Adherence of Actinomyces viscosus T14V and T14AV to Hydroxyapatite Surfaces In Vitro and Human Teeth In Vivo

Adsorption of Actinomyces viscosus strains T14V and T14AV to hydroxyapatite (HA) surfaces was studied, using an adsorption model based on the Langmuir adsorption isotherm. Data generally followed the adsorption model as judged by high correlation coefficients obtained for both strains to most of the treated surfaces studied. The number of binding sites for strains T14V and T14AV cells to human saliva-treated HA was similar to that for untreated HA. The affinity of strain T14V for saliva-treated HA was tenfold greater than the affinity of strain T14AV for that surface. To approximate the pellicle of the gingival crevice and margin and to determine whether adherence by strain T14V was to specific saliva or serum receptors, experimental pellicles were formed on HA by saliva/serum mixtures. The number of binding sites on the saliva/serum-treated HA remained the same as for the saliva-treated surface. Although the affinity of strain T14V cells for the saliva/serum HA surface remained generally the same as the affinity for the HA treated with saliva alone, the affinity of strain T14AV cells decreased further as the serum content increased. Strain T14V cell numbers adsorbed to serum-treated HA, and albumin-treated HA were less than those adsorbed to saliva-treated HA, indicating that the adherence by strain T14V was to specific saliva receptors. In vivo results from streptomycin-resistant mutants of both strains T14V and T14AV confirmed in vitro results using saliva-serum pellicles. Pretreatment of strain T14V with proteolytic enzymes and heat inhibited adherence to saliva-treated HA, suggesting that the adherence receptor(s) on the cell surface of strain T14V is protein in nature.     

Differences in the Adsorptive Behavior of Human Strains of Actinomyces viscosus and Actinomyces naeslundii to Saliva-Treated Hydroxyapatite Surfaces

Human strains of Actinomyces viscosus and A. naeslundii differ in the time of their appearance and in their patterns of colonization in the mouth. Strains of these organisms were found to differ in their abilities to adsorb to saliva-treated hydroxyapatite (S-HA) surfaces, thought to mimic the teeth, and these differences parallel their patterns of colonizing the dentition. Thus, strains of A. viscosus tended to adsorb in higher numbers to hydroxyapatite (HA) treated with saliva of older children and adults than with saliva of younger children (ages 6 to 11). These salivary changes may account for the increased frequency with which this organism can be isolated from the mouths of children as they grow older. In contrast, strains of A. naeslundii and Streptococcus mutans did not show a preference for attaching to either type of S-HA. Strains of A. viscosus also generally adsorbed in higher numbers than A. naeslundii to HA treated with adult saliva; this may explain why higher proportions of A. viscosus are usually recoverable from the teeth of adults, even though A. naeslundii is generally present in higher proportions in saliva. Significant variation was noted between strains and between saliva samples collected from different donors. The differences in adsorptive behavior of strains of these species suggests that they are binding to different receptors in the salivary glycoprotein coating on HA surfaces. Adsorption of A. naeslundii ATCC 12104 was enhanced when S-HA was pretreated with neuraminidase, but this had little effect upon the adsorption of other Actinomyces strains tested. Adsorption of strain ATCC 12104 to S-HA was also strongly inhibited by fructose and sucrose and weakly inhibited by glucose, maltose, galactose, and lactose. However, other strains of A. naeslundii tested were affected less, or not at all, by these sugars. Adsorption of two strains of A. viscosus was not affected by any of the sugars or amines tested.     

The Presence of CD14 Overcomes Evasion of Innate Immune Responses by Virulent Francisella tularensis in Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo

Francisella tularensis is a Gram-negative bacterium that causes acute, lethal disease following inhalation. We have previously shown that viable F. tularensis fails to stimulate secretion of proinflammatory cytokines following infection of human dendritic cells (hDC) in vitro and pulmonary cells in vivo. Here we demonstrate that the presence of the CD14 receptor is critical for detection of virulent F. tularensis strain SchuS4 by dendritic cells, monocytes, and pulmonary cells. Addition of soluble CD14 (sCD14) to hDC restored cytokine production following infection with strain SchuS4. In contrast, addition of anti-CD14 to monocyte cultures inhibited the ability of these cells to respond to strain SchuS4. Addition of CD14 or blocking CD14 following SchuS4 infection in dendritic cells and monocytes, respectively, was not due to alterations in phagocytosis or replication of the bacterium in these cells. Administration of sCD14 in vivo also restored cytokine production following infection with strain SchuS4, as assessed by increased concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-12p70, and IL-6 in the lungs of mice receiving sCD14 compared to mock-treated controls. In contrast to homogenous cultures of monocytes or dendritic cells infected in vitro, mice treated with sCD14 in vivo also exhibited controlled bacterial replication and dissemination compared to mock-treated controls. Interestingly, animals that lacked CD14 were not more susceptible or resistant to pulmonary infection with SchuS4. Together, these data support the hypothesis that the absence or low abundance of CD14 on hDC and in the lung contributes to evasion of innate immunity by virulent F. tularensis. However, CD14 is not required for development of inflammation during the last 24 to 48 h of SchuS4 infection. Thus, the presence of this receptor may aid in control of virulent F. tularensis infections at early, but not late, stages of infection.Daily insults of inhaled particulate and foreign antigens into the lungs could result in devastating inflammation. However, the lung counters these attacks by tightly regulating inflammatory responses. This regulation occurs both in the form of inhibitory molecules, such as surfactants that dampen macrophage and dendritic cell (DC) responsiveness, as well as production of immunosuppressive cytokines, such as transforming growth factor β (TGF-β) (1, 10, 15, 33, 34). Given the immunosuppressive nature of the lung environment, it is not surprising that pathogens capable of causing lethal disease following inhalation, such as Francisella tularensis, take advantage of this property for rapid replication while evading detection by the host immune response.Francisella tularensis is a Gram-negative, facultative intracellular bacterium and is the causative agent of tularemia. Pneumonic tularemia is an acute, lethal disease mediated by F. tularensis, following inhalation of as few as 10 to 15 bacteria in mice and humans (20, 49). Surprisingly, despite the rapidity by which pulmonary F. tularensis infections progress, there is little to no evidence of inflammation in the lung until the very end stage of infection (11, 20, 54). The mechanisms by which F. tularensis replicates within the lung while evading detection by the host are not well understood and represent an important hurdle for the development of novel therapeutics and vaccines directed against tularemia.Recently we have demonstrated that, similar to pulmonary cells, conventional human dendritic cells (hDC) derived from peripheral blood fail to secrete proinflammatory cytokines following infection with virulent F. tularensis strain SchuS4 (17). The absence of production of proinflammatory cytokines was not due to the inability of the cells to become infected or support replication of strain SchuS4, nor was it due to induction of apoptosis among infected hDC. There are multiple explanations for the lack of cytokine production in hDC and pulmonary cells following SchuS4 infection. One possibility is that these cells fail to detect F. tularensis during the initial phases of infection.CD14 is a glycosylphosphatidylinositol receptor that exists in both a membrane-bound form and a soluble form in vivo. CD14 is present on monocytes, most macrophages, fibroblasts, and neutrophils (39). Conventional human DC lose surface expression of CD14 following their differentiation from blood monocytes, primarily due to exposure to interleukin-4 (IL-4) (40). Similar to hDC, alveolar macrophages have been reported to have little to no CD14 present on their surface (9, 45). Soluble CD14 (sCD14) is abundant in serum. However, it is present at very low levels to nearly undetectable concentrations in the airways of mammals (47). CD14 (both in its soluble and membrane-bound forms) is best known as a coreceptor for lipopolysaccharide (LPS), facilitating optimal delivery of LPS to the Toll-like receptor 4 (TLR4)/MD-2 complex on the cell surface (35, 64). In addition to delivery of LPS to TLR4, CD14 has been described as an important coreceptor for delivery of other microbial antigens, including polyuronic acids from Pseudomonas, lipoteichoic acid (LTA) from Staphylococcus aureus, outer surface protein of Borrelia burgdorferi and WI-1 antigen of Blastomyces dermatitidis to TLR2 (reviewed in reference 58). Thus, CD14 is an important coreceptor for initiating inflammatory responses via TLR2 and TLR4 against a wide variety of bacterial and fungal diseases.In this report, we demonstrate that CD14 serves as a critical coreceptor for detection of F. tularensis SchuS4 during the initial stage of in vitro infection in hDC and primary human monocytes. Further, CD14 was also found to have an important role in the early in vivo detection and control of pulmonary infections with strain SchuS4. However, development of inflammatory responses associated with bacterial sepsis observed at late stages of SchuS4 infection were not dependent on the presence of CD14. Thus, in contrast to infections with other microbial pathogens where the presence of CD14 in the lungs is detrimental to control of infection, CD14 represents an important sensor to initiate protective host immune responses during pulmonary infections with strain SchuS4, but it is not required to elicit responses at the end of the disease process.     

Strains of Actinomyces naeslundii and Actinomyces viscosus exhibit structurally variant fimbrial subunit proteins and bind to different peptide motifs in salivary proteins

Oral strains of Actinomyces spp. express type 1 fimbriae, which are composed of major FimP subunits, and bind preferentially to salivary acidic proline-rich proteins (APRPs) or to statherin. We have mapped genetic differences in the fimP subunit genes and the peptide recognition motifs within the host proteins associated with these differential binding specificities. The fimP genes were amplified by PCR from Actinomyces viscosus ATCC 19246, with preferential binding to statherin, and from Actinomyces naeslundii LY7, P-1-K, and B-1-K, with preferential binding to APRPs. The fimP gene from the statherin-binding strain 19246 is novel and has about 80% nucleotide and amino acid sequence identity to the highly conserved fimP genes of the APRP-binding strains (about 98 to 99% sequence identity). The novel FimP protein contains an amino-terminal signal peptide, randomly distributed single-amino-acid substitutions, and structurally different segments and ends with a cell wall-anchoring and a membrane-spanning region. When agarose beads with CNBr-linked host determinant-specific decapeptides were used, A. viscosus 19246 bound to the Thr42Phe43 terminus of statherin and A. naeslundii LY7 bound to the Pro149Gln150 termini of APRPs. Furthermore, while the APRP-binding A. naeslundii strains originate from the human mouth, A. viscosus strains isolated from the oral cavity of rat and hamster hosts showed preferential binding to statherin and contained the novel fimP gene. Thus, A. viscosus and A. naeslundii display structurally variant fimP genes whose protein products are likely to interact with different peptide motifs and to determine animal host tropism.     

In vivo and in vitro studies on possible pathogenic mechanisms of Actinomyces viscosus.

Actinomycotic infections are characterized by long-term inflammatory lesions containing large numbers of polymorphonuclear leukocytes (PMNs) and mononuclear cells. The pathogenic mechanisms involved in these lesions are not understood. Homogenates of Actinomyces viscosus (AVIS) induce an acute inflammatory response with a predominance of PMNs within 6 h after injection into the footpads of nonimmunized mice. These homogenates, when tested in vitro, contain potent chemotactic activity for human PMNs. In vitro chemotactic activity for human monocytes is weak but statistically significant (P less than 0.025). Doses of AVIS, which alone have little chemotactic activity, cause the generation of PMN chemotactic activity in fresh, but not complement-inactivated, serum. The injection of AVIS into the footpads of immunized mice induces an acute inflammatory response followed within 48 h by a mononuclear cell infiltrate, suggesting that factors affecting monocyte accumulation are generated by the immune host in response to challenge with the bacterial antigens. These findings indicate that the pathogenicity of the Actinomyces may result in part from (i) their direct chemotactic effect on PMNs, (ii) their cytotaxigenic effects on serum, and (iii) their ability to stimulate host immune cells to produce and release mediators of inflammation.     

Anti-septic Effects of Pellitorine in HMGB1-Induced Inflammatory Responses In Vitro and In Vivo

High mobility group box 1 (HMGB1) acts as a late mediator of vascular inflammatory conditions. Pellitorine (PT), an active amide compound from Asarum sieboldii, is known to possess antibacterial and anticancer properties. In this study, we investigated the anti-septic effects of PT against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) induced by HMGB1 and the associated signaling pathways. According to our findings, treatment with PT resulted in inhibited release of HMGB1, down-regulation of HMGB1-dependent inflammatory responses in HUVECs, and inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with PT resulted in reduced cecal ligation and puncture (CLP)-induced release of HMGB1 and sepsis-related mortality. PT suppressed the production of tumor necrosis factor-α and interleukin 6 and the activation of nuclear factor-κB and extracellular regulated kinases 1/2 by HMGB1. Collectively, these results indicate the potential of PT as a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.     

Diazepam Inhibits Phagocytosis and Killing Exerted by Polymorphonuclear Cells and Monocytes from Healthy Donors. In Vitro Studies

The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte pha gocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10^-5 and 10^-6 M concentrations/ 4 × 10⁶ phagocytes. 10^-7 M con centration was not effective in all the instances.

These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.     

Morphological features and functional properties of human fibroblasts exposed to Actinomyces viscosus substances.

Connective tissue fibroblasts undergo cytopathic degenerative changes during certain long-term inflammatory diseases such as rheumatoid arthritis and periodontitis. The failure of inflamed tissues to repair properly may result from functional alterations of fibroblasts within the affected tissues. Numerous previous studies indicate that direct cytotoxicity by bacterial or other substances may be responsible for the cellular alterations observed in vivo. We have tested this hypothesis by exposing cultures of human diploid fibroblasts to homogenates of Actinomyces viscosus (a microorganism associated with periodontitis and capable of causing other chronic inflammatory diseases) and analyzing the effects on cell viability, morphology, and function. The cells bind and subsequently engulf relatively large quantities of the bacterial substances. These substances do not appear to be toxic to fibroblasts as determined by 51Cr release and microcytotoxicity assays, although there is a slight but significant decrease in protein synthesis (P less than 0.01) as measured by the incorporation of [14C]proline. However, collagen production was not altered, and the cytopathic alterations observed in diseased tissues in vivo did not occur in the exposed cells. These findings suggest that A. viscosus substances do not directly cause injury to connective tissue fibroblasts in periodontal disease but may, through cell-surface binding, mark these cells for subsequent immune-mediated damage.     

Quantitative fluorescent immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus.

Optimal conditions for a fluorescence immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus ATCC 19246 are described. In the standard fluorescence immunoassay, 10(8) colony-forming units of A. viscosus reacted with an antibody preparation, were washed, and then were treated with an excess of fluorescein-conjugated goat anti-rabbit immunoglobulin G. After another set of washes, fluorescence was determined in a spectofluorometer; in most cases excitation was at 485 nm, with emission measured at 525 nm. These conditions minimized interference from light scatter and stray light. Under appropriate conditions, antibodies to A. viscosus could be readily determined, with the fluorescence of the specific antibody-treated cells more than five times the fluorescence of controls treated with normal rabbit serum. Organisms coated with specific antibody could be detected at levels approaching 10(5) colony-forming units per ml. The standard fluorescence immunoassay procedure was readily adapted to the measurement of either particulate or soluble surface antigens of A. viscosus by competition of the antigen with a fixed amount of antibody in the standard assay system; the competition resulted in an antigen dose-dependent inhibition of fluorescence. The fluorescent immunoassay system thus appears to be a general one that could be applied to other microbial systems as well.     

In vivo biocompatibility studies. VII. Inflammatory response to polyethylene and to a cytotoxic polyvinylchloride

The cellular biocompatibility of low-density polyethylene and a cytotoxic polyvinylchloride were investigated using an in vivo cage implant system. Components of the inflammatory response (white cells, extra-cellular alkaline and acid phosphatase, the complement component C3, and total protein content) were monitored over a 21-day implantation period. Scanning electron microscopy was used to evaluate the morphologic condition of leukocytes adherent to the implanted polymers. Prior to implantation, each polymer was evaluated using an established primary acute toxicity screen. The results showed that the cytotoxic polyvinylchloride stimulated an intense acute phase inflammatory response, and at later observation periods, an intense and increasing chronic inflammatory response. In contrast, the polyethylene promoted relatively small increases in the acute and chronic phases of inflammation; the overall cellular response being essentially resolved by the third week after implantation. The initial toxicity screen of each polymer suggested that the observed differences in inflammation were primarily caused by the release from the polyvinylchloride of the added cytotoxic agent (dioctyltinbisoctylmercaptoacetate).     

Interaction of inflammatory cells and oral microorganisms. III. Modulation of rabbit polymorphonuclear leukocyte hydrolase release response to Actinomyces viscosus and Streptococcus mutans by immunoglobulins and complement.

In the absence of antiserum, rabbit polymorphonuclear leukocytes (PMNs) released lysosomal enzymes in response to Actinomyces viscosus (19246) but not to Streptococcus mutans (6715). Antibodies had a marked modulating influence on these reactions. PMN hydrolase release was significantly enhanced to both organisms when specific rabbit antiserum and isolated immunoglobulin G (IgG) were included in the incubations. Immune complex F(ab')2 fragments of IgG directed against S. mutans agglutinated bacteria. Immune complexes consisting of S. mutans and F(ab')2 fragments of IgG directed against this organism were not effective as bacteria-IgG complexes in stimulating PMN release. The intensity of the release response to bacteria-IgG complexes was also diminished when PMNs were preincubated with isolated Fc fragments derived from IgG. Fresh serum as a source of complement components had no demonstrable effect on PMN release either alone or in conjuction with antiserum in these experiments. These data may be relevant to the mechanisms and consequences of the interaction of PMNs and plaque bacteria in the pathogenesis of periodontal disease.     

Interaction of Human Polymorphonuclear Leukocyte (PMN) Elastase with Human 1gM. Production of a Factor Enhancing PMN Migration In Vitro

Digestion in vitro of human monoclonal 1gM by human polymorphonuclear leukocyte (PMN) elastase produces small peptides in addition to larger fragments. These small peptides were shown to enhance PMN locomotion in vitro, as tested by the migration-under-agarose assay. The small peptides from elastase degradation of the Fc5u portion of the IgM. nolecule also increase PMN migration in vitro. The migration-enhancing factor is stable at 60°C for 20 minutes but is inactivated by boiling for :0 minutes, and has a molecular weight which falls between 1,000 and 10,000 daltons.     

Oral Immunization in Experimental Salmonellosis III. Behavior of Virulent and Temperature-Sensitive Mutant Strains in the Intestinal Tissues of Rats

Infection of rats via a Peyer's patch has been used as a means of studying the behavior of Salmonella enteritidis in intestinal tissues. The course of infection in the Peyer's patch and draining mesenteric lymph node is characterized by multiplication of the organism over a period of 4 days followed by a gradual decline in numbers; the organism also passes to the liver and spleen and may be isolated from these organs as well as the intestinal tissues for at least 4 weeks. Temperature-sensitive mutants derived from the virulent strain are unable to multiply and do not pass to the liver and spleen; they remain viable for periods of less than 2 weeks. A quantitative technique based on the number of viable organisms remaining in the injected Peyer's patch 48 hr after infection has been used to assess the immune state of rats. The results have clearly demonstrated that Salmonella immunity can only be induced by living vaccines and that although viable organisms remain in the reticuloendothelial tissues, organisms given in a challenge infection are immediately subject to enhanced bactericidal activity within the intestinal tissues. Under the conditions used here, humoral antibody does not seem to offer any protective effect against Salmonella infection in the intestinal lymphoid tissues.     

Construction and use of integration plasmids to generate site-specific mutations in the Actinomyces viscosus T14V chromosome.

Stable transformants of Actinomyces viscosus T14V carrying heterologous DNA were obtained with the aid of integration plasmids. These plasmids contained a kanamycin resistance (Kmr) gene flanked by A. viscosus T14V genomic DNA, including parts of the type 1 structural fimbrial subunit gene (fimP) on one or both sides of the antibiotic marker. Significantly more Kmr transformants were obtained with a plasmid carrying longer segments of homologous strain T14V DNA. Integration of this plasmid into the A. viscosus T14V genome affected the expression and function of type 1 fimbriae in the transformants. In the transformant strain designated A. viscosus MY50D, the inactivated fimP replaced the wild-type fimP via allelic replacement. A. viscosus MY51S and MY52S each contained a copy of the plasmid integrated into the genome by a Campbell-like insertion mechanism. A. viscosus MY50D and MY51S lacked type 1 fimbriae and did not bind to proline-rich proteins (the fimbrial receptors) immobilized on nitrocellulose. In contrast, strain MY52S synthesized the structural subunit protein, as detected by immunostaining with anti-A. viscosus T14V type 1 fimbria antibodies. However, the high-molecular-weight proteins observed in sodium dodecyl sulfate-polyacrylamide gels of fimbriae from the cell wall of the wild-type strain T14V were absent in cell wall preparations of this strain. Moreover, A. viscosus MY52S failed to bind, in vitro, to proline-rich proteins. Thus, these results demonstrate that insertion of heterologous DNA at specific sites of the Actinomyces genome can be facilitated with integratable plasmids and that the transformants and mutants generated will aid in the delineation of the roles and contributions of specific genes to the structure and function of any macromolecule produced by these organisms.     

Human salivary acidic proline-rich proteins and statherin promote the attachment of Actinomyces viscosus LY7 to apatitic surfaces.

Actinomyces viscosus LY7 cells adsorbed in high numbers to experimental pellicles formed on hydroxyapatite (HA) from human parotid or submandibular saliva but not to pellicles prepared from human plasma or serum. To determine the nature of the salivary components responsible for promoting adhesion, pellicles were prepared from fractions of submandibular and parotid saliva obtained by chromatography on Trisacryl GF 2000 columns. Adsorption of LY7 cells was promoted by two groups of fractions. Each group was rechromatographed on DEAE-agarose. Fractions which promoted adsorption of LY7 cells were found by polyacrylamide gel electrophoresis to contain the acidic proline-rich proteins (PRPs) and statherin. Pellicles prepared from 12-micrograms/ml solutions of pure PRP-1, PRP-2, or parotid isoelectric focusing (PIF-slow) variant promoted maximal adsorption of A. viscosus LY7 cells. Somewhat higher concentrations of PRP-3 and PRP-4 were required for maximal adsorption, indicating that the 44-residue carboxy-terminal segment of PRP-1, PRP-2, and PIF-slow enhances LY7 binding but is not essential. Much higher concentrations of statherin were required to promote LY7 adsorption. Adsorption of LY7 cells to pellicles prepared from PRP-1 was not affected over the range of pH 5 to 8. Adsorption was also not inhibited by 50 mM lactose, which is consistent with the notion that type 1 fimbriae, rather than type 2 fimbriae, were responsible. A. viscosus T14, Actinomyces odontolyticus ATCC 17982, and Actinomyces israelii 12597 also adsorbed to PRP-1 pellicles, whereas Actinomyces naeslundii ATCC 12104 did not. Although A. viscosus cells bind strongly to adsorbed PRP-1, the presence of PRP-1 or PRP-3 in solution did not inhibit adhesion. Similarly, [3H]PRP-1 did not bind to LY7 cells, nor was it degraded when incubated with the organism. However, LY7 cells adsorbed to [3H]PRP-1 pellicles. These data suggest that hidden molecular segments of PRP become exposed when the protein adsorbs to HA; these segments then react with adhesins of LY7 cells. The apparent ability of A. viscosus cells to recognize segments of PRPs which are exposed only in surface-adsorbed molecules provides a novel mechanism which enables the organism to attach to teeth when suspended in salivary secretions.     

In Vitro Effect of an Acute Nonspecific Inflammatory Exudate on Tritiated-Thymidine Incorporation by Unstimulated and Pha-Stimulated Spleen Cells

The effect of an acute nonspecific inflammatory exudate with mitogenic activity for macrophages in culture has been tested on the spontaneous and PHA-induced DNA synthesis by spleen cells in vitro.

Stimulatory effect of this exudate was observed on the spontaneous DNA synthesis which was detectable over a range of 1:4 to 1:4096 concentrations. After optimal PHA stimulation, an inhibition of mitogen-induced DNA synthesis was observed when the cells were exposed to the highest concentrations (up to 1:128) of the exudate. Thereafter, the phenomenon could be reversed and the stimulation was maximal at the concentration of 1:2048. When a suboptimal dose of PHA was used, the stimulatory effect was more pronounced and detected from 1:8 up to 1:4096 concentrations.     

Characterization of the Interaction between Yersinia enterocolitica Biotype 1A and Phagocytes and Epithelial Cells In Vitro

Yersinia enterocolitica strains of biotype 1A are increasingly being recognized as etiological agents of gastroenteritis. However, the mechanisms by which these bacteria cause disease differ from those of highly invasive, virulence plasmid-bearing Y. enterocolitica strains and are poorly understood. We have investigated several biotype 1A strains of diverse origin for their ability to resist killing by professional phagocytes. All strains were rapidly killed by polymorphonuclear leukocytes but persisted within macrophages (activated with gamma interferon) to a significantly greater extent (survival = 40.5% +/- 17.4%) than did Escherichia coli HB101 (9.3% +/- 0.7%; P = 0.0001). Strains isolated from symptomatic patients were significantly more resistant to killing by macrophages (survival = 48.9% +/- 19.5%) than were strains obtained from food or the environment (survival = 32.1% +/- 10.3%; P = 0.04). Some strains which had been ingested by macrophages or HEp-2 epithelial cells showed a tendency to reemerge into the tissue culture medium over a period lasting several hours. This phenomenon, which we termed "escape," was observed in 14 of 15 strains of clinical origin but in only 3 of 12 nonclinical isolates (P = 0.001). The capacity of bacteria to escape from cells was not directly related to their invasive ability. To determine if escape was due to host cell lysis, we used a variety of techniques, including lactate dehydrogenase release, trypan blue exclusion, and examination of infected cells by light and electron microscopy, to measure cell viability and lysis. These studies established that biotype 1A Y. enterocolitica strains were able to escape from macrophages or epithelial cells without causing detectable cytolysis, suggesting that escape was achieved by a process resembling exocytosis. The observations that biotype 1A Y. enterocolitica strains of clinical origin are significantly more resistant to killing by macrophages and significantly more likely to escape from host cells than are strains of nonclinical origin suggest that these properties may account for the virulence of these bacteria.     

In Vitro Susceptibility to Gemifloxacin and Trovafloxacin of Streptococcus pneumoniae Strains Exhibiting Decreased Susceptibility to Ciprofloxacin

 The in vitro susceptibility to trovafloxacin and gemifloxacin of Streptococcus pneumoniae strains exhibiting decreased susceptibility to ciprofloxacin (MIC ≥2 μg/ml; 30 strains with intermediate resistance [MIC 2 μg/ml] and 43 strains with complete resistance [MIC ≥4 μg/ml]) was determined. Seventy-three strains collected in a surveillance study carried out from May 1996 to April 1997 in Spain (prior to commercialisation of trovafloxacin and gemifloxacin) from patients with respiratory tract infections were tested. The antibacterial activity of gemifloxacin was affected to a lesser extent than that of trovafloxacin by the increase in the MIC of ciprofloxacin, with gemifloxacin showing significantly (P≤0.001) better antibacterial activity than trovafloxacin in all ciprofloxacin MIC categories (MIC50/MIC90 values of 0.015/0.03, 0.015/0.06, 0.03/0.06 and 0.12/0.25 μg/ml for gemifloxacin vs 0.12/0.12, 0.12/1, 0.25/0.5 and 2/4 μg/ml for trovafloxacin in the 2, 4, 8 and ≥16 μg/ml ciprofloxacin MIC categories, respectively). Nine (12.3%) of these 73 strains exhibited decreased susceptibility to trovafloxacin (≥2 μg/ml), whereas all strains were inhibited by 0.25 μg/ml of gemifloxacin.     

Differentiation of Human Embryonic Stem Cells to Cardiomyocytes for In Vitro and In Vivo Applications

The ability of human embryonic stem cells to differentiate into spontaneously contracting cardiomyocyte-like cells has attracted substantial interest from the scientific community over the last decade. From having been difficult to control, human cardiomyogenesis in vitro is now becoming a process which, to a certain extent, can be effectively manipulated and directed. Although much research remains, new and improved protocols for guiding pluripotent stem cells to the cardiomyocyte lineage are accumulating in the scientific literature. However, the stem cell derived cardiomyocytes described to date, generally resemble immature embryonic/fetal cardiomyocytes, and they are in some functional and structural aspects different from adult cardiomyocytes. Thus, a future challenge will be to design strategies that eventually may allow the cells to reach a higher degree of maturation in vitro. Nevertheless, the cells which can be prepared using current protocols still have wide spread utility, and they have begun to find their way into the drug discovery platforms used in the pharmaceutical industry. In addition, stem cell derived cardiomyocytes and cardiac progenitors are anticipated to have a tremendous impact on how heart disease will be treated in the future. Here, we will discuss recent strategies for the generation of cardiomyocytes from human embryonic stem cells and recapitulate their features, as well as highlight some in vitro applications for the cells. Finally, opportunities in the area of cardiac regenerative medicine will be illustrated.     

Cytokine Responses to Parasite Antigens: In Vitro Cytokine Production to Promastigotes of L. aethiopica by Cells from Non-Leishmania Exposed Donors may Influence Disease Establishment

The various cytokine responses associated with stimulation by parasites is discussed with emphasis on Leishmania parasites.
Cells from normal individuals can respond to Leishmania antigen in vitro but the State of the antigen used for stimulation influences the outcome. We have used cells from non- Leishmania exposed donors and stimulated them in vitro with variously treated promastigotes of L. aethiopica. The levels of some cytokines released into the supernatant were measured. All the Leishmania preparations tested induced high levels of IL-6. whereas IFN-_γ production to the different stimuli was variable in the individual donors. The ability of these supernatants to inhibit intracellular forms of L. aethiopica was sometimes stronger in L. aethiopica -induced than in PHA-induced cultures. Such strong non- Leishmania specific responses, if they exist in vivo, may influence whether disease is established when the host encounters Leishmania parasites.