Effects of various resin composite (co)monomers and extracts on two caries-associated micro-organisms in vitro.

Previous studies have shown that extracts of various filling materials, e.g., resin composites, may influence the growth of cariogenic micro-organisms. The purpose of this study was to examine the effects of important resin composite (co)monomers (Bis-GMA, UDMA, TEGDMA, EGDMA) and extracts of two commercial dental composites with similar composition (composite A, Arabesk; composite S, Superlux) on the growth of the two cariogenic bacterial pathogens Streptococcus sobrinus and Lactobacillus acidophilus. It was found that neither the monomers Bis-GMA and UDMA, nor the comonomer EGDMA, nor the extract of composite A influenced the growth of S. sobrinus in the log phase. The comonomer TEGDMA and the extract of composite S were found to stimulate growth in the log phase, but this stimulation was not statistically significant. However, EGDMA, TEGDMA, and the extract of composite S did stimulate the total growth of S. sobrinus. In the assays with L. acidophilus, Bis-GMA, UDMA, and the extract of composite A inhibited the growth in the log phase, whereas TEGDMA stimulated it. Furthermore, EGDMA, TEGDMA, and the extract of composite S stimulated the biomass production of L. acidophilus. We conclude from our results that a release of EGDMA and TEGDMA from resin composites should be avoided due to their growth-stimulating effects on the caries-associated micro-organisms S. sobrinus and L. acidophilus.

Response of germfree rat colonic mucous cells to peroral endotoxin application.

During microbial colonization, mucin-releasing goblet cells of germ-free (GF) rats proliferate and upregulate their mucin synthesis, thus improving the intestinal mucus barrier. The present study determined the significance of bacterial membrane constituents for this development. A single dose of lipopolysaccharide (LPS) (35 micrograms/100 g body weight) and lipid A (3.5 micrograms/100 g body weight, respectively), was perorally administered to GF AS/Ztm rats. One, 3 and 5 days later, sections of the proximal and distal colon served for characterization of mucin-secreting goblet cells, released mucins were isolated in parallel. Maximal goblet cell diameters were evidenced at day 3. LPS generated a maximal goblet cell hyperplasia one day after challenge, lipid A stimulated the goblet cell proliferation continuously up to day 5. Three days after challenge with one of the stimuli, either, intracellular mucins had shifted significantly to neutral constituents. In addition, mucins, adherent to the colon mucosa and submerged to the luminal content, respectively, then were augmented. At day 5, adherent mucins were similar to the controls, while luminal, soluble constituents had further increased. Histometrical and biochemical methods evidenced a transient, inflammatory response of mucin-secreting cells, followed by an upregulated release of immature mucins.

Interaction between Helicobacter pylori and human gastric epithelial cells in culture: effect of antiulcer drugs.

A human in vitro model to study the interaction between Helicobacter pylori and gastric epithelial cells was developed using primary cultures of gastric mucosal cells (isolated from gastric biopsies or operative specimen and maintained in culture for 2 weeks) as well as the well-differentiated human gastric carcinoma cell line HM02, the undifferentiated gastric tumour cell line HM51, and the laryngeal epithelial cell line HEp-2. Primary cultures and all cell lines were exposed to seven isolates of H. pylori isolated from gastritis and duodenal ulcer patients. Microbial adherence was assessed by microscopical evaluation of Giemsa-stained preparations and by culturing the viable bacteria attached to the epithelial cells. All H. pylori isolates adhered to the gastric cells in primary culture, to HM02 cells, and to HEp-2 cells with the greatest binding affinity found in primary gastric cells. No adherence was detected in HM51 cells. H. pylori adherence was dependent on bacterial load, incubation time, and temperature. There was no difference in microbial binding between H. pylori isolates derived from gastritis and duodenal ulcer patients. The effect of antiulcer drugs on H. pylori adherence was investigated by pre-incubating isolates of H. pylori with omeprazole, cimetidine, and bismuth subcitrate. Omeprazole and cimetidine failed to significantly influence microbial adherence. In contrast, bismuth subcitrate already in concentrations below the MIC range decreased H. pylori adherence in gastric epithelial cells and in HEp-2 cells substantially. Our study shows that primary cultured human gastric mucosal cells and the human gastric carcinoma cell line HM02 provide suitable in vitro models for the study of the interactions between H. pylori and the gastric epithelium. This gastric cell model is characterized by a high affinity for H. pylori binding.

Selected cryopreservatives for long term storage of Helicobacter pylori at low temperatures.

To meet the need for information on cryopreservation, a study was done on 32 Helicobacter pylori strains, comparing different cryopreservative media. Sheep blood, horse blood, horse serum with and without glycerol, and mineral oil media were used for long term storage of H pylori at -70 degrees C or in liquid nitrogen. Procedures were developed which permitted recovery of 87.5% of the strains included in the study after they had been stored for 24 months. Of those strains stored for more than three years, 60% were recovered. It is concluded that most strains of H pylori can be stored for up to one year or longer, under refrigeration, at -70 degrees C or in liquid nitrogen.

Immunopathophysiology of gastrointestinal disease in HIV infection.

In summary, gastrointestinal manifestations are a major complication of HIV infection. These manifestations present clinically as early, intermediate, and late-phase enteric illness due to HIV itself or the acquisition of opportunistic infections and neoplasms. HIV-induced defects in lymphocytes and mononuclear phagocytes, which circulate through and populate the gastrointestinal mucosa, and a sequence of HIV-dependent alterations in mucosal integrity represent the fundamental immunopathophysiologic basis for these clinical manifestations.

Surface proteins from Helicobacter pylori exhibit chemotactic activity for human leukocytes and are present in gastric mucosa.

The mechanism by which Helicobacter pylori, a noninvasive bacterium, initiates chronic antral gastritis in humans is unknown. We now show that H. pylori releases products with chemotactic activity for monocytes and neutrophils. This chemotactic activity was inhibited by antisera to either H. pylori whole bacteria or H. pylori-derived urease. Moreover, surface proteins extracted from H. pylori and purified H. pylori urease (a major component of the surface proteins) exhibited dose-dependent, antibody-inhibitable chemotactic activity. In addition, a synthetic 20-amino acid peptide from the NH2-terminal portion of the 61-kD subunit, but not the 30-kD subunit, of urease exhibited chemotactic activity for monocytes and neutrophils, localizing the chemotactic activity, at least in part, to the NH2 terminus of the 61-kD subunit of urease. The ability of leukocytes to chemotax to H. pylori surface proteins despite formyl-methionyl-leucyl-phenylalanine (FMLP) receptor saturation, selective inhibition of FMLP-mediated chemotaxis, or preincubation of the surface proteins with antiserum to FMLP indicated that the chemotaxis was not FMLP mediated. Finally, we identified H. pylori surface proteins and urease in the lamina propria of gastric antra from patients with H. pylori-associated gastritis but not from uninfected subjects. These findings suggest that H. pylori gastritis is initiated by mucosal absorption of urease, which expresses chemotactic activity for leukocytes by a mechanism not involving N-formylated oligopeptides.

Evaluation of liquid media for growth of Helicobacter pylori.

Helicobacter pylori has routinely been isolated and grown on solid media. Recently, we have succeeded in obtaining growth of this organism in several liquid media in large volumes, including tryptic soy broth, Mueller-Hinton broth, brucella broth, brain heart infusion broth, and Columbia broth. Growth was tested in the media with and without supplementation. Growth was obtained after incubation under microaerobic conditions and with CO2 enrichment. Growth in a stationary system versus that in an agitated system was evaluated. Results from these experiments show that H. pylori can be grown in any of the liquid media tested except buffered yeast extract-alpha-ketoglutarate if serum is added. No growth was observed on buffered yeast extract-alpha-ketoglutarate even with serum and other supplementation. Growth of H. pylori in most of the liquid media with supplements was improved if the culture was incubated in a CO2 atmosphere. The findings reported here may be useful in clinical, industrial, and research laboratories that require harvests of large quantities of H. pylori cells.

Type I transforming growth factor-beta receptors on neutrophils mediate chemotaxis to transforming growth factor-beta.

Participation of human polymorphonuclear neutrophils in the inflammatory response is mediated, in part, by soluble factors such as chemotactic peptides and cytokines. Although the cytokine, transforming growth factor beta (TGF-beta), has been shown to recruit monocytes and promote the inflammatory process, its effects on neutrophils are unknown. In this investigation, [125I]TGF-beta 1 affinity binding studies were employed to show that neutrophils express TGF-beta receptors (350 +/- 20 receptors/cell), which exhibit high affinity for the ligand (dissociation constant, 50 pM). Affinity cross-linking studies identified the receptors to be primarily of the type I class. In contrast to the receptors on monocytes, neutrophil TGF-beta receptors were not down-regulated by exposure to specific inflammatory mediators. Additional studies examined whether exposure of neutrophils to TGF-beta could enhance specific functions, as occurs with monocytes. TGF-beta was shown to cause directed migration of neutrophils at femtomolar concentrations, thus it is the most potent neutrophil chemotactic factor yet identified. Neutrophil production of reactive oxygen intermediates was not stimulated by TGF-beta, nor did TGF-beta enhance or depress subsequent PMA- or FMLP-stimulated superoxide production. However, the stable expression of neutrophil TGF-beta receptors, and the capacity of this cytokine to stimulate neutrophil chemotaxis, suggest that the pro-inflammatory effects of TGF-beta are mediated by neutrophils in addition to monocytes.

Macrophage- and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome.

The multifunctional cytokine, transforming growth factor beta (TGF-beta), was identified by immunocytochemistry in the brain tissues of four patients with acquired immune deficiency syndrome (AIDS), but not in control brain tissue. The TGF-beta staining was localized to cells of monocytic lineage as well as astrocytes, especially in areas of brain pathology. In addition, the brain tissues from the AIDS patients contained transcripts for human immunodeficiency virus 1 (HIV-1) by in situ hybridization, suggesting a correlation between the presence of HIV-1 in the brain and the expression of TGF-beta. However, the expression of TGF-beta was not limited to HIV-1-positive cells, raising the possibility of alternative mechanisms for the induction of TGF-beta in these AIDS patients' brains. To investigate these mechanisms, purified human monocytes were infected in vitro with HIV-1 and were shown to secrete increased levels of TGF-beta. In addition, HIV-1-infected monocytes released a factor(s) capable of triggering cultured astrocytes that are not infected with HIV-1 to secrete TGF-beta. The release of TGF-beta, which is an extremely potent chemotactic factor, may contribute to the recruitment of HIV-1-infected monocytic cells, enabling viral spread to and within the central nervous system (CNS). Moreover, TGF-beta augments cytokine production, including cytokines known to be neurotoxic. The identification of TGF-beta within the CNS implicates this cytokine in the immunopathologic processes responsible for AIDS-related CNS dysfunction.

Soluble surface proteins from Helicobacter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism.

The inflammatory lesions associated with Helicobacter pylori gastritis and duodenitis contain large numbers of mononuclear cells. The close proximity of H. pylori to gastric mucosa suggests that the organism interacts with mononuclear cells, thereby modulating the inflammatory response. To investigate the role of monocytes/macrophages in this response, we examined the effect of whole H. pylori bacteria, H. pylori surface proteins, and H. pylori lipopolysaccharide (LPS) on purified human monocytes. Whole H. pylori and the extracted LPS induced expression of the monocyte surface antigen HLA-DR and interleukin-2 receptors, production of the inflammatory cytokines interleukin 1 and tumor necrosis factor (peptide and messenger RNA), and secretion of the reactive oxygen intermediate superoxide anion. Since H. pylori in vivo does not invade mucosal tissue, we determined whether soluble constituents of the bacteria could activate monocytes. Soluble H. pylori surface proteins, which are enriched for urease and do not contain LPS, stimulated phenotypic, transcriptional, and functional changes consistent with highly activated monocytes. These findings indicate that H. pylori is capable of activating human monocytes by an LPS-independent as well as an LPS-dependent mechanism. H. pylori activation of resident lamina propria macrophages and monocytes trafficking through the mucosa, leading to the secretion of increased amounts of inflammatory cytokines and reactive oxygen intermediates, could play an important role in mediating the inflammatory response associated with H. pylori gastritis and duodenitis.