Peripheral insulin resistance and impaired insulin action are the primary characteristics of type 2 diabetes. The first observable defect in this major disorder occurs in muscle, where glucose disposal in response to insulin is impaired. We have developed a transgenic mouse with a dominant-negative insulin-like growth factor-I receptor (KR-IGF-IR) specifically targeted to the skeletal muscle. Expression of KR-IGF-IR resulted in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby abrogating the normal function of these receptors and leading to insulin resistance. Pancreatic ␤-cell dysfunction developed at a relative early age, resulting in diabetes. These mice provide an excellent model to study the molecular mechanisms underlying the development of human type 2 diabetes.

Maladaptive inflammation is a major suspect in progressive neurodegeneration, but the underlying mechanisms are difficult to envisage in part because reactive glial cells at lesion sites secrete both proinflammatory and anti-inflammatory mediators. We now report that astrocytes modulate neuronal resilience to inflammatory insults through the phosphatase calcineurin. In quiescent astrocytes, inflammatory mediators such as tumor necrosis factor-␣ (TNF-␣) recruits calcineurin to stimulate a canonical inflammatory pathway involving the transcription factors nuclear factor B (NFB) and nuclear factor of activated T-cells (NFAT). However, in reactive astrocytes, local anti-inflammatory mediators such as insulin-like growth factor I also recruit calcineurin but, in this case, to inhibit NFB/NFAT. Proof of concept experiments in vitro showed that expression of constitutively active calcineurin in astrocytes abrogated the inflammatory response after TNF-␣ or endotoxins and markedly enhanced neuronal survival. Furthermore, regulated expression of constitutively active calcineurin in astrocytes markedly reduced inflammatory injury in transgenic mice, in a calcineurin-dependent manner. These results suggest that calcineurin forms part of a molecular pathway whereby reactive astrocytes determine the outcome of the neuroinflammatory process by directing it toward either its resolution or its progression.

bWe investigated the mechanism of carbapenem resistance in 10 Acinetobacter baumannii strains isolated from the United States and Mexico between 2005 and 2009. The detection of known metallo-␤-lactamase or carbapenem-hydrolyzing oxacillinase (OXA) genes by PCR was negative. The presence of plasmid-encoded carbapenem resistance genes was investigated by transformation of A. baumannii ATCC 17978. Shotgun cloning experiments and sequencing were performed, followed by the expression of a novel ␤-lactamase in A. baumannii. Three novel OXA enzymes were identified, OXA-235 in 8 isolates and the amino acid variants OXA-236 (Glu173-Val) and OXA-237 (Asp208-Gly) in 1 isolate each. The deduced amino acid sequences shared 85% identity with OXA-134, 54% to 57% identities with the acquired OXA-23, OXA-24, OXA-58, and OXA-143, and 56% identity with the intrinsic OXA-51 and, thus, represent a novel subclass of OXA. The expression of OXA-235 in A. baumannii led to reduced carbapenem susceptibility, while cephalosporin MICs were unaffected. Genetic analysis revealed that bla OXA-235 , bla OXA-236 , and bla OXA-237 were bracketed between two ISAba1 insertion sequences. In addition, the presence of these acquired ␤-lactamase genes might result from a transposition-mediated mechanism. This highlights the propensity of A. baumannii to acquire multiple carbapenem resistance determinants.

Objective. To compare the efficacy of combination therapy with methotrexate (MTX) and hydroxychloroquine (HCQ), MTX and sulfasalazine (SSZ), and MTX, HCQ, and SSZ in patients with rheumatoid arthritis (RA).Methods. RA patients (n ‫؍‬ 171) who had not previously been treated with combinations of the study medications were randomized to receive 1 of the 3 treatment combinations in this 2-year, double-blind, placebo-controlled protocol. HCQ was given at a dosage of 200 mg twice a day. The dosage of MTX was accelerated from 7.5 mg/week to 17.5 mg/week in all patients who were not in remission. Similarly, the dosage of SSZ was escalated from 500 mg twice a day to 1 gm twice a day in patients who were not in remission. The primary end point of the study was the percentage of patients who had a 20% response to therapy according to the American College of Rheumatology (ACR) criteria at 2 years.Results. Intent-to-treat analysis revealed that patients receiving the triple combination responded best, with 78% achieving an ACR 20% response at 2 years, compared with 60% of those treated with MTX and HCQ (P ‫؍‬ 0.05) and 49% of those treated with MTX and SSZ (P ‫؍‬ 0.002). Similar trends were seen for the ACR 50% response, with 55%, 40%, and 29% of patients in the 3 treatment groups, respectively, achieving these results at 2 years (P ‫؍‬ 0.005 for the triple combination group versus the MTX and SSZ group). All combination treatments were well-tolerated. Fourteen patients (evenly distributed among the 3 groups) withdrew from the protocol because of symptoms that were potentially related to the study medication.Conclusion. The triple combination of MTX, SSZ, and HCQ is well-tolerated, and its efficacy is superior to that of the double combination of MTX and SSZ and is marginally superior to that of the double combination of MTX and HCQ.

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Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, Ec⌬acrA and Ec⌬tolC and Jc⌬acrA and Jc⌬tolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/Ec⌬acrA, 0.24 and 0.38 for EcDC64/Ec⌬tolC, 0.15 and 0.11 for Jc194/Jc⌬acrA, and 0.38 and 0.39 for Jc194/Jc⌬tolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.

IntroductionIncreasing evidence supports the view that the detection of circulating tumor cells (CTCs) predicts outcomes of nonmetastatic breast cancer patients. CTCs differ genetically from the primary tumor and may contribute to variations in prognosis and response to therapy. As we start to understand more about the biology of CTCs, we can begin to address how best to treat this form of disease.MethodsNinety-eight nonmetastatic breast cancer patients were included in this study. CTCs were isolated by immunomagnetic techniques using magnetic beads labelled with a multi-CK-specific antibody (CK3-11D5) and CTC detection through immunocytochemical methods. Estrogen receptor, progesterone receptor and epidermal growth factor receptor (EGFR) were evaluated by immunofluorescence experiments and HER2 and TOP2A by fluorescence in situ hybridization. We aimed to characterize this set of biomarkers in CTCs and correlate it with clinical-pathological characteristics.ResultsBaseline detection rate was 46.9% ≥ 1 CTC/30 ml threshold. CTC-positive cells were more frequent in HER2-negative tumors (p = 0.046). In patients younger than 50 years old, HER2-amplified and G1-G2 tumors had a higher possibility of being nondetectable CTCs. Heterogeneous expression of hormonal receptors (HRs) in samples from the same patients was found. Discordances between HR expression, HER2 and TOP2A status in CTCs and their primary tumor were found in the sequential blood samples. Less that 35% of patients switched their CTC status after receiving chemotherapy. EGFR-positive CTCs were associated with Luminal tumors (p = 0.03).ConclusionsThis is the largest exploratory CTC biomarker analysis in nonmetastatic BC patients. Our study suggests that CTC biomarkers profiles might be useful as a surrogate marker for therapeutic selection and monitoring since heterogeneity of the biomarker distribution in CTCs and the lack of correlation with the primary tumor biomarker status were found. Further exploration of the association between EGFR-positive CTCs and Luminal tumors is warranted.

CD133 has been associated with cell properties such as self renewal, migration and vasculogenic mimicry, potentially involved in generation of circulating tumor cells (CTCs). We characterized CD133 expression in CTCs of 98 nometastatic breast cancer (BC) patients. CTCs were isolated by immunomagnetic techniques using magnetic beads labeled with a multicytokeratin(CK)-specific antibody (CK3-11D5) and CTCs and CD133 detection through immunocytochemical methods. CK 1 /CD133 1 CTCs were identified in 65% of patients at baseline and 47.8% after systemic therapy (p 5 0.53). Correlation of CD133 status in CTCs with classical clinicopathological characteristics and response to therapy was performed. Her2 not amplified and low Ki-67 index were positively correlated with presence of CK 1 /CD133 1 CTCs. Before any treatment, CK 1 /CD133 1 CTCs were more frequently isolated in patients with luminal BC subtype. No statistically significant differences were found between proportion of CK 1 /CD133 1 CTCs and BC subtypes after systemic therapy, implying a relative enrichment of CK 1 /CD133 1 CTCs in triple negative and HER2-amplified tumors. While CK 1 /CTCs decreases after chemotherapy when analyzing the whole population, CK 1 / CD133 1 CTCs were enriched in post-treatment samples in nonluminal BC subtypes. These findings suggest the potential role of CD133 as a promising marker of chemoresistance in nonluminal BC patients. Further prospective studies and extensive preclinical modeling will be needed to confirm whether CD133 is a marker of resistance to chemotherapy, and its role as a target for novel anticancer therapies targeting cancer stem cells and tumor vasculature.Breast cancer (BC) is a complex and heterogeneous disease comprising multiple tumor entities associated with distinctive different biological feature, clinical behaviors and response to therapy. The current classification of BC is based on the pattern of expression of the hormone receptors (HR) estrogen receptor and/or progesterone receptor, and human epidermal receptor 2 (Her2) that identify three major BC subtypes: luminal tumors, which are HR positive and Her2 negative; Her2 amplified tumors; and those tumors with lack of expression of the three receptors, referred to as triple negative (TN) BC. 1 Despite advances in BC treatments, primary and acquired resistance to cancer therapies remains a challenge especially in nonmetastatic patients. Cancer stem cells (CSC) have been