Toll-like receptor activation by sino-nasal mucus in chronic rhinosinusitis.

BACKGROUND: The sino-nasal disease chronic rhinosinusitis (CRS) is primarily an inflammatory condition that manifests in several ways. However, the aetiology of this complex disease is poorly understood. The aim of this study was to explore the association between toll-like receptor (TLR) activation, host immune response and sino-nasal mucus in healthy and diseased patients. METHODS: The activation of TLR2/1 and TLR4 by sino-nasal mucus from 26 CRS patients and 10 healthy controls was measured. In addition, 7 inflammatory cytokines, bacterial community composition and bacterial abundance within the sino-nasal mucus were measured using molecular and diagnostic tools. RESULTS: TLR activity was observed in 9/36 samples, including 2 healthy controls. There was a strong, positive correlation between members of the Gammaproteobacteria (Haemophilus, Enterobacter, Pseudomonas) and TLR2/1 and TLR4 activity. Bacterial abundance and cytokine (tumour necrosis factor) abundance were also positively correlated with TLR activity. CONCLUSIONS: These findings suggest that a small proportion (20-30%) of individuals in each sub-group are more predisposed to TLR activity, which may be related to bacterial composition, diversity and abundance in the sinuses.

The in vitro effect of xylitol on chronic rhinosinusitis biofilms.

INTRODUCTION: Biofilms have been implicated in chronic rhinosinusitis (CRS) and may explain the limited efficacy of antibiotics. There is a need to find more effective, non-antibiotic based therapies for CRS. This study examines the effects of xylitol on CRS biofilms and planktonic bacteria. METHODS: Crystal violet assay and spectrophotometry were used to quantify the effects of xylitol (5% and 10% solutions) against Staphylococcus epidermidis, Pseudomonas aeruginosa, and Staphylococcus aureus. The disruption of established biofilms, inhibition of biofilm formation and effects on planktonic bacteria growth were investigated and compared to saline and no treatment. RESULTS: Xylitol 5% and 10% significantly reduced biofilm biomass (S. epidermidis), inhibited biofilm formation (S. aureus and P. aeruginosa) and reduced growth of planktonic bacteria (S. epidermidis, S. aureus, and P. aeruginosa). Xylitol 5% inhibited formation of S. epidermidis biofilms more effectively than xylitol 10%. Xylitol 10% reduced S. epidermidis planktonic bacteria more effectively than xylitol 5%. Saline, xylitol 5% and 10% disrupted established biofilms of S. aureus when compared with no treatment. No solution was effective against established P. aeruginosa biofilm. CONCLUSIONS: Xylitol has variable activity against biofilms and planktonic bacteria in vitro and may have therapeutic efficacy in the management of CRS.

Effect of low-dose antigen exposure on development of immunity to Helicobacter pylori infection in mice.

The effect of low-dose antigen exposure on the development of immunity to Helicobacter pylori infection was studied in outbred mice. Animals that were primed with a subinfectious number of H. pylori bacteria exhibited significantly lower bacterial loads after challenge with an infectious dose of pathogen (versus controls, P < 0.05).

Migration of Langerhans cells and gammadelta dendritic cells from UV-B-irradiated sheep skin.

Depletion of dendritic cells from UV-B-irradiated sheep skin was investigated by monitoring migration of these cells towards regional lymph nodes. By creating and cannulating pseudoafferent lymphatic vessels draining a defined region of skin, migrating cells were collected and enumerated throughout the response to UV-B irradiation. In the present study, the effects of exposing sheep flank skin to UV-B radiation clearly demonstrated a dose-dependent increase in the migration of Langerhans cells (LC) from the UV-B-exposed area to the draining lymph node. The range of UV-B doses assessed in this study included 2.7 kJ/m2, a suberythemal dose; 8 kJ/m2, 1 minimal erythemal dose (MED); 20.1 kJ/m2; 40.2 kJ/m2; and 80.4 kJ/m2, 10 MED. The LC were the cells most sensitive to UV-B treatment, with exposure to 8 kJ/m2 or greater reproducibly causing a significant increase in migration. Migration of gammadelta+ dendritic cells (gammadelta+ DC) from irradiated skin was also triggered by exposure to UV-B radiation, but dose dependency was not evident within the range of UV-B doses examined. This, in conjunction with the lack of any consistent correlation between either the timing or magnitude of migration peaks of these two cell types, suggests that different mechanisms govern the egress of LC and gammadelta+ DC from the skin. It is concluded that the depression of normal immune function in the skin after exposure to erythemal doses of UV-B radiation is associated with changes in the migration patterns of epidermal dendritic cells to local lymph nodes.

Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions.

Experimental infection of mice with Helicobacter felis reproduces many aspects of the gastritis observed in Helicobacter pylori-infected humans. The development of gastric inflammatory lesions in chronically infected inbred mice is host-dependent; in BALB/c mice, gastric B-cell MALT lymphomas were observed, whilst other murine hosts (e.g. C57BL/6) developed severe glandular hyperplasia. The aims of this investigation were to characterize and immunophenotype Helicobacter-induced inflammatory lesions in mice with an outbred genetic background. Swiss mice (n=10 per group) were either inoculated with a suspension of H. felis or left untreated. H. felis-inoculated mice and age-matched control animals were killed 13 months later. The severity of gastric inflammatory lesions in the animals was graded and the number and distribution of B (CD45R(+)) and T (CD3(+)) lymphocytes in lymphoid tissues was determined by immunohistochemistry. Compared with control mice, animals with long-term H. felis infection developed severe hyperplastic gastritis (0.80+/-0.63 vs. 2.7+/-0.68), with epithelial dedifferentiation (0. 40+/-0.52 vs. 2.3+/-0.82) and lengthening of the pits and glands (0. 46+/-0.05 vs. 0.8+/-0.19). Gastric CD45R(+) and CD3(+) lymphocyte scores were significantly elevated (r=0.803) in infected animals, while lymphoepithelial lesions and polymorphonuclear leucocyte infiltrates were absent. Although prominent lymphoid follicles were present in the tissues of all infected animals, and in one control animal, only a proportion (55%) of the mucosal follicles had a dominant B-cell phenotype (defined as > or =75% CD45R(+) labelling), and all were poorly labelled with anti-mouse immunoglobulin antibodies. It was concluded that the lesions in outbred Swiss mice differed from B-cell MALT lymphomas. In contrast to inbred mice, outbred animals developed both glandular and lymphoid tissue lesions to chronic H. felis infection. It is suggested that the default T-helper phenotype of the host influences glandular lesion formation or B-cell lymphomagenesis in this model of infection.

Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity.

Arginase of the Helicobacter pylori urea cycle hydrolyzes L-arginine to L-ornithine and urea. H. pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved in H. pylori nitrogen metabolism. The roles of arginase in the physiology of H. pylori were investigated in vitro and in vivo, since arginase in H. pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The H. pylori gene hp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H. pylori strains were made by using two different constructs: 236-2 and rocF::aphA3. In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of H. pylori, the rocF::aphA3 mutant was approximately 1, 000-fold more sensitive to acid exposure. The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of L-arginine, in contrast to the WT, and yielded a approximately 10, 000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro. Finally, H. pylori mouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of H. pylori recovered from these mice (in log(10) CFU) were 6.1, 5.5, and 4.1, respectively. Thus, H. pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.

Isolation of recombinant protective Helicobacter pylori antigens.

A total of seven clones producing both new and previously described Helicobacter pylori proteins were isolated from a library of H. pylori genomic DNA. The screening approach by which these proteins were detected relied on the use of antisera raised in mice vaccinated with Helicobacter felis sonicate plus cholera toxin, a regimen which protects mice from H. pylori challenge. This strategy was designed to maximize the possibility of obtaining antigens which might be capable of conferring protection from H. pylori infection. Two of the clones were shown to encode the urease enzyme and the heat shock protein HspB, which have already been identified as protective antigens. The other five clones were sequenced, protein coding regions were deduced, and these sequences were amplified by PCR for incorporation into Escherichia coli expression vectors. The proteins produced from these expression systems were purified to allow testing for protective efficacy in an H. pylori mouse model. All five proteins were able to facilitate the clearance of a challenge with H. pylori, as judged by an assay of gastric urease activity and light microscopy on stomach sections. These results clearly indicate that the screening strategy has successfully identified candidate vaccine antigens.

Catalase, a novel antigen for Helicobacter pylori vaccination.

The efficacy of an orogastric vaccine comprised of purified Helicobacter pylori catalase plus the mucosal adjuvant cholera toxin (CT) was examined with both the Helicobacter felis and H. pylori mouse models with BALB/c mice. Native H. pylori catalase (200 microg) plus CT was initially used as a vaccine antigen in the H. felis mouse model and protected 80% (8 of 10) of the challenged animals, while all control animals were infected (20 of 20). In a follow-up experiment, recombinant H. pylori catalase plus CT was used for immunization, and groups of mice were challenged with the Sydney strain of H. pylori. Immunization with recombinant catalase protected a significant proportion (9 of 10) of the mice from H. pylori challenge, indicating that this enzyme should be considered as a candidate for a future vaccine. This study provides the first available data on the efficacy of protective immunization with the new Sydney strain of H. pylori in a mouse model. These data also provide indirect evidence that proteins which are normally intracellular, such as catalase, may be present on the surface of H. pylori and thus may provide targets for immunization.

The endotoxin of Helicobacter pylori is a modulator of host-dependent gastritis.

Atrophic gastritis caused by Helicobacter pylori is the precursor lesion in the development of intestinal-type gastric adenocarcinoma. In animal models, atrophic gastritis induced by Helicobacter felis has been shown to be host dependent, developing in some mouse strains and not in others. The lipopolysaccharide (LPS) of H. pylori has been suggested to play a role in the induction of gastritis. The goal of this study was to compare the inflammation induced by long-term infection of the C3H/He and the C3H/HeJ strains of mice with H. felis. C3H/HeJ mice are unresponsive to LPS. Six months after infection, severe atrophic gastritis had developed in the body mucosae of all infected C3H/He mice, with replacement of parietal and chief cells. Atrophy was associated with a loss of the H. felis from the antral mucosa. In contrast, no atrophy was seen in the infected C3H/HeJ non-LPS responder animals, and heavy colonization of the antrum remained. There were no significant differences between both the quantitative and qualitative serum immunoglobulin G (IgG) and salivary IgA levels in both strains of mice. The main difference between the two strains of long-term-infected mice was a lack of macrophage infiltration in the lamina propria. Immunization induced good protective immunity to challenge with viable H. felis. Helicobacter-induced, host-dependent gastritis is likely to be cell mediated. The C3H/He and C3H/HeJ mouse model provides an excellent opportunity to investigate the cellular basis of atrophic gastritis.

Protective immunization against Helicobacter stimulates long-term immunity.

Immunization with an oral vaccine composed of whole cell sonicates of Helicobacter felis plus cholera toxin (CT) can protect mice from H. felis challenge. The aim of this study was to determine whether this protective immunity was long-lived. Mice were given the vaccine then left for up to 15 months before challenge. After 15 months, all mice were still protected from H. felis infection, indicating that oral immunization using bacterial antigens plus CT can stimulate long-term local immunological memory.

Bacterial flora of Tasmanian SIDS infants with special reference to pathogenic strains of Escherichia coli.

The general bacterial flora of 38 Tasmanian SIDS infants was examined together with faecal flora of 134 comparison infants ranging in age from birth to 6 months. The microflora of all specimens received was investigated with special emphasis on the toxigenic Escherichia coli (TEC). Samples were examined for verocytotoxigenic E. coli, free faecal verocytotoxin (FVT), heat labile toxin (LT) and heat stable toxin (ST) producers with the use of a Vero cell assay and commercial kits. The findings of this study revealed a high isolation rate (39%) of TEC from SIDS infants as compared to 1.5% from the healthy comparison infants. Atypical E. coli strains were also identified during the study, including E. coli A-D. An analysis of the same specimens for rotaviral and adenoviral antigens indicated that 30% of the SIDS cases were positive as compared to 20% in the comparison group.

Estimating fat in feces by near-infrared reflectance spectroscopy.

We describe use of near-infrared reflectance spectroscopy (NIRRS) to estimate the amount of fat in feces, for diagnosis of steatorrhea. After sample homogenization, the spectrum of the fecal homogenate is scanned over the near-infrared region. Assay of 94 samples of feces having a known concentration of fat showed the appropriate wavelengths for the NIRRS procedure to be 1734, 1778, 1818, 2270, and 2310 nm. The reflectance output of 47 fecal samples subsequently measured at these wavelengths was used to compute the reflectance scaling factors (F values) by the instrument's microprocessor. Assay of fat content in a further 124 fecal samples, by both hydrolysis/titration (J Biol Chem 1949;177:347) and the NIRRS procedure at the wavelengths and corresponding F values previously determined, gave results that correlated highly satisfactorily. However, the NIRRS procedure demonstrated much better precision.