Frequent recovery of a single clonal type of multidrug-resistant Staphylococcus aureus from patients in two hospitals in Taiwan and China

One hundred thirty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from patients with S. aureus infections between January 1998 and February 1999 in two hospitals, one located in Taipei, Taiwan, and another in Nanjing, People's Republic of China, were examined for antibiotic susceptibility and for clonal type by a combination of three methods: hybridization of ClaI restriction digests with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis of chromosomal SmaI digests. Selected isolates representing each clonal type were also analyzed by spaA typing, multilocus sequence typing, and a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. The overwhelming majority of isolates (126 of 132 or 95%) belonged to minor variants of a single clonal type resembling the Brazilian and Hungarian epidemic MRSA clones, which showed a common spaA type and which were either sequence type 239 (ST239) or ST241 (a single-locus variant of ST239) in association with SCCmec type III or IIIA.     

CD4+ T-cell memory: generation and multi-faceted roles for CD4+ T cells in protective immunity to influenza

Summary: We have outlined the carefully orchestrated process of CD4⁺ T‐cell differentiation from naïve to effector and from effector to memory cells with a focus on how these processes can be studied in vivo in responses to pathogen infection. We emphasize that the regulatory factors that determine the quality and quantity of the effector and memory cells generated include (i) the antigen dose during the initial T‐cell interaction with antigen‐presenting cells; (ii) the dose and duration of repeated interactions; and (iii) the milieu of inflammatory and growth cytokines that responding CD4⁺ T cells encounter. We suggest that heterogeneity in these regulatory factors leads to the generation of a spectrum of effectors with different functional attributes. Furthermore, we suggest that it is the presence of effectors at different stages along a pathway of progressive linear differentiation that leads to a related spectrum of memory cells. Our studies particularly highlight the multifaceted roles of CD4⁺ effector and memory T cells in protective responses to influenza infection and support the concept that efficient priming of CD4⁺ T cells that react to shared influenza proteins could contribute greatly to vaccine strategies for influenza.     

Methylglyoxal chronic administration promotes diabetes-like cardiac ischaemia disease in Wistar normal rats

Background and aimsThe influence of lifestyle is well documented, especially the diet regime, in the development of type 2 diabetes (T2D) and associated cardiovascular diseases. Diabetic patients have increased risk of suffering cardiac ischemia and impaired response to such accidents. Methylglyoxal (MG) circulates at high concentration in diabetics' blood and is linked to the development of diabetes chronic complications. We propose that besides promoting the cardiovascular disease, MG may also negatively regulate the endogenous cardioprotection pathways after ischemia.Methods and resultsWe performed a comparative study between three animal groups: normal Wistar (W), type 2 diabetic non-obese Goto-Kakizaki (GK) and normal rats submitted to MG chronic administration (3months) with gradually enhanced concentration, up to 75 mg/Kg (WMG). Hearts were submitted to different experimental conditions: control, ischemia and ischemia-reperfusion. Levels of oxidative stress markers, advanced glycation end-products (AGEs) and their receptors (RAGEs) were evaluated. The serine/threonine protein kinase Akt (Akt), crucial for cardiomyocytes recovery after ischemia, and apoptosis markers were also assessed.Levels of MG, systemic and cardiac oxidative stress markers, AGEs and RAGEs were similar in GK and WMG groups. Akt protein was negatively regulated by MG, leading to impaired apoptotic markers.ConclusionChronic MG administration to normal rodents mimicked most diabetic alterations, being associated with the development of cardiovascular disease and the impairment of survival pathways. Our results demonstrate the negative effect of MG rich diet in healthy animals and suggest the potential of methylglyoxal as a therapeutic target in diabetes.     

Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and immature myeloid elements, whereas erythroid precursors and lymphoid cells lacked VEGF and receptor expression. Foci of abnormal localized immature myeloid precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting autocrine cytokine interaction. Antibody neutralization of VEGF inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony formation in 12 patients. Neutralization of VEGF activity suppressed the generation of tumor necrosis factor-alpha and interleukin-1beta from MDS BM-mononuclear cells and BM-stroma and promoted the formation of CFU-GEMM and burst-forming unit-erythroid in methylcellulose cultures. These findings indicate that autocrine production of VEGF may contribute to leukemia progenitor self-renewal and inflammatory cytokine elaboration in CMML and MDS and thus provide a biologic rationale for ALIP and its adverse prognostic relevance in high-risk MDS.     

Population genetic implications from sequence variation in four Y chromosome genes

Some insight into human evolution has been gained from the sequencing of four Y chromosome genes. Primary genomic sequencing determined gene SMCY to be composed of 27 exons that comprise 4,620 bp of coding sequence. The unfinished sequencing of the 5' portion of gene UTY1 was completed by primer walking, and a total of 20 exons were found. By using denaturing HPLC, these two genes, as well as DBY and DFFRY, were screened for polymorphic sites in 53-72 representatives of the five continents. A total of 98 variants were found, yielding nucleotide diversity estimates of 2.45 x 10(-5), 5. 07 x 10(-5), and 8.54 x 10(-5) for the coding regions of SMCY, DFFRY, and UTY1, respectively, with no variant having been observed in DBY. In agreement with most autosomal genes, diversity estimates for the noncoding regions were about 2- to 3-fold higher and ranged from 9. 16 x 10(-5) to 14.2 x 10(-5) for the four genes. Analysis of the frequencies of derived alleles for all four genes showed that they more closely fit the expectation of a Luria-Delbrück distribution than a distribution expected under a constant population size model, providing evidence for exponential population growth. Pairwise nucleotide mismatch distributions date the occurrence of population expansion to approximately 28,000 years ago. This estimate is in accord with the spread of Aurignacian technology and the disappearance of the Neanderthals.     

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and β cells has not been elucidated. Here we implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. Our results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.The endoplasmic reticulum (ER) takes center stage for protein production, redox regulation, calcium homeostasis, and cell death (1, 2). It follows that genetic or acquired ER dysfunction can trigger a variety of common diseases, including neurodegenerative diseases, metabolic disorders, and inflammatory bowel disease (3, 4). Breakdown in ER function is also associated with genetic disorders such as Wolfram syndrome (5–8). It is challenging to determine the exact effects of ER dysfunction on the fate of affected cells in common diseases with polygenic and multifactorial etiologies. In contrast, we reasoned that it should be possible to define the role of ER dysfunction in mechanistically homogenous patient populations, especially in rare diseases with a monogenic basis, such as Wolfram syndrome (9).Wolfram syndrome (OMIM 222300) is a rare autosomal recessive disorder characterized by juvenile-onset diabetes mellitus and bilateral optic atrophy (7). Insulin-dependent diabetes usually occurs as the initial manifestation during the first decade of life, whereas the diagnosis of Wolfram syndrome is invariably later, with onset of symptoms in the second and ensuing decades (7, 10, 11). Two causative genes for this genetic disorder have been identified and named Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2) (12, 13). It has been shown that multiple mutations in the WFS1 gene, as well as a specific mutation in the WFS2 gene, lead to β cell death and neurodegeneration through ER and mitochondrial dysfunction (5, 6, 14–16). WFS1 gene variants are also associated with a risk of type 2 diabetes (17). Moreover, a specific WFS1 variant can cause autosomal dominant diabetes (18), raising the possibility that this rare disorder is relevant to common molecular mechanisms altered in diabetes and other human chronic diseases in which ER dysfunction is involved.Despite the underlying importance of ER malfunction in Wolfram syndrome, and the identification of WFS1 and WFS2 genes, a molecular mechanism linking the ER to death of neurons and β cells has not been elucidated. Here we show that the calpain protease provides a mechanistic link between the ER and death of neurons and β cells in Wolfram syndrome.     

Evaluation of Immunogenicity and Efficacy of Anthrax Vaccine Adsorbed for Postexposure Prophylaxis

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.