contain alpha 1,3 fucosyltransferase and beta 1,4 galactosyltransferase activities that synthesize the Lewis X structure by the transfer of monosaccharides from GDP-fucose and UDP-galactose donors, respectively. The enzyme reaction products that formed were characterized by capillary zone electrophoresis and by 1H-NMR spectroscopy. The biosynthetic pathway is therefore identical to that found in humans. In the three strains, the fucosyltransferase and galactosyltransferase activities differed in various cellular fractions. Km values for their donor and acceptor substrates also differed.

Diagnostic difficulties caused by a nonclamped Schizophyllum commune isolate in a case of fungus ball of the lung.

The presence of clamp connections on hyphae and the development of fruiting bodies in culture are primary characters which allow identification of the basidiomycete Schizophyllum commune in cases of human infection. The diagnostic problems presented by a nonclamped, nonfruiting isolate from a dense mass in the right upper lobe of the lung in a female with a past history of pulmonary tuberculosis and diabetes are described. Several features of the isolated fungus, including rapid growth rate and white, dense, cottony colonies, tolerance to the fungicide benomyl at a concentration of 10 micrograms/ml, and susceptibility to cycloheximide at 400 micrograms/ml, suggested that it might be a basidiomycete. Transmission electron microscopy showed the presence of a dolipore septum with perforate pore cap characteristic of fungi in the class Holobasidiomycetes. However, species identification remained elusive until compatibility tests with known single-basidiospore isolates confirmed the identification of the sterile lung isolate as S. commune. Sequence analysis of the 5' internal transcribed spacer region of ribosomal DNA further supported conspecificity.

Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice.

The disulfide loop domain of Pseudomonas aeruginosa PAO pilin was altered by insertion of a chloramphenicol acetyltransferase gene into the pilin gene so that the C-terminal nine amino acids were replaced with 11 new amino acids. The altered pilin gene was transferred into wild-type PAO by recombination, where it did not affect normal piliation as observed by transmission electron microscopy or change of sensitivity to f116, PO4, B9, and Pf1 pilus-specific bacteriophages. However, the binding to human pneumocyte A549 cells was markedly reduced when tested in an in vitro binding assay (2 to 6 bacteria bound per A549 cell for the mutant bacteria compared with 50 bacteria per A549 cell for the wild-type bacteria). Additionally, when susceptible A.BY/SnJ mice were challenged with wild-type P. aeruginosa PAO and with P. aeruginosa PAO-MP (altered pilin gene), a 50% lethal dose of 3 x 10(6) bacteria per mouse was observed for PAO-MP compared with 7 x 10(4) bacteria per mouse for PAO. Approximately 90 of the adherence capability of P. aeruginosa PAO is seemingly attributable to the C-terminal disulfide loop adherence domain of pili. The pilus adherence function contributes significantly to the virulence of P. aeruginosa PAO in the A.BY/SnJ mouse infection model.

The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids.

The effects of defined mutations in the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating-pair formation in liquid media by the transfer systems of the F-like plasmids pOX38 (F), ColB2 and R100-1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responsible for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. ColB2 transfer was more strongly affected by mutations in the heptose II-heptose III region of the LPS (rfaF) whereas R100-1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating-pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F-like plasmids (F, ColB2, R100-1) were used to complement this mutation. Unexpectedly, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit.

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM1) and, to a lesser extend, II3N-acetylneuraminosylgangliotetraosyl ceramide (GM1) in solid-phase binding assays. Asialo-GM1, but not GM1, inhibited both PAK and PAO pili binding to immobilized asialo-GM1 on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM1, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM1 and pili occurs at the pilus tip as asialo-GM1 coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134-140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128-144)ox-OH and PAO(128-144)ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM1 and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM1.(ABSTRACT TRUNCATED AT 250 WORDS)

H-pilus assembly kinetics determined by electron microscopy.

The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnlacZ insertion derivative of the IncHI1 plasmid R27, which was derepressed for transfer. IncHI1 plasmids are thermosensitive for transfer. The pili specified by pDT1942 were examined by transmission electron microscopy after the pilus had been labeled with the H-pilus-specific bacteriophage Hgal, which had been inactivated with RNase A. H pili were extended by extrusion from the cell surface and not by the addition of pilin subunits to the pilus tip. After pili were removed by vortexing, the outgrowth of full-length pili (2 microns long) required 20 min. H pili expressed at the transfer optimal temperature (27 degrees C) remained stable after incubation at the transfer inhibitory temperature (37 degrees C), but the formation of mating aggregates was inhibited at 37 degrees C. Within 1 min of exposure of the host cell to a heat stimulus of 50 degrees C, pili vanished. Pili were observed in straight and flexible forms with a field emission scanning electron microscope, which may indicate a dynamic role for the pilus in conjugation.

Characterization of an immunodominant Giardia lamblia protein antigen related to alpha giardin.

The trophozoites of Giardia lamblia possess several protein antigens, predominant among them a protein of approximately 32,000 Da. In the present study, we used monospecific antibodies that recognize this protein to demonstrate its presence on a variety of G. lamblia isolates from human and animal sources. Immune electron microscopy was used to localize 32-kDa antigen on the trophozoite membrane and disk. Immunofluorescent assays employing monospecific antibodies confirmed the presence of 32-kDa antigen on the membrane and disk and its absence on flagella or nuclei. The N-terminal 17 amino acids of the 32-kDa antigen are identical to alpha-1-giardin, a protein component of microribbons on the ventral disk. These results suggest that the 32-kDa immunodominant trophozoite antigen is alpha-1-giardin.

Bacteriophages for incompatibility group H plasmids: morphological and growth characteristics.

Two independently isolated temperature-sensitive bacteriophage that are specific for enterobacterial hosts harboring HI and HII plasmids were characterized to determine if any identifiable differences existed between them. The traits examined included adsorption pattern of phage to H pili, bacteriophage size, sensitivity to chloroform, RNA strandedness, reaction with F-specific antiphage serum, virion protein pattern, temperature range of lytic ability, and plaque morphology. No differences between the phages were observed for any of the features analyzed. Ecological questions on the origin and maintenance of temperature-sensitive phages are discussed.

Hemadsorption by colonies of Ureaplasma urealyticum.

Hemadsorption by colonies of Ureaplasma urealyticum and Mycoplasma pneumoniae differed quantitatively and qualitatively. Using standard methodology, few strains of U. urealyticum hemadsorbed; with a modified method, most strains hemadsorbed, indicating a second type of association. Scanning electron microscopy of tannin-osmium-stained preparations showed guinea pig erythrocytes embedded in ureaplasma colonies and craters left when erythrocytes were dislodged.