Phage types and genotypes of shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in spain: identification and characterization of two predominating phage types (PT2 and PT8)

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused.     

Mechanical Analysis of Atherosclerotic Plaques Based on Optical Coherence Tomography

Finite element analysis is a powerful tool for investigating the biomechanics of atherosclerosis and has thereby provided an improved understanding of acute myocardial infarction. Structural analysis of arterial walls is traditionally performed using geometry contours derived from histology. In this paper we demonstrate the first use of a new imaging technique, optical coherence tomography (OCT), as a basis for finite element analysis. There are two primary benefits of OCT relative to histology: 1) imaging is performed without excessive tissue handling, providing a more realistic geometry than histology and avoiding structural artifacts common to histologic processing, and 2) OCT imaging can be performed in vivo, making it possible to study disease progression and the effect of therapeutic treatments in animal models and living patients. Patterns of mechanical stress and strain distributions computed from finite element analysis based on OCT were compared with those from modeling based on "gold standard" histology. Our results indicate that vascular structure and composition determined by OCT provides an adequate basis for investigating the biomechanical factors relevant to atherosclerosis and acute myocardial infarction.     

Inter-laboratory comparison of qualitative and quantitative detection of hepatitis C (HCV) virus RNA in diagnostic virology: a multicentre study (MS) in Italy

BACKGROUND: The importance of the standardisation of nucleic acid amplification technology (NAT) assays for the detection of hepatitis C virus RNA is well known today, as many studies carried out in different European countries attest. The results of a previous study performed in Italy (J. Clin. Virol. 1 (2003) 83) by the Italian Society of Clinical Microbiology (AMCLI) showed that the use of external reference standards and of multicentre collaborative studies significantly improves laboratory performance for the qualitative evaluation of HCV RNA. OBJECTIVES: the AMCLI organised a new study on the standardisation of both the qualitative and the quantitative evaluation of HCV RNA with NAT in order to improve the implementation of the diagnostic methods for HCV RNA detection. STUDY DESIGN: seventeen diagnostic centres of major Italian Hospitals participated in this quality control study. The study consisted of testing three panels, each made up of 10 coded samples including negative and positive samples. Positive samples contained four levels of HCV RNA (genotype 1). RESULTS AND CONCLUSION: Seven out of 510 qualitative results obtained were incorrect (1.4%), two false negative and five false positive. The results gave a sensitivity of 99.5% and a specificity of 95.8%. Regarding quantitative tests, the geometric mean (GM) and standard deviation (S.D.) could be calculated only for the three highest HCV RNA levels. The percentage of results within the range of GM +/- 0.5 log(10) varied from 91% to 100%. Some laboratories had some difficulty in the exact quantification of the lowest (3.00 log IU/ml) as well as of the highest viral levels (6.35 log IU/ml) values, very near to the limits of the dynamic range of the assays. The comparison of the results of this study with that previously carried out one confirms that a regular participation in external quality assessment (EQA) assures the achievement of a high proficiency level in the diagnosis of HCV infection.     

Brucella lumazine synthase elicits a mixed Th1-Th2 immune response and reduces infection in mice challenged with Brucella abortus 544 independently of the adjuvant formulation used

The immunogenicity and protective efficacy of recombinant lumazine synthase from Brucella spp. (rBLS) administered with different adjuvants was evaluated in mice. Mice were immunized with rBLS in the absence or the presence of aluminum hydroxide gel (BLS-Al), monophosphoryl lipid A (BLS-MPA), or incomplete Freund's adjuvant (BLS-IFA). rBLS per se induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. All the adjuvants increased this response; the BLS-IFA formulation was the most effective at inducing BLS-specific IgG antibodies. In addition, after in vitro stimulation with rBLS, spleen cells from BLS-IFA-, BLS-Al-, or BLS-MPA-immunized mice proliferated and produced interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-10, and IL-4, suggesting the induction of a mixed Th1-Th2 response. Immunization with rBLS protected mice against challenge with B. abortus 544. The levels of protection in the spleen were similar for all adjuvants, but only BLS-Al and BLS-IFA were effective in the liver. Our results indicate that BLS might be a useful candidate for the development of subunit vaccines against brucellosis, since it elicits antigen-specific cellular responses, with production of IFN-gamma and protection, independently of the adjuvant formulation used.     

Cavitary pneumonia in an AIDS patient caused by an unusual Bordetella bronchiseptica variant producing reduced amounts of pertactin and other major antigens

Although Bordetella bronchiseptica can infect and colonize immunocompromised humans, its role as a primary pathogen in pneumonia and other respiratory processes affecting those patients remains controversial. A case of cavitary pneumonia caused by B. bronchiseptica in an AIDS patient is presented, and the basis of the seemingly enhanced pathogenic potential of this isolate (designated 814) is investigated. B. bronchiseptica was the only microorganism recovered from sputum, bronchoalveolar lavage fluid, and samples taken through the protected brush catheter. Unlike previous work reporting the involvement of B. bronchiseptica in cases of pneumonia, antibiotic treatment selected on the basis of in vitro antibacterial activity resulted in clearance of the infection and resolution of the pulmonary infiltrate. Although isolate 814 produced reduced amounts of several major antigens including at least one Bvg-activated factor (pertactin), the molecular basis of this deficiency was found to be BvgAS independent since the defect persisted after the bvgAS locus of isolate 814 was replaced with a wild-type bvgAS allele. Despite its prominent phenotype, isolate 814 displayed only a modest yet a significant deficiency in its ability to colonize the respiratory tracts of immunocompetent rats at an early time point. Interestingly, the antibody response elicited by isolate 814 in these animals was almost undetectable. We propose that isolate 814 may be more virulent in immunocompromised patients due, at least in part, to its innate ability to produce low amounts of immunogenic factors which may be required at only normal levels for the interaction of this pathogen with its immunocompetent natural hosts.     

NADPH oxidase-mediated oxidative stress: genetic studies of the p22(phox) gene in hypertension

Increased vascular production of reactive oxygen species, especially superoxide anion, significantly contributes to the oxidative stress associated with hypertension. An enhanced superoxide production causes an increased inactivation of nitric oxide that diminishes nitric oxide bioavailability, thus contributing to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that NADPH oxidases play a major role as the most important sources of superoxide anion in phagocytic and vascular cells. Several experimental observations have described an enhanced superoxide generation as a result of NADPH oxidase activation in hypertension. Although these enzymes respond to stimuli such as vasoactive factors, growth factors, and cytokines, recent data suggest a significant role of the genetic background in the modulation of the expression of its different components. Several polymorphisms have been identified in the promoter and in the coding region of CYBA, the gene that encodes the essential subunit of the NADPH oxidase p22phox, some of which seem to influence significantly the activity of these enzymes in the context of cardiovascular diseases. Among CYBA polymorphisms, genetic investigations have provided a novel marker, the -930(A/G) polymorphism, which determines the genetic susceptibility of hypertensive patients to oxidative stress.     

Induction of micronucleated cells in the shed skin of salamanders (Ambystoma sp.) treated with colchicine or cyclophosphamide

The micronucleus (MN) assay can be used to detect the genotoxic effects of chemical agents in virtually any cell that divides frequently. Salamanders (Ambystoma sp.) are amphibians that can be easily maintained and bred in the laboratory and spontaneously shed their skin every 2.5-4 days. In this present study, we have evaluated the usefulness of this shed skin for the MN assay. We exposed salamanders to different concentrations of both the aneugen colchicine (COL) and the clastogen cyclophosphamide (CP) and we determined the frequency of micronucleated cells (MNCs) in their sheds. Fragments of shed skin were placed on clean slides, fixed, stained, observed with a light microscope, and the number of MNCs was counted. The MNC frequency was increased significantly by all doses of COL and CP tested, administered either as single or repeated exposures. The presence of MNCs in the shed skin and the speed of sloughing lead us to propose that the sheds of Ambystoma sp., or other amphibians that slough their skin, are suitable alternative models for detecting genotoxic exposures relevant to aquatic environments.     

Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity

The activity of the homeobox gene Prox1 is necessary and sufficient for venous blood endothelial cells (BECs) to acquire a lymphatic endothelial cell (LEC) fate. We determined that the differentiated LEC phenotype is a plastic, reprogrammable condition that depends on constant Prox1 activity for its maintenance. We show that conditional down-regulation of Prox1 during embryonic, postnatal, or adult stages is sufficient to reprogram LECs into BECs. Consequently, the identity of the mutant lymphatic vessels is also partially reprogrammed as they acquire some features typical of the blood vasculature. siRNA-mediated down-regulation of Prox1 in LECs in culture demonstrates that reprogramming of LECs into BECs is a Prox1-dependent, cell-autonomous process. We propose that Prox1 acts as a binary switch that suppresses BEC identity and promotes and maintains LEC identity; switching off Prox1 activity is sufficient to initiate a reprogramming cascade leading to the dedifferentiation of LECs into BECs. Therefore, LECs are one of the few differentiated cell types that require constant expression of a certain gene to maintain their phenotypic identity.