Two Novel CMY-2-Type β-Lactamases Encountered in Clinical Escherichia Coli Isolates

BACKGROUND: Chromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.

Influenza A(H1N1)pdm09 Resistance and Cross-Decreased Susceptibility to Oseltamivir and Zanamivir Antiviral Drugs

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011. A total of 144 cases of A(H1N1)pdm09 virus infection from community and hospitalized patients were studied, including three suspected cases of clinical resistance to oseltamivir. Oseltamivir resistance was confirmed for two of the suspected cases. Neuraminidase (NA) H275Y resistant marker was found in viruses from both cases but for one it was only present in 26.2% of virus population, raising questions about the minimal percentage of resistant virus that should be considered relevant. Cross-decreased susceptibility to oseltamivir and zanamivir (2-4 IC50 fold-change) was detected on viruses from two potentially linked community patients from 2009. Both viruses harbored the NA I223V mutation. NA Y155H mutation was found in 18 statistical non-outlier viruses from 2009, having no impact on virus susceptibility. The mutations at NA N369K and V241I may have contributed to the significantly higher baseline IC50 value obtained to oseltamivir for 2010/2011 viruses, compared to viruses from the pandemic period. These results may contribute to a better understanding of the relationship between phenotype and genotype, which is currently challenging, and to the global assessment of A(H1N1)pdm09 virus susceptibility profile and baseline level to NAIs.

Adult B-Cell Acute Lymphoblastic Leukemia Cells Display Decreased PTEN Activity and Constitutive Hyperactivation of PI3K/Akt Pathway Despite High PTEN Protein Levels

Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.

Brazilian multicenter study on efficacy and tolerability of trandolapril in mild-to-moderate essential arterial hypertension. EMBATHE substudy with ambulatory blood pressure monitoring

OBJECTIVE: A double-blind, placebo-controlled multicenter study involving 34 centers from different Brazilian regions was performed to evaluate the antihypertensive efficacy and tolerability of trandolapril, an angiotensin I converting enzyme inhibitor, in the treatment of mild-to-moderate systemic arterial hypertension. METHODS: Of 262 patients enrolled in this study, 127 were treated with trandolapril 2 mg/day for 8 consecutive weeks, and the remaining 135 patients received placebo for the same period of time. Reduction in blood pressure (BP) and the occurrence of adverse events during this period were evaluated in both groups. RESULTS: Significant reductions in both systolic and diastolic pressures were observed in patients treated with trandolapril when compared with those on placebo. Antihypertensive efficacy was achieved in 57.5% of the patients on trandolapril and in 42% of these normal values of BP were obtained. The efficacy of trandolapril was similar in all centers, regardless of the area of the country. In a subset of 30 patients who underwent ABPM, responders showed a significant hypotensive effect to trandolapril throughout the 24 hour day. The adverse event profile was similar in both trandolapril and placebo groups. CONCLUSION: Our results demonstrate, for the first time in a large group of hypertensive patients from different regions in Brazil, good efficacy and tolerability of trandolapril during treatment of mild-to-moderate essential systemic hypertension.

4-Second Exercise Test: Reference Values for Ages 18–81 Years

Background: Physiological reflexes modulated primarily by the vagus nerve allow the heart to decelerate and accelerate rapidly after a deep inspiration followed by rapid movement of the limbs. This is the physiological and pharmacologically validated basis for the 4-s exercise test (4sET) used to assess the vagal modulation of cardiac chronotropism. Objective: To present reference data for 4sET in healthy adults. Methods: After applying strict clinical inclusion/exclusion criteria, 1,605 healthy adults (61% men) aged between 18 and 81 years subjected to 4sET were evaluated between 1994 and 2014. Using 4sET, the cardiac vagal index (CVI) was obtained by calculating the ratio between the duration of two RR intervals in the electrocardiogram: 1) after a 4-s rapid and deep breath and immediately before pedaling and 2) at the end of a rapid and resistance-free 4-s pedaling exercise. Results: CVI varied inversely with age (r = -0.33, p < 0.01), and the intercepts and slopes of the linear regressions between CVI and age were similar for men and women (p > 0.05). Considering the heteroscedasticity and the asymmetry of the distribution of the CVI values according to age, we chose to express the reference values in percentiles for eight age groups (years): 18–30, 31–40, 41–45, 46–50, 51–55, 56–60, 61–65, and 66+, obtaining progressively lower median CVI values ranging from 1.63 to 1.24. Conclusion: The availability of CVI percentiles for different age groups should promote the clinical use of 4sET, which is a simple and safe procedure for the evaluation of vagal modulation of cardiac chronotropism.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

The impact of penicillinase on cefamandole treatment and prophylaxis of experimental endocarditis due to methicillin-resistant Staphylococcus aureus.

Beta-lactams active against methicillin-resistant Staphylococcus aureus (MRSA) must resist penicillinase hydrolysis and bind penicillin-binding protein 2A (PBP 2A). Cefamandole might share these properties. When tested against 2 isogenic pairs of MRSA that produced or did not produce penicillinase, MICs of cefamandole (8-32 mg/L) were not affected by penicillinase, and cefamandole had a > or =40 times greater PBP 2A affinity than did methicillin. In rats, constant serum levels of 100 mg/L cefamandole successfully treated experimental endocarditis due to penicillinase-negative isolates but failed against penicillinase-producing organisms. This suggested that penicillinase produced in infected vegetations might hydrolyze the drug. Indeed, cefamandole was slowly degraded by penicillinase in vitro. Moreover, its efficacy was restored by combination with sulbactam in vivo. Cefamandole also uniformly prevented MRSA endocarditis in prophylaxis experiments, a setting in which bacteria were not yet clustered in the vegetations. Thus, while cefamandole treatment was limited by penicillinase, the drug was still successful for prophylaxis of experimental MRSA endocarditis.

Altered NKG2D function in NK cells induced by chronic exposure to NKG2D ligand-expressing tumor cells.

NKG2D is an activation receptor that allows natural killer (NK) cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the receptor and reduced function upon subsequent receptor engagement. However, it is not clear whether in addition to modulation the NKG2D receptor complex and/or its signaling capacity is preserved. We show here that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of cell-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can be activated via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive interferon gamma (IFNgamma) production, suggesting sustained signaling. The functional changes are associated with a reduced presence of the relevant signal transducing adaptors DNAX-activating protein of 10 kDa (DAP-10) and killer cell activating receptor-associated protein/DNAX-activating protein of 12 kDa (KARAP/DAP-12). That is likely the consequence of constitutive NKG2D engagement and signaling, since NKG2D function and adaptor expression is restored to normal when the stimulating tumor cells are removed. Thus, the chronic exposure to tumor cells expressing NKG2D ligand alters NKG2D signaling and may facilitate the evasion of tumor cells from NK cell reactions.

Metoprolol prevents sodium retention induced by lower body negative pressure in healthy men.

BACKGROUND: Lower body negative pressure (LBNP) has been shown to induce a progressive activation of neurohormonal systems, and a renal tubular and hemodynamic response that mimics the renal adaptation observed in congestive heart failure (CHF). As beta-blockers play an important role in the management of CHF patients, the effects of metoprolol on the renal response were examined in healthy subjects during sustained LBNP. METHODS: Twenty healthy male subjects were randomized in this double blind, placebo versus metoprolol 200 mg once daily, study. After 10 days of treatment, each subject was exposed to 3 levels of LBNP (0, -10, and -20 mbar) for 1 hour, each level of LBNP being separated by 2 days. Neurohormonal profiles, systemic and renal hemodynamics, as well as renal sodium handling were measured before, during, and after LBNP. RESULTS: Blood pressure and heart rate were significantly lower in the metoprolol group throughout the study (P < 0.01). GFR and RPF were similar in both groups at baseline, and no change in renal hemodynamic values was detected at any level of LBNP. However, a reduction in sodium excretion was observed in the placebo group at -20 mbar, whereas no change was detected in the metoprolol group. An increase in plasma renin activity was also observed at -20 mbar in the placebo group that was not observed with metoprolol. CONCLUSION: The beta-blocker metoprolol prevents the sodium retention induced by lower body negative pressure in healthy subjects despite a lower blood pressure. The prevention of sodium retention may be due to a blunting of the neurohormonal response. These effects of metoprolol on the renal response to LBNP may in part explain the beneficial effects of this agent in heart failure patients.

D-amphetamine fails to increase extracellular dopamine levels in mice lacking alpha 1b-adrenergic receptors: relationship between functional and nonfunctional dopamine release.

It was found recently that locomotor and rewarding effects of psychostimulants and opiates were dramatically decreased or suppressed in mice lacking alpha1b-adrenergic receptors [alpha1b-adrenergic receptor knock-outs (alpha1bAR-KOs)] (Drouin et al., 2002). Here we show that blunted locomotor responses induced by 3 and 6 mg/kg d-amphetamine in alpha1bAR-KO mice [-84 and -74%, respectively, when compared with wild-type (WT) mice] are correlated with an absence of d-amphetamine-induced increase in extracellular dopamine (DA) levels in the nucleus accumbens of alpha1bAR-KO mice. Moreover, basal extracellular DA levels in the nucleus accumbens are lower in alpha1bAR-KO than in WT littermates (-28%; p < 0.001). In rats however, prazosin, an alpha1-adrenergic antagonist, decreases d-amphetamine-induced locomotor hyperactivity without affecting extracellular DA levels in the nucleus accumbens, a finding related to the presence of an important nonfunctional release of DA (Darracq et al., 1998). We show here that local d-amphetamine releases nonfunctional DA with the same affinity but a more than threefold lower amplitude in C57BL6/J mice than in Sprague Dawley rats. Altogether, this suggests that a trans-synaptic mechanism amplifies functional DA into nonfunctional DA release. Our data confirm the presence of a powerful coupling between noradrenergic and dopaminergic neurons through the stimulation of alpha1b-adrenergic receptors and indicate that nonfunctional DA release is critical in the interpretation of changes in extracellular DA levels. These results suggest that alpha1b-adrenergic receptors may be important therapeutic pharmacological targets not only in addiction but also in psychosis because most neuroleptics possess anti-alpha1-adrenergic properties.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines.

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.