Isolation of alpha-toxin-producing Staphylococcus aureus from the skin of highly sensitized adult patients with severe atopic dermatitis.

BACKGROUND: Staphylococcus aureus (S. aureus) is a well-known trigger factor of atopic dermatitis (AD). Besides staphylococcal superantigens, alpha-toxin may influence cutaneous inflammation via induction of T-cell proliferation and cytokine secretion. OBJECTIVES: To investigate the association between sensitization to inhalant allergens and skin colonization with alpha-toxin-producing S. aureus in AD. PATIENTS AND METHODS: We investigated 127 patients with AD, aged 14-65 years, who were on standard anti-inflammatory and antiseptic treatment before investigation. We evaluated skin colonization, medical history, severity of AD and sensitization to inhalant allergens. RESULTS: Forty-eight of 127 patients were colonized with S. aureus, suffered from more severe AD, had asthma more often and showed higher sensitization levels to inhalant allergens. Thirty of 48 patients with S. aureus skin-colonizing strains produced alpha-toxin and had higher total IgE and specific IgE to birch pollen and timothy grass pollen. CONCLUSIONS: Under topical treatment with antiseptic and anti-inflammatory agents the colonization of lesional skin with S. aureus was clearly lower than commonly found in untreated patients with AD. Colonization with S. aureus was associated with a higher severity of AD, higher degree of sensitization, and a higher frequency of asthma. The proportion of patients whose skin was colonized with alpha-toxin-producing S. aureus was higher than expected from a former study. Cutaneous colonization with alpha-toxin-producing S. aureus was associated with a higher sensitization level to birch pollen allergen in AD. This may point to a higher susceptibility of patients with higher T-helper 2 polarization towards alpha-toxin-producing S. aureus.

DNA overwinds when stretched.

DNA is often modelled as an isotropic rod, but its chiral structure suggests the possible importance of anisotropic mechanical properties, including coupling between twisting and stretching degrees of freedom. Simple physical intuition predicts that DNA should unwind under tension, as it is pulled towards a denatured structure. We used rotor bead tracking to directly measure twist-stretch coupling in single DNA molecules. Here we show that for small distortions, contrary to intuition, DNA overwinds under tension, reaching a maximum twist at a tension of approximately 30 pN. As tension is increased above this critical value, the DNA begins to unwind. The observed twist-stretch coupling predicts that DNA should also lengthen when overwound under constant tension, an effect that we quantitatively confirm. We present a simple model that explains these unusual mechanical properties, and also suggests a possible origin for the anomalously large torsional rigidity of DNA. Our results have implications for the action of DNA-binding proteins that must stretch and twist DNA to compensate for variability in the lengths of their binding sites. The requisite coupled DNA distortions are favoured by the intrinsic mechanical properties of the double helix reported here.

Alpha-toxin is produced by skin colonizing Staphylococcus aureus and induces a T helper type 1 response in atopic dermatitis.

BACKGROUND: Staphylococcus aureus is a well known trigger factor of atopic dermatitis (AD). Besides the superantigens, further exotoxins are produced by S. aureus and may have an influence on the eczema. OBJECTIVE: To explore the impact of staphylococcal alpha-toxin on human T cells, as those represent the majority of skin infiltrating cells in AD. METHODS: Adult patients with AD were screened for cutaneous colonization with alpha-toxin producing S. aureus. As alpha-toxin may induce necrosis, CD4(+) T cells were incubated with sublytic alpha-toxin concentrations. Proliferation and up-regulation of IFN-gamma on the mRNA and the protein level were assessed. The induction of t-bet translocation in CD4(+) T cells was detected with the Electrophoretic Mobility Shift Assay. RESULTS: Thirty-four percent of the patients were colonized with alpha-toxin producing S. aureus and alpha-toxin was detected in lesional skin of these patients by immunohistochemistry. Sublytic alpha-toxin concentrations induced a marked proliferation of isolated CD4(+) T cells. Microarray analysis indicated that alpha-toxin induced particularly high amounts of IFN-gamma transcripts. Up-regulation of IFN-gamma was confirmed both on the mRNA and the protein level. Stimulation of CD4(+) T cells with alpha-toxin resulted in DNA binding of t-bet, known as a key transcription factor involved into primary T helper type 1 (Th1) commitment. CONCLUSION: alpha-toxin is produced by S. aureus isolated from patients with AD. We show here for the first time that sublytic alpha-toxin concentrations activate T cells in the absence of antigen-presenting cells. Our results indicate that alpha-toxin is relevant for the induction of a Th1 like cytokine response. In AD, this facilitates the development of Th1 cell dominated chronic eczema.

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.

Use of autoradiography to assess viability of Helicobacter pylori in water.

Autoradiographic methods have been developed to detect metabolic activity of viable but nonculturable cells of Helicobacter pylori in water. Four strains of H. pylori were studied by using microcosms containing suspensions of 72-h cultures in water. The suspensions of aged, nonculturable cells of H. pylori were incubated with [3H]thymidine for 24 to 72 h, after which the cell suspensions were exposed to Kodak NTB2 emulsion for 3 to 28 days. Each sample was processed with three separate controls to rule out false-positive reactions. The organism remains viable and culturable under these conditions for up to 48 h and, in some cases, 20 to 30 days, depending on physical conditions of the environment. We found that temperature was a significant (P < or equal to 0.01) environmental factor associated with the viability of H. pylori cells in water. Autoradiographs of tritium-labeled cells of H. pylori revealed aggregations of silver grains associated with uptake by H. pylori of radiolabelled substrate. Findings based on the autoradiographic approach give strong evidence supporting the hypothesis that there is a waterborne route of infection for H. pylori. The possibility that H. pylori may persist in water in a metabolically active stage but not actively growing and dividing is intriguing and relevant to public health concerns.

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.

[Serodiagnosis of Campylobacter pylori]. / Die Serodiagnostik von Campylobacter pylori.

The colonization of gastric mucosa with Campylobacter pylori can be detected by serological methods. ELISA and immunoblot methods are currently being employed for detection of antibodies against Campylobacter pylori. In general, both tests will differentiate between Campylobacter pylori positive and Campylobacter pylori negative patients. However, 5-10% of persons with negative cultures for Campylobacter pylori have positive serological tests, but only very few patients with Campylobacter pylori associated with chronic gastritis have negative serological tests. This is true for tests detecting IgG and IgA antibodies. Tests for IgM antibodies have not been found to be useful. Immunoblot analyses have shown that detection of antibodies against a 100-120 KD antigen has a high specificity for Campylobacter pylori infection. In a small study we evaluated the possible use of serological testing for follow-up studies on patients after Campylobacter pylori therapy. We found that patients who became Campylobacter pylori negative after therapy showed a significant decline of serum IgG titers against Campylobacter pylori.

[Campylobacter pylori: clinical correlations and prospective comparative studies of various diagnostic techniques]. / Campylobacter pylori: Klinische Korrelationen und prospektive vergleichende Untersuchungen zur Wertigkeit mehrerer Testverfahren.

The diagnostic performance of two different urease tests and of histologic search after modified Giemsa staining to detect Campylobacter pylori (CP) colonization of the upper gastrointestinal tract was prospectively investigated in 215 esophagogastroduodenoscopies, by using a sensitive culture technique as reference. Single antral urease tests had a high specificity of 95-96%, but a limited sensitivity of 78-83%, which increased to 91-94%, when one antral and one additional body biopsy were submitted to the biochemical tests. Giemsa stains were very sensitive, but less specific. The rate of colonization was similar in antrum and body biopsies, and increased with age. There was a close association of Campylobacter pylori colonization with duodenal and to a lower degree with gastric ulcer disease, but especially with gastritic mucosal changes. CP was never detected in patients without gastritis. Therefore, submitting one antral and one body biopsy specimen to validated urease tests represents a sensitive (91-94%) and specific (93%) method to detect Campylobacter pylori colonization, which appears to be a diffuse phenomenon affecting antral and body mucosae with similar frequency.

Ultrastructure and chemical analysis of Campylobacter pylori flagella.

Flagella of Campylobacter pylori were analyzed by electron microscopy and purified, and the molecular weight of the flagellin was determined. Isolation of flagella was performed by mechanical shearing from the cell surface, sucrose density gradient centrifugation, and Sepharose CL-4B gel chromatography. The flagella of C. pylori differ from those of other Campylobacter species and of most other bacteria by the presence of a flagellar sheath. The sheath narrows at the end and is linked to a club-shaped terminal structure. The molecular weight of C. pylori flagellin was 51,000.

Are dyspeptic symptoms in patients with Campylobacter pylori-associated type B gastritis linked to delayed gastric emptying?

In this paper, the association of gastric Campylobacter pylori (CP) colonization with histologic findings and gastric emptying of a mixed solid-liquid meal was prospectively investigated in 43 consecutive patients with essential non-ulcer dyspepsia (ENUD). Gastric emptying was also measured in 30 symptom-free control subjects. The frequency of CP infection in patients with ENUD was 44.2%. We found a strong association between gastric CP colonization and chronic antral type B gastritis. Although gastric emptying was significantly prolonged in patients with ENUD, compared with the control group (p less than 0.05), we could not detect significant differences of gastric emptying between CP-negative and CP-positive ENUD patients, both groups disclosing a similar proportion of patients with significantly delayed gastric emptying (29.2% vs 31.6%). We therefore conclude that delayed gastric emptying of a mixed solid-liquid meal is not correlated with CP-positive chronic antral type B gastritis, and does not help to explain dyspeptic symptoms, which may possibly arise in relation to gastric CP colonization.

[Microbiology of Campylobacter pylori]. / Zur Mikrobiologie von Campylobacter pylori.

Campylobacter pylori is a hitherto unknown spiral bacterium. It is implicated in the pathogenesis of chronic antral type B gastritis and also of duodenal and gastric ulcer disease. However, its causal role has not yet been unequivocally established. Taxonomically, C.pylori appears to belong to a genus different from genuine campylobacters, but a precise classification is likewise lacking. The in vitro sensitivity analyses of various antibiotics are complicated by a number of technical difficulties. This is one of the reasons, that results of in vivo eradication trials have to be interpreted with considerable caution.