Molecular characterization of Pasteurella multocida isolates from rabbits

Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.     

Aspects of constitutive and acquired antibioresistance in Aeromonas hydrophila strains isolated from water sources

Over the last three decades, the literature pointed out the implications of Aeromonas species in human pathology. These species were described as being involved in intestinal (several outbreaks of acute gastroenteritis of choleric/dysenteric form or chronic diarrhoea, ulcerative colitis, etc.) in normal adults or children, as well as in extraintestinal infections in immunocompromised hosts. This last aspect included a large range of cutaneous injuries (micronecrosis, abscesses, bums, cellulites, furunculosis), joint, bones, respiratory, urinary tract, ocular infections up to meningitis, endocarditis, peritonitis, hepatobilliary disease, endotoxic shock and septicemia (as consequence of leech microvascular surgery). During the last decade, the literature reported a high mortality in Aeromonas infections determined by certain phenospecies (A. hydrophila and A. veronii) especially in extraintestinal infections in immunocompromised patients. In microbiologists' opinion this high rate of mortality was probably due to poor knowledge concerning the aspects of antibioresistance in Aeromonas strains, to empiric treatments with antibiotics to which these bacteria exhibiting constitutive resistance lead to insuccessful results, and at last to the increasing trend of aeromonads resistance to certain antibiotics after 1996. The literature mentioned also that for a great number of Beta-lactamase producing Aeromonas strains, the use of microdilution method (by comparison to disk diffusion in agar medium) giving false results made more difficult the true knowledge of Aeromonas antibioresistance patterns. At the same time, in 2002, the literature mentioned 4 ecological compartments considered as "reservoirs for dissemination and transfer of microbial antibioresistance i.e. humans, animals, plants and natural soil and water. In the last time, more and more data of the literature revealed that some bacteria with role of reservoir of antibioresistance in the natural environment, even without a direct medical impact, however they could play an indirect one remaining permanent sources of R genes for bacterial strains with pathogenic abilities implicated in human pathology (i.e. Aeromonas infections in man related to different professional activities such as fishing, surfing, swimming, diving, etc.). The purpose of this work was to determine the aspects related to constitutive and acquired antibioresistance in 35 A. hydrophila strains isolated in aquatic environment of Danube Delta (10 salmaster waters, 5 aquatic plants, 5 fish intestinal content, 5 fish sapling, 5 snake and oyster shells). The strains were biochemically identified by using API20E and API20NE kits. The antibioresistance spectrum was determined by disk diffusion method following NCCLS 2000 recommendations. The choice and disposal of antibiotics on the Mueller Hinton plate was done to allow the interpretive reading and the phenotypic detection of different antibioresistance mechanisms, as follows: beta-lactamases (PEN, ME, AMX, AMC, CAZ) and carbapenemase (IMP) production; porin deficiency (FOX); efflux mechanism (C, TE, NOR). All tested strains exhibited high resistance to penicillin, aspect pleading for constitutive penicillinase production in Aeromonas strains. With reference to other penicillins (ME, AMX, AMC) and cephalosporins (CAZ, FOX) the tested strains exhibited 2 different antibioresistance patterns: AMX-R, AMC-S, CAZ-S (65%) indicating the presence of beta-lactamase sensitive to inhibitors and AMX-R, AMC-R, CAZ-S (22%) indicating the presence of beta-lactamase resistant to inhibitors. Resistance to FOX in 8% of strains signifies a phenotypical marker for the presence of porin deficiency. Only one Aeromonas strain (2.8%) was resistant to IMP. Three strains (8%) were simultaneous resistant to TE and TMP/SMX, NOR and CHL probably due to the presence of a resistance plasmid (codifying an efflux/ enzymatic mechanism). These aspects are pleading for the necessity to investigate the bacterial antibioresistance patterns of bacterial strains isolated from the environment, in the purpose to identify the factors responsible for the spreading of certain antibioresistance mechanisms in the external medium as risk factors for the colonization process with possible impact upon the human pathology.     

A flavodiiron protein and high molecular weight rubredoxin from Moorella thermoacetica with nitric oxide reductase activity

A five-gene "oxidative stress protection" cluster has recently been described from the strictly anaerobic, acetogenic bacterium, Moorella thermoacetica [Das, A., et al. (2001) J. Bacteriol. 183, 1560-1567]. Within this cluster are two cotranscribed genes, fprA (for A-type flavoprotein) and hrb (for high molecular weight rubredoxin) whose encoded proteins have no known functions. Here we show that FprA and Hrb are expressed in M. thermoacetica under normal anaerobic growth conditions and report characterizations of the recombinant FprA and Hrb. FprA contains flavin mononucleotide (FMN) and a non-heme diiron site. M?ssbauer spectroscopy shows that the irons of the diferric site are antiferromagnetically coupled, implying a single-atom, presumably solvent, bridge between the irons. Hrb contains FMN and a rubredoxin-like [Fe(SCys)4] site. NADH does not directly reduce either the FMN or the diiron site in FprA, whereas Hrb functions as an efficient NADH:FprA oxidoreductase. Substitution of zinc for iron in Hrb completely abolished this activity. The observation that homologues of FprA from other organisms show O2 and/or anaerobic NO consumption activity prompted an examination of these activities for M. thermoacetica FprA. The Hrb/FprA combination does indeed have both NADH:O2 and NADH:NO oxidoreductase activities. The NO reductase activity, however, was significantly more efficient due to a lower Km for NO (4 M) and to progressive and irreversible inactivation of FprA during O2 reductase turnover but retention of activity during NO reductase turnover. Substitution of zinc for iron in FprA completely abolished these reductase activities. The stoichiometry of 1 mol of NADH oxidized:2 mol of NO consumed implies reduction to N2O. Fits of an appropriate rate law to the kinetics data are consistent with a mechanism in which 2NO's react at each FprA active site in the committed step. Expression of FprA in an Escherichia coli strain deficient in NO reductase restored the anaerobic growth phenotype of cultures exposed to otherwise toxic levels of exogenous NO. The accumulated results indicate that Hrb/FprA is fully capable of functioning in nitrosative stress protection in M. thermoacetica.     

Apoptosis in the immune system: 1. Fas-induced apoptosis in monocytes-derived human dendritic cells

Dendritic cells (DC) are cells of the hematopoietic system specialized in capturing antigens and initiating T cell-mediated immune responses. We show here that human DC generated from adherent peripheral blood mononuclear cells (PBMC) after in vitro stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) express Fas antigen (APO-1, CD95) and can undergo apoptosis upon triggering of Fas by monoclonal antibodies. Immature monocytes-derived dendritic cells (MDDC) upregulate CD86 and HLA-DR expression and develop dendrites and veiled processes. Flow cytometry analysis revealed CD95 expression in approx. 40% of these MDDC and incubation with anti-CD95 mAb (0.5μg/ml) induced apoptosis when compared to untreated controls. The extent of apoptosis induced by the agonist anti-Fas antibody strongly related to the percentage of cells expressing CD 95. Upon tumor necrosis factor α (TNF-α) additional stimulation, MDDC assumed a characteristic mature dendritic cells morphology showing prolonged veils, CD83 expression, and high levels of HLA-DR. These cells have downregulated their Fas receptors (to approx. 20%) and undergo apoptosis to a lesser extent when treated with anti-CD 95, as demonstrated by the hardly noticeable effect of this antibody on the viability of cultured cells as compared to controls. Thus, upon TNF-α induced maturation, MDDC became resistant to Fas-induced apoptosis. The apoptotic episodes surrounding the earlier stage of DC differentiation appeared to be mediated by Fas. In contrast, a Fas independent pathway is probably responsible for the apoptotic events associated with terminally differentiated DC.     

Age-associated reduction of nuclear protein import in human fibroblasts

Age-dependent decreases in the protein concentrations of the nucleocytoplasmic transport factors karyopherin alpha2, CAS, and RanBP1 were found by comparing fibroblast cultures obtained from young, mature, and old human donors. Karyopherin beta1 levels do not change with age and present very little variation among donors. The decrease in the concentration of transport factors is accompanied by a reduction in the protein import rate in fibroblasts from old donors, as detected by a change in the intracellular localization of a test transport substrate that shuttles between the cytoplasm and the nucleus. Measurements of concentrations of the same import factors in organs and tissues of old mice revealed a decrease of CAS in kidney, lung, and spleen. The import reduction in old age is expected to lead to impaired activity of proteins whose functions depend on timely import into the nuclei.     

Phenotypical changes during in vitro cultivation of monocytes derived immature dendritic cells

Dendritic cells (DC) form a link between the first line of host defence and cellular immunity. In the present study we investigated the effect of cultivation time in generation of immature dendritic cells (iDCs) in vitro from human peripheral blood (PB) monocytes and the influence of iDCs on the oxygen free radicals (OFR) release by polymorphonuclear granulocytes (PMN) stimulated with opsonized zymozan (OZ) and concanavalin A (ConA). Our data suggest that the differentiation in vitro of PB monocytes in iDCs is influenced by the health status of the cell donor, and more by the concentration of cytokines GM-CSF and IL-4 in the system than by the time of cultivation. The in vitro experiments performed demonstrated the interactions between iDC and PMN granulocytes, evaluated by enhanced release of OFR.     

PTEN induces cell cycle arrest by decreasing the level and nuclear localization of cyclin D1

PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancers that acts as a phosphatase on phosphatidylinositol-3,4,5-trisphosphate, antagonizing the activity of the phosphatidylinositol 3'-OH kinase. PTEN manifests its tumor suppressor function in most tumor cells by inducing G(1)-phase cell cycle arrest. To study the mechanism of cell cycle arrest, we established a tetracycline-inducible expression system for PTEN in cell lines lacking this gene. Expression of wild-type PTEN but not of mutant forms unable to dephosphorylate phosphoinositides reduced the expression of cyclin D1. Cyclin D1 reduction was accompanied by a marked decrease in endogenous retinoblastoma (Rb) protein phosphorylation on cyclin D/CDK4-specific sites, showing an early negative effect of PTEN on Rb inactivation. PTEN expression also prevented cyclin D1 from localizing to the nucleus during the G(1)- to S-phase cell cycle transition. The PTEN-induced localization defect and the cell growth arrest could be rescued by the expression of a nucleus-persistent mutant form of cyclin D1, indicating that an important effect of PTEN is at the level of nuclear availability of cyclin D1. Constitutively active Akt/PKB kinase counteracted the effect of PTEN on cyclin D1 translocation. The data are consistent with an oncogenesis model in which a lack of PTEN fuels the cell cycle by increasing the nuclear availability of cyclin D1 through the Akt/PKB pathway.     

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.