The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo

In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.     

Effects of adaptation to carvacrol on Staphylococcus aureus in the planktonic and biofilm phases

The effect of exposure to sub-minimum inhibitory concentrations of carvacrol, for either 3–10 days, on direct (carvacrol) or cross-protection (cinnamaldehyde, eugenol, antibiotics) and the influence on planktonic and biofilm growth of four Staphylococcus aureus strains were reported. The sequential exposure to carvacrol resulted in a direct protection that was more evident in two of the four strains after 10 days. No significant cross-protection against cinnamaldehyde, eugenol and antibiotics was detected. An adaptive response was associated with a prolonged lag phase, a lower yield of bacteria, a colony phenotype likely to be associated to small colony variants and an increase in biofilm production. Generally, the biofilm of the adapted strains was less susceptible to subMICs of carvacrol compared to the biofilms of non-adapted strains. In contrast, it was demonstrated that in the case of mature biofilms the susceptibility was similar. The exposure of S. aureus to carvacrol at concentrations above the MIC resulted in a very low mutation frequency.     

A model of Helicobacter pylori persistence in a case of gastric cancer

The aim of this work was to analyze several clones of Helicobacter pylori isolated from a patient with gastric cancer, to evaluate i) genetic variability ii) virulence factors profile and iii) antimicrobial susceptibility against the drugs commonly used in the H. pylori therapy. A total of 32 H. pylori clones isolated from a biopsy sample coming from a patient with gastric cancer previously treated for H. pylori infection, were analyzed for: the genetic variability by amplified fragment polymorphism analysis; the vacA, cagA virulence status by PCR; the antimicrobial susceptibility by minimum inhibitory concentrations with the agar dilution method towards amoxicillin, clarithromycin, levofloxacin and tinidazole. The patient showed a mixed infection with the presence of at least 3 different strains. The clones isolated possessed the vacA, cagA virulence factors with a different allelic combination (vacA s1/i1/m1; s1/i1i2/m1; s2/i2/m2; s2/i1i2/m2) together with repeated cagA EPIYA motif pattern P1P2P3P3P3. Moreover, a pattern of multi-drug resistance was disclosed in the different clones. The presence of multiple H. pylori strains colonizing the same patient, with the main virulence factors displaying a different allelic combination and a different multi-drug resistance among isolates, point out the role of genetic variability generating, in time, more virulent and adapted strains.     

Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis

When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress.     

Enhanced clearing of Helicobacter pylori after omeprazole plus roxithromycin treatment

The in vitro antibacterial activity of omeprazole against eight strains of Helicobacter pylori was evaluated. Minimum inhibitory concentration (MIC) values were 32 micrograms/ml and 64 micrograms/ml (MIC50 and MIC90 respectively). We performed a randomized single blind study comparing the efficacy of omeprazole alone (for 4 weeks) or combined with roxithromycin (for 2 weeks) in the treatment of duodenal ulcer and chronic active gastritis associated with H. pylori infection, H. pylori was eradicated in 75% of patients treated with omeprazole alone whereas the patients treated with the combination of these drugs were completely free from H. pylori at the end of the therapy.     

Immunity to fungal infections

Fungal diseases represent an important paradigm in immunology, as they can result from either a lack of recognition by the immune system or overactivation of the inflammatory response. Research in this field is entering an exciting period of transition from studying the molecular and cellular bases of fungal virulence to determining the cellular and molecular mechanisms that maintain immune homeostasis with fungi. The fine line between these two research areas is central to our understanding of tissue homeostasis and its possible breakdown in fungal infections and diseases. Recent insights into immune responses to fungi suggest that functionally distinct mechanisms have evolved to achieve optimal host-fungus interactions in mammals.     

Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus

A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, 1H and 13C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates.     

Cryopreservation and fertility of ejaculated and epididymal stallion sperm

The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides was flushed to obtain EP-0h sperm. The contralateral epididymis was stored at 5°C for 24h before being flushed to obtain EP-24h sperm. The sperm samples were analyzed at three different times: immediately after sperm recovery, after dilution in the freezing extender, and post-thawing. A fertility trial was performed using 39 estrous cycles. After ovulation induction with 1mg of deslorelin acetate (i.m.), mares were inseminated with 800×10(6) sperm. The total number of sperm recovered was 7.8±4.7×10(9) for EJ-0h sperm, 12.9±9.2×10(9) for EP-0h sperm and 12.0±8.0×10(9) for EP-24h sperm. The sperm motility, evaluated by total motility, progressive motility and the percentage of rapid cells, was similar among the samples before and after freezing (P>0.05). However, the plasma membrane integrity was different between EJ-0h and EP-0h pre-freezing and between EJ-0h and EP-24h post-thawing (P<0.05). The conception rates were similar between groups inseminated with sperm recovered from the epididymal cauda immediately after orchiectomy (EP-0h), after 24h of storage at 5°C of the epididymal cauda (EP-24h) and with ejaculated sperm (EJ-0h) (P>0.05). In conclusion, the viability and fertility of cauda epididymal sperm are similar to those of ejaculated sperm.     

The interaction pattern of murine serum ficolin-A with microorganisms

The ficolins are soluble pattern recognition molecules in the lectin pathway of complement, but the spectrum and mode of interaction with pathogens are largely unknown. In this study, we investigated the binding properties of the murine serum ficolin-A towards a panel of different clinical relevant microorganisms (N = 45) and compared the binding profile with human serum ficolin-2 and ficolin-3. Ficolin-A was able to bind Gram-positive bacteria strains including E. faecalis, L. monocytogenes and some S. aureus strains, but not to the investigated S. agalactiae (Group B streptococcus) strains. Regarding Gram-negative bacteria ficolin-A was able to bind to some E. coli and P. aeruginosa strains, but not to the investigated Salmonella strains. Of particular interest ficolin-A bound strongly to the pathogenic E. coli, O157:H7 and O149 strains, but it did not bind to the non-pathogenic E. coli, ATCC 25922 strain. Additionally, ficolin-A was able to bind purified LPS from these pathogenic strains. Furthermore, ficolin-A bound to a clinical isolate of the fungus A. fumigatus. In general ficolin-2 showed similar selective binding spectrum towards pathogenic microorganisms as observed for ficolin-A indicating specific pathophysiological roles of these molecules in host defence. In contrast, ficolin-3 did not bind to any of the investigated microorganisms and the anti-microbial role of ficolin-3 still remains elusive.