IL-10 underlies distinct susceptibility of BALB/c and C57BL/6 mice to Mycobacterium avium infection and influences efficacy of antibiotic therapy

Increased production of IL-10 has been frequently associated with augmented susceptibility to infection. However, the correlation between IL-10 activity and susceptibility to mycobacterial infection is still uncertain. Although studies using transgenic mice overexpressing IL-10 consistently showed an increased susceptibility to mycobacterial infection, experimental approaches in which IL-10 activity was reduced or abrogated originated inconclusive data. We show here that this controversy might be due to the mouse strains used in the various experimental procedures. Our results show that BALB/c mice are more susceptible than C57BL/6 to Mycobacterium avium infection. This increased susceptibility of BALB/c mice is, to a great extent, due to distinct activity of IL-10 between the two mouse strains. In accordance, reduction of IL-10 activity through the administration of anti-IL-10R mAb, or the absence of IL-10 as studied in IL-10 knockout mice, clearly decreased the susceptibility of BALB/c mice to M. avium but had a less obvious effect in C57BL/6 mice. Moreover, abrogation of IL-10 activity in infected BALB/c mice increased the efficacy of antimycobacterial therapy, whereas for the C57BL/6 mice it produced no effect. These observations show that the activity of IL-10 in response to the same mycobacterial stimulus influences not only the susceptibility to infection but also the efficacy of antimycobacterial therapy. This should now be considered in the context of human response to mycobacterial infection, particularly as a possible strategy to improve treatment against infections by mycobacteria.     

MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (K(i) = 0.2 nm) over TIMP-2 (K(i) = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.     

The genetic structure of fermentative vineyard-associated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> populations revealed by microsatellite analysis

From the analysis of six polymorphic microsatellite loci performed in 361 Saccharomyces cerevisiae isolates, 93 alleles were identified, 52 of them being described for the first time. All these isolates have a distinct mtDNA RFLP pattern. They are derived from a pool of 1620 isolates obtained from spontaneous fermentations of grapes collected in three vineyards of the Vinho Verde Region in Portugal, during the 2001–2003 harvest seasons. For all loci analyzed, observed heterozygosity was 3–4 times lower than the expected value supposing a Hardy–Weinberg equilibrium (random mating and no evolutionary mechanisms acting), indicating a clonal structure and strong populational substructuring. Genetic differences among S. cerevisiae populations were apparent mainly from gradations in allele frequencies rather than from distinctive “diagnostic” genotypes, and the accumulation of small allele-frequency differences across six loci allowed the identification of population structures. Genetic differentiation in the same vineyard in consecutive years was of the same order of magnitude as the differences verified among the different vineyards. Correlation of genetic differentiation with the distance between sampling points within a vineyard suggested a pattern of isolation-by-distance, where genetic divergence in a vineyard increased with size. The continuous use of commercial yeasts has a limited influence on the autochthonous fermentative yeast population collected from grapes and may just slightly change populational structures of strains isolated from sites very close to the winery where they have been used. The present work is the first large-scale approach using microsatellite typing allowing a very fine resolution of indigenous S. cerevisiae populations isolated from vineyards.     

Caspofungin reduces the incidence of fungal contamination in cell culture

Fungal contamination is a major problem in cell culture, and the antifungal compounds currently in use can affect cultured cells. Echinocandins are antifungal drugs that inhibit fungal cell wall synthesis by targeting an enzyme that has no counterpart in mammalian cells. We evaluated whether the echinocandin caspofungin affected the growth or morphology of six murine cell lines (a macrophage-like cell line (J774.16) and five hybridoma lines), or primary human endothelial cells. The antifungal did not influence cellular characteristics at concentrations less than 512 μg/ml, while effectively reducing the incidence of fungal contamination. Also, caspofungin did not affect the production of antibody by hybridoma cells, or alter the cytokine production of J774.16 cells, although modest increases in IL-4 and IFN-γ occurred upon LPS stimulation. Hence, echinocandins appear to be relatively non-toxic, and protect against fungal contamination in cell culture.     

Range-wide phylogeography and gene zones in Pinus pinaster Ait. revealed by chloroplast microsatellite markers

Some 1339 trees from 48 Pinus pinaster stands were characterized by five chloroplast microsatellites, detecting a total of 103 distinct haplotypes. Frequencies for the 16 most abundant haplotypes (p(k) > 0.01) were spatially interpolated over a lattice made by 430 grid points. Fitting of spatially interpolated values on raw haplotype frequencies at the same geographical location was tested by regression analysis. A range-wide 'diversity map' based on interpolated haplotype frequencies allowed the identification of one 'hotspot' of diversity in central and southeastern Spain, and two areas of low haplotypic diversity located in the western Iberian peninsula and Morocco. Principal component analysis (PCA) carried out on haplotypes frequency surfaces allowed the construction of a colour-based 'synthetic' map of the first three PC components, enabling the detection of the main range-scale genetic trends and the identification of three main 'gene pools' for the species: (i) a 'southeastern' gene pool, including southeastern France, Italy, Corsica, Sardinia, Pantelleria and northern Africa; (ii) an 'Atlantic' gene pool, including all the western areas of the Iberian peninsula; and (iii) a 'central' gene pool, located in southeastern Spain. Multivariate and AMOVA analyses carried out on interpolated grid point frequency values revealed the existence of eight major clusters ('gene zones'), whose genetic relationships were related with the history of the species. In addition, demographic models showed more ancient expansions in the eastern and southern ranges of maritime pine probably associated to early postglacial recolonization. The delineation of the gene zones provides a baseline for designing conservation areas in this key Mediterranean pine.     

Brazilian green propolis on Helicobacter pylori infection. a pilot clinical study

Recent in vitro studies suggest that propolis and some of its phenolic components are able to inhibit Helicobacter pylori growth. To date, there are no clinical studies. AIMS: To evaluate the effect of Brazilian green propolis on H. pylori-infected individuals. PATIENTS AND METHODS: Eighteen (11 females, 7 males, mean age 47 years) participants were included. Before treatment, all participants were submitted to gastroscopy, and H. pylori infection was confirmed by histology, urease test, and (13)C-urea breath test (UBT). Participants with UBT showing a delta over baseline (DOB) value higher than 4 per thousand were considered positive for H. pylori infection. Twenty drops from an alcoholic preparation of Brazilian green propolis were administered three times a day for 7 days. Clinical evaluation and UBT were performed at 1-3 days and at 40 days after the end of therapy to evaluate H. pylori suppression or eradication, respectively. RESULTS: All participants took all medication and completed the study. Eighty-three percent of the subjects did not succeed in suppressing or eradicating H. pylori. Two participants reached partial suppression after treatment, but became positive again at UBT performed 40 days after treatment. Another participant presented negative at UBT 40 days after treatment, not confirmed by a second UBT performed 100 days after treatment. CONCLUSIONS: Brazilian green propolis used in popular dose showed minimal effect on H. pylori infection. Larger studies with longer duration, larger dose, and different frequency of administration of propolis extract should be undertaken to define its role on H. pylori therapy.     

The volume and hydration of the Cryptococcus neoformans polysaccharide capsule

We present a new method to measure capsule size in the human fungal pathogen Cryptococcus neoformans that avoids the limitations and biases inherent in India ink measurements. The method is based on the use of gamma-radiation, which efficiently releases the capsule from the cell. By comparing the volume of irradiated and non-irradiated cells, one can accurately estimate the relative size of the capsule per cell. This method was also used to obtain an estimate of the capsule weight and water content. The C. neoformans capsule is a highly hydrated structure in all the conditions measured. However, after capsule enlargement, the amount of capsular polysaccharide significantly increases, suggesting a that capsule growth has a high energy cost for the cell.     

Path tortuosity of Eurasian badgers (<Emphasis Type="Italic">Meles meles</Emphasis>) in a heterogeneous Mediterranean landscape

Movement is the process by which individual organisms are displaced over time to eat, reproduce and defend resources. Fractal analysis is a technique used to study animal movement that measures spatial complexity of path tortuosity; here, we apply it to characterize the movement patterns of the Eurasian badger (Meles meles) in a Mediterranean landscape. We calculated path tortuosity overall and seasonally, and for individuals of different sexes and social groups. The influence of variables related to badgers’ resources (food, shelter, water), human infra-structures and weather conditions were analysed with respect to the tortuosity of each badger’s path. A total of 55 search paths from six badgers were considered for this study. Although badgers generally displayed convoluted movement, there were two exceptions: (a) males overall and (b) all badgers in summer; for both, movements had a lower fractal value, i.e. were less tortuous. The convoluted movement pattern generally observed is probably adapted to the clumped distribution of food in the study area. Nevertheless, our results suggest that the use of dens and latrines were the principal determinants of tortuosity of badgers’ paths while foraging.