Accumulation of carbapenem resistance mechanisms in VIM-2-producing Pseudomonas aeruginosa under selective pressure.

Pseudomonas aeruginosa has the potential to achieve resistance to carbapenems via the acquisition of carbapenemase-encoding genes, the downregulation of the OprD porin, the overexpression of efflux systems and the overproduction of cephalosporinases. One hundred and fifty carbapenem-non-susceptible isolates from 2008 to 2010 were screened for carbapenemase production, OprD porin loss, efflux pumps overexpression and inducible AmpC beta-lactamase production. For comparison reasons, the presence of the same mechanisms was also assessed in a previous collection of 30 carbapenem-non-susceptible P. aeruginosa isolated between 2003 and 2005. Results showed the accumulation of various resistance mechanisms among VIM-2 producers isolated between 2008 and 2010 with a parallel considerable increase in imipenem MIC90 and the geometric mean of the MIC values of imipenem and meropenem between the two study groups. The accumulation of carbapenem resistance mechanisms highlights the potential of this formidable pathogen for evolutionary success under antibiotic selective pressure.

Biomarkers in pre-eclampsia: a novel approach to early detection of the disease.

Pre-eclampsia is a unique disorder of human pregnancy with a great impact on maternal and perinatal morbidity and mortality worldwide and especially in developing countries. The aetiology is still unknown and the pathophysiology of the disease is the subject of extensive investigation. Recently, much of the interest of the investigators for the prediction of pre-eclampsia has been aimed at measurable manifestations of abnormal placentation, endothelial dysfunction and feto-maternal unit perfusion. Biomarkers constitute a novel approach to an early detection of the disease. Low maternal serum levels of PAPP-A and PP13 early in pregnancy are predictive for emerging pre-eclampsia. On the other hand, increased levels of homocysteine, ADMA, sEng, leptin and sFlt-1 in the 1st trimester, signal the onset of the disease later in pregnancy. After the onset of pre-eclampsia, increased serum levels of PAPP-A, ADMA, homocysteine and sFlt-1 are associated with the severity of the disease. The identification of biomarkers which can contribute to the early detection of pre-eclampsia is essential. It could then be possible to apply better surveillance and treatment protocols in such patients.

Mechanisms responsible for the emergence of carbapenem resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen associated with a range of nosocomial infections. This microorganism is noted for its intrinsic resistance to antibiotics and for its ability to acquire genes encoding resistance determinants. Among the beta-lactam antibiotics, carbapenems with antipseudomonal activity are important agents for the therapy of infections due to P. aeruginosa. The development of carbapenem resistance among P. aeruginosa strains is multifactorial. Plasmid or integron-mediated carbapenemases, increased expression of efflux systems, reduced porin expression and increased chromosomal cephalosporinase activity have all been defined as contributory factors. Phenotypic tests and molecular techniques are used for the characterization of the resistance determinants. The isolation of carbapenem resistant strains is alarming and requires the implementation of strict infection control measures in order to prevent the spread of carbapenemase encoding genes to unrelated clones or to other bacterial species.

Vaccination with Trichinella spirallis antigens increases CD8+ peripheral T cells and enhances the Th2 immune response in Leishmania infantum challenged mice.

In this study we investigate the effect of Trichinella spiralis vaccination on immune responses elicited in BALB/c mice challenged subcutaneously with 0.5 x 10 6 of Leishmania infantum promastigotes. Secretion of specific anti-L. infantum antibodies and changes in the number of CD4+, CD8+ T cell and CD19+ B cells in the peripheral blood were tested for the evaluation of immune responses. Immunization with low amounts of T. spiralis antigens induced depression in anti-Leishmania specific antibodies of the IgG1 isotype, while no changes in the number of CD4+ and CD8+ T cell subpopulations or CD19+ B cells were observed. In contrast, high amounts of T. spiralis antigens induced an enhancement in anti-Leishmania specific antibodies of total IgG and IgG1 isotype, increase of CD8+ T cell number and activation of CD19+ B cells, indicated by the co-expression of CD69 marker. Our results suggest that immunization with a certain dose of T. spiralis antigens in experimentally challenged mice with L. infantum leads to an increase of peripheral CD8+ T cells which are responsible for the control of L. infantum infection, although a simultaneous enhancement in Th2-type of immune response is also observed.

Cyclosporine enhances liver regeneration: the role of hepatocyte MHC expression and PGE2--a study relevant to graft immunogenicity.

AIM: We have investigated CsA induced liver hyperplasia to explore the potential effects on the immunogenicity of the regenerating liver within the clinical context of rejection after transplantation. MATERIALS AND METHODS: Flow cytometry analysis of hepatocytes, isolated 48 hours after 2/3 partial hepatectomy (PH2/3) or sham operation in rats, was performed to determine the effect of CsA on DNA synthesis and MHC molecule expression. The possible role of PGE2 was evaluated by the administration of SC-19220, an EP1-PGE2 receptor antagonist. RESULTS: CsA augmented liver regeneration and this was partially attenuated by SC-19220. The moderate expression of class I MHC expression, as well as the very low class II MHC expression detected in normal hepatocytes by flow cytometry was augmented after PH2/3 and reduced by CsA. The CsA-mediated decrease of hepatocyte immunogenicity was not SC-19220 dependent. CONCLUSIONS: It is proposed that the enhancing effect of CsA on hepatocyte proliferation is by means of an indirect mechanism that can be attributed to a) reduced immunogenicity of the regenerating liver as a result of inhibition of class I and II MHC hepatocyte expression and b) increased PGE2 synthesis in the liver mediated by its action on EP1 receptor.

Expression of multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1), lung resistance protein (LRP), and breast cancer resistance protein (BCRP) genes and clinical outcome in childhood acute lymphoblastic leukemia.

The aim of this prospective study was to analyze the expression of messenger RNA of genes, such as MDR1, MRP1, BCRP, and LRP, implicated in the mechanism of multidrug resistance (MDR) in relation to the response to induction chemotherapy and relapse and these genes' correlation with each other and with pretreatment laboratory and clinical characteristics. We prospectively studied 49 children (26 boys and 23 girls) with acute lymphoblastic leukemia (ALL) (median age, 5.5 years; range, 15 months to 12.5 years) who were treated with the BFM95 chemotherapy protocol. We used bone marrow mononuclear cells from 7 healthy children as controls. The expression of MDR genes and the beta-actin housekeeping gene was detected by the reverse transcription-polymerase chain reaction with the appropriate primers. The mean expression of each MDR gene was significantly higher in the patients than in the control group (P < .01). We found statistically significant correlations between MRP1 and LRP expression and between MRP1 or LRP expression and MDR1 expression (P < .05). High expression for the MDR1 gene was found in 18 patients (36.7%), and their prognoses were significantly worse than those with low expression (event-free survival, 55.56% versus 86.67%; P = .03, log-rank test). Expression of each of the MDR genes was independent of the initial white blood cell count, immunophenotype, National Cancer Institute risk classification, and prednisone response. Interestingly, MDR1 expression was significantly higher at relapse than at diagnosis for 4 sample pairs. Evaluation of MDR1 expression at diagnosis of childhood ALL may contribute to the early identification of patients at risk of treatment failure.

Genetic heterogeneity underlies juvenile hemochromatosis phenotype: analysis of three families of northern Greek origin.

Hereditary hemochromatosis is a genetically heterogeneous disease. Common HFE mutations (C282Y and H63D) are related to the majority of hereditary hemochromatosis cases in populations of Northern European ancestry (HFE1). Juvenile hemochromatosis (JH) is a more severe iron overload disorder, usually presenting at the second decade of life. The gene responsible for JH lies on a genetic locus at chromosome 1q. We have performed a genetic linkage study in three families of Northern Greek origin with typical clinical features of JH. In two families results were in accordance with linkage to chromosome 1q. In one family linkage of the disease to the genetic loci at 1q21, 7q22, and 6p22 was excluded. We suggest that more than one gene may underlie the JH phenotype. This genetic type of hemochromatosis may be designated 1q unlinked juvenile hemochromatosis. Family studies are necessary to establish the genetic diagnosis of JH.

Increased frequency of the rare PstI allele (P2) in a population of CAD patients in northern Greece.

Restriction fragment length polymorphisms (RFLPs) at the apolipoprotein AI-CIII-AIV gene cluster and their association with coronary artery disease (CAD) and lipid levels were studied in a Northern Greek population. Ninety-five patients with CAD and fifty-four normal controls, angiographically proven, were included in this study. Using genomic hybridization techniques, three polymorphic restriction sites were identified at this locus: the PstI at the 3' end of the apoAI gene, the SacI at the 3' non-coding region of the apoCIII gene and the PvuII at the intergenic region between the apoCIII-AIV genes. The rare allele (P2) arising from the absence of the PstI restriction site was observed with a significantly higher frequency (p < 0.01) in patients compared to normals (0.11 vs 0.02). In contrast, the rare allele for the SacI polymorphic site had a similar distribution among patients and controls (0.12 vs 0.16). The same was observed for the PvuII RFLP (0.04 vs 0.05). Correlation of lipid and apolipoprotein AI levels with the three RFLPs revealed no significant association, although apo AI and HDL were lower in patients with the P2 allele. Thus, in this Greek population, only the PstI polymorphism, among the polymorphic restriction sites examined, appears to be associated with CAD.

HLA class II DNA-RFLP typing in 102 individuals from Northern Greece.

The serological identification of HLA class II alleles is often doubtful. Since accurate HLA typing is essential for the matching of donor-recipient pairs in allogeneic transplantation, an effort was made to establish DNA restriction fragment length polymorphism (RFLP) typing and to assess the correlation between the serological and RFLP techniques in the population of Northern Greece. One hundred and two healthy individuals (204 HLA-DR alleles) from Northern Greece were HLA-DR, DQ typed with both the microcytotoxicity and the Taq I RFLP method, using three exon-specific probes. DNA-RFLP typing revealed (1) concordant results with serology in 69.9% (142/204) of the alleles and (2) at least one HLA-DR allele discrepant to serology in 30.4% (62/204) of the alleles. Incorrect serological DR types (weak reactions or inability to distinguish between two alleles with a common epitope) were identified in 54 alleles (26.5%), while 3.9% (8/204) of serological "blank" alleles turned out to be definable alleles by RFPL. Of the individuals tested, 10.8% (11/102) were DR-homozygous by RFLP. This comparison of results obtained by serology and RFLP demonstrated the necessity of the clinical application of DNA typing, especially for organ transplantation where accurate HLA typing has an important influence on graft survival.

A comparison of sickle cell syndromes in northern Greece.

Haematological and clinical characteristics have been examined in 30 patients with homozygous sickle cell (SS) disease, 28 with sickle cell-beta zero thalassaemia, and 21 with sickle cell-beta+ thalassaemia. The latter could be divided into three groups on their molecular basis and HbA levels, four subjects with an IVS-2 nt 745 mutation having 3-6% HbA (designated S beta+ thalassaemia type I), 14 subjects with an IVS-1 nt 110 mutation having 8-15% HbA (designated S beta+ thalassaemia type II), and three subjects with an IVS-1 nt 6 mutation having 20-25% HbA (designated S beta+ thalassaemia type III). Comparisons were conducted between SS disease, S beta zero thalassaemia, and S beta+ thalassaemia type II. Compared to SS disease, both thalassaemia syndromes had higher HbA2 levels and red cell counts and lower mean cell haemoglobin content (MCHC), mean cell volume (MCV) and MCH, and S beta zero thalassaemia had higher HbF and reticulocyte counts. Compared to S beta zero thalassaemia, S beta+ thalassaemia had a higher haemoglobin and MCHC. Clinically, persistence of splenomegaly was more common in S beta zero and S beta+ thalassaemia type II compared to SS disease. Few significant differences occurred between SS disease, S beta zero and S beta+ thalassaemia type II in Northern Greece suggesting that the 8-15% HbA in the latter condition was insufficient to modify the clinical course.

Cellular and tissue distribution of a single-strand-specific nuclease.

1. Specific antibodies which were raised against a single-strand-specific nuclease isolated from rat liver microsomes were used for characterizing this enzyme and determining its cellular and tissue distribution. 2. The single-strand-specific nuclease does not show any homology with other known nucleolytic enzymes. 3. It is mainly localized in microsomes and cytosol; traces of it are also found in nuclei, but it could not be detected in mitochondria. 4. Using the same specific antibodies we attempted to detect this nuclease in some other tissues which exhibit high metabolic rates, namely kidneys, heart and spleen. 5. Thus, with the aid of immunological techniques we were able to determine that at least part of the total poly(U) nucleolytic activity observed in kidney and heart is due to a nuclease immunologically identical to the enzyme under study. Kidneys were the best source for this enzyme.

Comparison of homozygous sickle cell disease in northern Greece and Jamaica.

The clinical and haematological features of homozygous sickle cell (SS) disease were compared in 30 Greek and 310 Jamacian patients. Deletional alpha-thalassaemia, which modifies SS disease, is rare among Greek patients, so only Jamacian patients with four alpha-globin genes were included in the control group. Greek patients had higher total haemoglobin concentration and red cell counts, and lower mean cell haemoglobin concentration (MCHC) and reticulocyte counts. They also had a more normal body build and more adults had persistent splenomegaly. Fewer had a history of leg ulceration or priapism but more reported acute chest syndrome. The comparatively mild disease in Greek patients is consistent with less haemolysis and sickling and therefore less bone marrow expansion. In the absence of amelioriating factors such as high HbF concentration or alpha-thalassaemia, these findings may be explained by the low MCHC.

Comparison of homozygous sickle cell disease in Northern Greece

The clinical and hematological features of homozygous sickle cell (SS) disease were compared in 30 Greek and 310 Jamaican patients. Deletional O-thallassaemia, which modifies SS disease, is rare among Greek patients, so only Jamaican patients with four O-globin genes were included in the control group. Greek patients had higher total haemoglobin concentration and red cell counts, and lower mean cell haemoglobin concentration (MCHC) and reticulocyte counts. They also had a more normal body build and more adults had persistent splenomegaly. Fewer had a history of leg ulceration or priapism but more reported acute chest syndrome. The comparitively mild disease in Greek patients is consistent with less haemolysis and sickling and therefore less bone marrow expansion. In the absence of amelioriating factors such as high HbF concentration or O-thalassaemia, these findings may be explained by the low MCHC. (AU)

Single-strand-specific nuclease from rat liver endoplasmic reticulum: characterization and mode of action.

1. The characteristics and mode of action of a single-strand-specific nuclease isolated from rat liver endoplasmic reticulum are investigated with respect to its DNA and RNA substrates. 2. The RNase activity of the enzyme is slightly influenced by the presence of divalent cations but the DNase activity is enhanced by divalent cations particularly Mn2+. 3. Activity is partially inhibited by the presence of EGTA; this effect is reversed most efficiently by the addition of Mn2+. 4. The enzyme exhibits small pH dependence between pH 6-9 and maximum activity is observed at pH 7-7.5 for both DNase and RNase activities. 5. Sulfhydryl group reagents do not affect its action but histidyl group reagents exert a small but definite effect. 6. The enzyme degrades DNA and RNA endonucleolytically producing fragments which possess 3'-OH and 5'-phosphate termini. 7. Monomers are not produced even after prolonged degradation. 8. The end product of poly(U)degradation ranges between two and four building blocks but the DNA product is longer probably due to considerable percentage of secondary structure.