Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.     

Retinoic acid treatment elevates matrix metalloproteinase-2 protein and mRNA levels in avian growth plate chondrocyte cultures

Matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling. In growth plate (GP) cartilage, extensive remodeling occurs at the calcification front. To study the potential involvement of MMPs in retinoic acid (RA) regulation of skeletal development, we studied the effect of all-trans-RA on MMPs levels in mineralizing chicken epiphyseal chondrocyte primary cultures. When treated for 4 day periods on days 10 and 17, RA increased levels of an ∼70 kDa gelatinase activity. The N-terminal sequence of the first 20 amino acid residues of the purified enzyme was identical to that deduced from chicken MMP-2 cDNA. Time-course studies indicated that RA elevated MMP-2 activity levels in the cultures within 16 h. This increase was inhibited by cycloheximide and was enhanced by forskolin. The increase in MMP-2 activity induced by RA was accompanied by an increase in MMP-2 mRNA levels and was abolished by treatment with cycloheximide. This upregulation of MMP levels by RA in GP chondrocytes is consistent with its effects on osteoblasts and osteosarcoma cells and opposite its inhibitory effects on fibroblasts and endothelial cells. It may well be related to the breakdown of the extracellular matrix in the GP and would be governed by the availability of RA at the calcification front where extensive vascularization also occurs. J. Cell. Biochem. 68:90–99, 1998. © 1998 Wiley-Liss, Inc.     

Break zones in the distributions of alleles and species in alpine plants

Aim We test for the congruence between allele‐based range boundaries (break zones) in silicicolous alpine plants and species‐based break zones in the silicicolous flora of the European Alps. We also ask whether such break zones coincide with areas of large elevational variation. Location The European Alps. Methods On a regular grid laid across the entire Alps, we determined areas of allele‐ and species‐based break zones using respective clustering algorithms, identifying discontinuities in cluster distributions (breaks), and quantifying integrated break densities (break zones). Discontinuities were identified based on the intra‐specific genetic variation of 12 species and on the floristic distribution data from 239 species, respectively. Coincidence between the two types of break zones was tested using Spearman’s correlation. Break zone densities were also regressed on topographical complexity to test for the effect of elevational variation. Results We found that two main break zones in the distribution of alleles and species were significantly correlated. Furthermore, we show that these break zones are in topographically complex regions, characterized by massive elevational ranges owing to high mountains and deep glacial valleys. We detected a third break zone in the distribution of species in the eastern Alps, which is not correlated with topographic complexity, and which is also not evident from allelic distribution patterns. Species with the potential for long‐distance dispersal tended to show larger distribution ranges than short‐distance dispersers. Main conclusions We suggest that the history of Pleistocene glaciations is the main driver of the congruence between allele‐based and species‐based distribution patterns, because occurrences of both species and alleles were subject to the same processes (such as extinction, migration and drift) that shaped the distributions of species and genetic lineages. Large elevational ranges have had a profound effect as a dispersal barrier for alleles during post‐glacial immigration. Because plant species, unlike alleles, cannot spread via pollen but only via seed, and thus disperse less effectively, we conclude that species break zones are maintained over longer time spans and reflect more ancient patterns than allele break zones.     

Antagonistic Antiviral Activity between IFN-Lambda and IFN-Alpha against Lethal Crimean-Congo Hemorrhagic Fever Virus In Vitro

MethodsHuman A549 and HuH7 cells were treated with increasing amounts of IFN-λ1, or IFN-α or a combination of them, infected with CCHF; the extent of virus yield inhibition and the induction of MxA and 2’-5’OAS mRNA was measured.

Results and Conclusions

Our study pointed out that type III IFN possess an antiviral activity against CCHFV, even if lower than type I IFN. Moreover, a clear antagonism between IFN-λ and IFN–α was observed in both cell lines (A549 and HuH7 cells), in terms of antiviral effect and activation of pivotal ISGs, i.e. MxA and 2’-5’OAS. Elucidating the interplay between type I and III IFNs will help to better understand innate defence mechanisms against viral infections and may provide novel scientific evidence for a more rational planning of available and future treatments, particularly against human diseases caused by high concern viruses.     

Structure-Guided Mutational Analysis of a Yeast DEAD-Box Protein Involved in Mitochondrial RNA Splicing

DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context of current models of Mss116p-facilitated splicing.     

A phase II trial of vaccination with autologous, tumor-derived heat-shock protein peptide complexes Gp96, in combination with GM-CSF and interferon-α in metastatic melanoma patients

The aim of this study was to determine the immunogenicity and antitumor activity of autologous, tumor-derived heat shock protein gp96-peptide complex vaccine (HSPPC-96; Oncophage®) given with GM-CSF and IFN-α in pre-treated metastatic (AJCC stage IV) melanoma patients. Patients underwent surgical resection of metastatic lesions for HSPPC-96 production. HSPPC-96 was administered subcutaneously (s.c.) in four weekly intervals (first cycle). Patients with more available vaccine and absence of progressive disease received four additional injections in 2-week intervals (second cycle) or more. GM-CSF was given s.c. at the same site at days –1, 0 and +1, while IFN-α (3 MU) was administered s.c. at a different site at days +4 and +6. Antigen-specific anti-melanoma T and NK lymphocyte response was assessed by enzyme-linked immunospot assay on peripheral blood mononuclear cells obtained before and after vaccination. Thirty-eight patients were enrolled, 20 received at least four injections (one cycle) of HSPPC-96 and were considered assessable. Toxicity was mild and most treatment-related adverse events were local erythema and induration at the injection site. Patients receiving at least four injections of HSPPC-96 were considered evaluable for clinical response: of the 18 patients with measurable disease post surgery, 11 showed stable disease (SD). The ELISPOT assay revealed an increased class I HLA-restricted T and NK cell-mediated post-vaccination response in 5 out of 17 and 12 out of the 18 patients tested, respectively. Four of the five class I HLA-restricted T cell responses fall in the group of SD patients. Vaccination with autologous HSPPC-96 together with GM-CSF and IFN-α is feasible and accompanied by mild local and systemic toxicity. Both tumor-specific T cell-mediated and NK cell responses were generated in a proportion of patients. Clinical activity was limited to SD. However, both immunological and clinical responses were not improved as compared with those recorded in a previous study investigating HSPPC-96 monotherapy.L.P. and R.P. have equally contributed to the work.     

Human lymphocyte activation gene-3 molecules expressed by activated T cells deliver costimulation signal for dendritic cell activation

Data have been reported on the in vivo adjuvant role of soluble lymphocyte activation gene-3 (LAG-3) recombinant protein in mouse models and on its ability to support the in vitro generation of human, tumor-specific CTLs. In this study, we show that soluble human rLAG-3 protein (hLAG-3Ig) used in vitro as a single maturation agent induces phenotypic maturation of monocyte-derived dendritic cells and promoted the production of chemokines and TNF-alpha inflammatory cytokine. When given in association with optimal or suboptimal doses of CD40/CD40L, hLAG-3Ig functions as a strong costimulatory factor and induces full functional activation of monocyte-derived dendritic cells that includes the production of high level of IL-12p70. Moreover, evidence is here provided that this costimulatory function licensing dendritic cells to produce IL-12p70 is also a functional property of LAG-3 molecules when expressed in a physiological context by CD4(+) activated T cells. Altogether, these data show for the first time a role of LAG-3 in mediating dendritic cell activation when expressed on the T cell surface or released after specific Ag stimulation in the interspaces of immunological synapses.