Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol. 87:779–784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics 61:171–177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.     

Epithelial cell association and hydrophobicity of Yersinia enterocolitica and related species

Six of 11 test strains of Yersinia enterocolitica and related species that carried other markers of pathogenicity were found to associate with Henle 407 epithelial cells in vitro. All Henle-positive strains were hydrophobic when tested by hydrophobic interaction chromatography with phenyl-Sepharose and by partitioning in an aqueous-hexadecane mixture. Hydrophobicity was also exhibited by some of the Henle-negative strains. None of the test strains aggregated in low concentrations of ammonium sulphate, suggesting that protein structures such as fimbriae were not involved in hydrophobicity or epithelial cell association.     

The pathogenic potential of Yersinia enterocolitica 1A

Yersinia enterocolitica 1A strains are generally considered apathogenic. However, besides environmental sources, foods and animals, they are repeatedly isolated from patients with gastrointestinal symptoms typical to those evoked by Yersinia of the virulent 1B and 2–4 biotypes. Also, at least 2 gastrointestinal outbreaks associated with 1A strains have been reported. There is a general controversy concerning the pathogenic potential of 1A isolates of clinical and non-clinical origin. To address the 1A puzzle, we have determined the genome sequences of 2 1A strains, a nosocomial O:5 and environmental O:36 isolates, and compared them to each other and to O:8/1B and O:3/4 representatives of the virulent serobiotypes.1A isolates have mosaic genomes and share genes both with serobiotypes O:8/1B and O:3/4 that implies their common descent. Besides the pYV virulence plasmid, 1A strains lack the classical virulence markers, like the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. However, they still possess genes encoding such known and suspect virulence-associated determinants like the YstB enterotoxin, the InvA invasin, the mucoid Yersinia factor MyfA, and the enterochelin utilisation fepBDGC/fepA/fes gene cluster. In contrast to previous studies, we have found that the strains of the 1A group possess the MyfA antigen although with limited similarity to the highly conserved MyfA in the virulent serobiotypes. In turn, the MyfB chaperone coevolved with the MyfA fibrillae, while the MyfC usher retains 90% identity to its MyfC counterparts in O:3/O:8 group. The only notable difference between clinical and non-clinical 1A strains was the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital O:5 isolate.Taken together, Y. enterocolitica BT 1A group represents opportunistic pathogens whose opportunity to establish infection seems to rely mainly on the state of the host defence system. However, presence of known and putative virulence-associated features shared with the pathogenic serobiotypes compels to reconsider properly the pathogenic potential of this group of emerging pathogens.     

The enigma of Yersinia enterocolitica biovar 1A

Yersinia enterocolitica, an important food- and water-borne enteropathogen causes acute diarrhea, terminal ileitis, and mesenteric lymphadenitis. It is represented by six biovars (1A, 1B, 2-5). The biovar 1A strains are generally regarded as avirulent as they lack pYV plasmid and major chromosomal virulence genes. Despite this, some biovar 1A strains produce disease symptoms indistinguishable from that produced by known pathogenic biovars (1B, 2-5). Suggested prospective studies to understand pathogenic potential of biovar 1A should focus on role of insecticidal toxins, urease, protease, superoxide dismutase, and host responses. These studies should also take into account the clonal groups of biovar 1A.     

Semaphorin 3A Negatively Affects Proliferation of Mouse Thymus Epithelial Cells In Vitro

Bulletin of Experimental Biology and Medicine - We studied effects of semaphorin 3A, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and their combinations on the proliferative...     

In Vitro and In Vivo Interactions of Haemophilus ducreyi with Host Phagocytes

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10⁶ CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted throughout the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.     

Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets.

Yersinia enterocolitica is a bacterium capable of growth at 4 degrees C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry, enterocolitica did not cause platelet aggregation or activation, not even when grown at 22 degrees C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37 degrees C but not with pYV- strains nor with strains grown at 22 degrees C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22 degrees C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37 degrees C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.     

Reprogramming of Human Retinal Pigment Epithelial Cells under the Effect of bFGF In Vitro

Bulletin of Experimental Biology and Medicine - We studied the effect of bFGF on human retinal pigment epithelial cells in vitro In ARPE-19 cells, enhanced expression of KLF4 mRNA and reduced...     

Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis.

Yersinia enterocolitica isolates harboring a particular species of plasmid deoxyribonucleic acid showed a high degree of lethality for gerbils and caused the detachment of HEp-2 tissue cell monolayers. Strains cured of their plasmid deoxyribonucleic acid showed loss of these properties. However, invasiveness of HEp-2 cells was shown not to be a plasmid-mediated property. The expression of plasmid-associated properties, including at least three major outer membrane polypeptides, occurred during growth at 37 but not at 25 degrees C and was related to the concentration of calcium in the growth medium. The plasmid species associated with these properties ranged in molecular mass from 40 x 10(6) to 48 x 10(6) daltons and comprised a family of related plasmids.     

Inducible and Constitutive In Vitro Neutrophil Chemokine Expression by Mammary Epithelial and Myoepithelial Cells

Previously, our laboratory showed that bovine and caprine mammary secretions are chemotactic and that chemoattractants found in these secretions are qualitatively different according to infection status and/or lactation stage. However, the cellular source of the chemoattractants has not been defined. In this study we used a modified Boyden chamber assay to examine the ability of previously established caprine mammary epithelial cell (CMEC) and myoepithelial cell (CMMyoEC) lines to produce chemoattractants for neutrophils. We found that CMEC culture supernatants, but not those of CMMyoEC cultures, induced in vitro neutrophil chemotaxis. Further characterization showed that chemotactic activity was produced when the cells underwent contact-induced differentiation. Neutrophil migration was chemotactic, not chemokinetic, and was augmented when the epithelial and myoepithelial cells were cocultured. Additionally, chemotactic activity was inducible by Staphylococcus aureus plus alpha-toxin, Escherichia coli, and interleukin-1β (IL-1β) in CMEC cultures. However, CMMyoEC cultures could not be induced to produce chemotactic activity. Anti-IL-8 antibody was able to block some constitutively produced chemotactic activity and chemotactic activity induced by IL-1β and S. aureus plus alpha-toxin. These results indicate that epithelial cells may play a major role in producing chemoattractants, specifically IL-8, in the mammary gland.     

Interaction of Human Dendritic Cells with Graphene Oxide Nanoparticles In Vitro

Bulletin of Experimental Biology and Medicine - We studied the effect of graphene oxide nanoparticles on the differentiation of human dendritic cells and uptake of nanoparticles by these cells in...     

In Vitro and In Situ Characterization of Fish Thymic Nurse Cells

We present an enzyme- and immuno-cytochemical, and ultrastructural characterization of trout thymic nurse cells (TNCs). Our data suggest that isolated trout thymic multicellular complexes are epithelial cells with acidic compartments that may be involved in the processing of antigens and in the generation of the MHC-II proteins that these cell express, and also that isolated TNCs are the In Vitro equivalent of the pale and intermediate electronlucent epithelial cells located in the inner zone of the trout thymus, constituting indirect evidence of the phylogenetical relationships of the inner zone of the teleost thymus with the thymic cortex of higher vertebrates.     

Relationship between clinical and milk isolates of Yersinia enterocolitica.

In the summer of 1987-1988, an outbreak of 11 cases of Yersinia enterocolitica enteritis caused by 2 serogroups (0:3, 0:6,30) occurred prompting an investigation into possible environmental sources. Symptoms were present for a mean of 9 days and occurred in 2 distinct age groups--toddlers (7) who presented with diarrhea, and young adults (4), 3 of whom presented clinically with appendicitis. In a survey of 39 randomly chosen pasteurized milk samples, 9 were positive for growth of Y. enterocolitica and 1 each for Y. fredericksenii and Y. intermedia. An association between clinical and milk isolates of Y. enterocolitica was thus sought by comparison of biogroups, serogroups, virulence markers and biochemical and outer membrane profiles. All milk isolates belonged to biogroup 1, serogroup 0:6,30. Pathogenicity studies on the 0:6,30 serogroup isolates from feces and milk were performed with 3 in-vitro tests (Ca2+ dependency, autoagglutination, & serum resistance). The human isolates were positive in most of the 3 tests whilst none of the milk isolates were positive. Outer membrane protein analysis of 0:6,30 from human and milk isolates showed similar profiles suggesting a possible association, however the environmental source of the majority of isolates (0:3) remains unknown.     

In Vitro Studies of Human Prostatic Epithelial Cells: Attempts to Identify Distinguishing Features of Malignant Cells

Abstract

Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-β and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.     

Beta-lactamase expression in Yersinia enterocolitica biovars 1A, 1B, and 3

Characteristic patterns of beta-lactam susceptibility are associated with different biovars of Yersinia enterocolitica. In a previous study differences in beta-lactam susceptibility among biovar 2, 4 and 5 strains were largely attributed to differences in expression of beta-lactamase A (BlaA) and beta-lactamase B (BlaB). The basis for differences in beta-lactam susceptibility of strains of biovars 1A, 1B and 3 is now considered. All the strains examined had blaB; nine of 31 biovar 3 strains and two of 13 biovar 1B strains had blaA, but PCR did not amplify blaA from biovar 1A strains. Nevertheless, inhibition data indicated that the majority of uninduced biovar 1A strains expressed BlaA and BlaB in similar amounts. Strong inducibility was seen in all these strains. Biovar 1B strains (which were less inducible than strains of biovar 1A) predominantly produced BlaA without induction; ticarcillin-sensitive strains of biovar 3 produced only BlaB but were not inducible; without induction biovar 3 strains resistant to ticarcillin and amoxycillin/clavulanate produced either predominantly BlaA, predominantly BlaB or exclusively BlaB and induction was demonstrated except for strains producing BlaB alone; biovar 3 strains resistant to ticarcillin but sensitive to amoxycillin/clavulanate predominantly produced BlaA without induction and were inducible for beta-lactamase activity. After induction, nearly all strains predominantly or exclusively produced BlaB. Although PCR amplification fragments with primers specific for blaA were obtained only from some strains, the induction and inhibition data suggest that all Y. enterocolitica strains possess enzymes related to BlaA- as well as BlaB. Nevertheless, expression of the beta-lactamase is regulated differently in different biovars and varies within most biovars. Failure to predict beta-lactamase expression profiles from MIC data indicates the presence of additional mechanisms contributing to differences in susceptibility.     

In Vitro Induction and Characterization of Mast Cells from Murine Peyer''s Patches

The present study was carried out in vitro to determine whether murine Peyer's patches (PP) can be a source of mucosal mast cells, using a well-defined mast cell growth factor, interleukin 3 (IL-3). The non-T, non-B, non-adherent (null) cell population of PP contained the precursor cells for mast cells. The mast cells induced possessed toluidine blue and alcian blue (pH 3)-positive granules, which were heterogeneous and contained histamine. The IL-3-induced mast cells were IL-3-dependent, did not express Ia, T-cell, B-cell or macrophage markers, and released histamine in response to Ca ionophore, but not in response to compound 48/80. These cells bore IgE-specific surface receptors and intracellular 20 alpha hydroxysteroid dehydrogenase. The latter enzyme activity was induced by IL-3 in null cells. All these features of the mast cells support the view that these cells belong to a lineage of mucosal mast cells (MMC) and are no different from those of other lymphoid tissues, such as the bone marrow and spleen. In the analysis of MMC precursor frequency, PP had a similar frequency to that of other gut-associated lymphoid tissues (mesenteric lymph nodes and lamina propria), but was much lower when compared to those of the bone marrow and spleen. Furthermore, like IL-2, PP are capable of producing IL-3 in vitro by activating their helper marker-bearing T cells. Thus, under certain conditions, such as the initial participation by PP in MMC-associated intestinal immune responses, PP are likely to provide MMC in their microenvironment, like mucosal T and B cells.     

In vitro assessment of virulence in Yersinia enterocolitica and related species.

We have examined 136 isolates of Yersinia species, comprising 112 strains of Yersinia enterocolitica, 12 of Y. frederiksenii, 8 of Y. intermedia, and 5 of Y. kristensenii, for the presence of 40- to 50-megadalton virulence-associated plasmids and expression of the following plasmid-associated characteristics: Congo red pigmentation (CR), calcium dependence, autoagglutination, hydrophobicity, resistance to normal human serum, and pathogenicity in mice. All 136 strains yielded both pigmented (CR+) and nonpigmented (CR-) variants. Only CR+ variants, however, were virulent for iron-overloaded, desferrioxamine B-treated mice (R. M. Robins-Browne and J. K. Prpic, Infect. Immun. 47:744-779, 1985). Although the in vitro virulence-associated characteristics generally occurred together, each one could be expressed independently. Strains of Y. frederiksenii, Y. intermedia, and Y. kristensenii also expressed individual virulence-associated properties. Of 53 Y. enterocolitica strains which were virulent for iron-overloaded, desferrioxamine-treated mice, all but one expressed every virulence-associated characteristic. Several strains which were avirulent for mice, however, demonstrated these characteristics in various combinations. Because many Yersinia strains, particularly environmental isolates, carried plasmids of 40 to 50 megadaltons, detection of plasmids provided little information about bacterial pathogenicity unless virulence-associated properties were also sought. The best in vitro predictor of virulence was autoagglutination, followed by calcium dependence. Because only CR+ variants expressed virulence-associated determinants, Congo red pigmentation is useful for selecting potentially virulent strains.     

Characterization of a Yersinia enterocolitica antigen common to enterocolitis-associated serotypes.

Yersinia enterocolitica synthesized an exocellular antigen common to the serotypes associated with enterocolitis but absent from other serotypes or from other Yersinia species. Both virulent Ca2+-dependent and avirulent Ca2+-independent isogenic pairs derived from the enterocolitis-associated serotypes synthesized the common antigen. Requirements for the synthesis of this common antigen were (i) the presence of metabolizable sugars and (ii) growth on a solid medium at 37 degrees C. The antigen was identified as a 24,000-dalton protein loosely associated with the cell surface but absent from either the cell envelope or the cytoplasmic fraction.