Evalution of methods for the detection of MRSA in patients of Botucatu Medical School Hospital, São Paulo, Brazil

Oxacillin is the main drug of choice for the treatment of S. aureus infections. However, S. aureus resistance to oxacillin has become a major problem in the recent decades. The study aimed assess the rates of oxacillin resistance in S. aureus samples obtained at the Botucatu Medical School Hospital, UNESP, and to compare phenotypic techniques for the detection of MRSA against the gold standard method (mecA gene detection) in these samples. A total of 102 samples, previously isolated between 2002 and 2006, and kept at the Culture Collection of the Department of Microbiology and Immunology, in the Botucatu Biosciences Institute, UNESP, were included. Oxacillin resistance was assessed by oxacillin and cefoxitin disk diffusion and agar dilution tests, screening tests using Mueller-Hinton agar with 6 mu g/mL of oxacillin and 4% NaCl, E-test, and mecA gene detection. of the samples analyzed, 46 (45.1%) were mecA-positive. Oxacillin disk sensitivity and specificity were 86.9% and 91.1%, respectively. Cefoxitin disk sensitivity and specificity were respectively 91.3% and 91.1%. The screening test with the cefoxitin disk showed almost the same level of sensitivity (91.3%) and specificity (91.1%). With E-test strips, sensitivity was higher (97.8%) and specificity was comparable to that found with the other methods (91.1%). Ninety-three percent of the samples produced beta-lactamase and five of them were mecA-negative. There was a gradual increase in the number of oxacillin-resistant S. aureus samples between 2002 and 2004. However, from 2004 to 2006, the number of resistant samples dropped from 55% of MRSA in 2004, to 45% in 2005 and 34.6% in 2006. The data obtained reveal that, among phenotypic methods, the E-test yielded the best results, with higher sensitivity levels when compared to the other methods. The decreased resistance rate observed over the most recent years may be explained by the rational use of antimicrobial agents associated with good practices in the control of hospital infection, or may be related to the diminished use of oxacillin as a treatment option.

Genomics and proteomics approaches to the study of cancer-stroma interactions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of CDKN1A polymorphisms: markers of cancer susceptibility?

The CDKN1A (TP21)(2) gene encodes a 21-kD protein that is a critical downstream mediator of wild-type TP53 and an important regulator of the cell cycle. Failure in the function of this gene would be expected to result in abnormal cell proliferation and transformation. Tumor-associated mutations of the coding region of the TP21 are rare. on the other hand, some TP21 polymorphisms have been identified and characterized by single base substitutions. In the present study, we investigated the potential role of TP21 gene polymorphisms in skin, head, and neck tumorigenesis. A total of 261 samples were examined by polymerase chain reaction single-strand conformational analysis, and one mutation at codon 31 and four polymorphisms in exons 2 (codon 55) and 3 [nucleotide (nt)590] and in promoter region (nt2298) were identified. In conclusion, this investigation confirmed the rarity of mutations in this gene, arguing against a role for TP21 mutations in skin, head, and neck cancers. Also, our results show significant differences in nt2298 allele frequencies between normal individuals and skin malignant tumors (P < 0.05). The results suggest that this polymorphism affects TP21 transactivator binding and may be important during the pathogenesis of skin cancer. (C) 2003 Elsevier B.V. All rights reserved.

Peripheral kappa and delta opioid receptors are involved in the antinociceptive effect of crotalphine in a rat model of cancer pain

Cancer pain is an important clinical problem and may not respond satisfactorily to the current analgesic therapy. We have characterized a novel and potent analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in a rat model of cancer pain induced by intraplantar injection of Walker 256 carcinoma cells. Intraplantar injection of tumor cells caused the development of hyperalgesia and allodynia, detected on day 5 after tumor cell inoculation. Crotalphine (6 μg/kg), administered p.o., blocked both phenomena. The antinociceptive effect was detected 1 h after treatment and lasted for up to 48 h. Intraplantar injection of nor-binaltorphimine (50 g/paw), a selective antagonist of κ-opioid receptors, antagonized the antinociceptive effect of the peptide, whereas N,N-diallyl-Tyr-Aib-Phe-Leu (ICI 174,864, 10 μg/paw), a selective antagonist of δ-opioid receptors, partially reversed this effect. On the other hand, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP, 20 g/paw), an antagonist of μ-opioid receptors, did not modify crotalphine-induced antinociception. These data indicate that crotalphine induces a potent and long lasting opioid-mediated antinociception in cancer pain. © 2013 Elsevier Inc.

Brief Report: The lincRNA Hotair Is Required for Epithelial-to-Mesenchymal Transition and Stemness Maintenance of Cancer Cell Lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Physical activity, adiposity and hypertension among patients of public healthcare system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nanoparticles increase the efficacy of cancer chemopreventive agents in cells exposed to cigarette smoke condensate

Lung cancer is a leading cause of death in developed countries. Although smoking cessation is a primary strategy for preventing lung cancer, former smokers remain at high risk of cancer. Accordingly, there is a need to increase the efficacy of lung cancer prevention. Poor bioavailability is the main factor limiting the efficacy of chemopreventive agents. The aim of this study was to increase the efficacy of cancer chemopreventive agents by using lipid nanoparticles (NPs) as a carrier. This study evaluated the ability of lipid NPs to modify the pharmacodynamics of chemopreventive agents including N-acetyl-L-cysteine, phenethyl isothiocyanate and resveratrol (RES). The chemopreventive efficacy of these drugs was determined by evaluating their abilities to counteract cytotoxic damage (DNA fragmentation) induced by cigarette smoke condensate (CSC) and to activate protective apoptosis (annexin-V cytofluorimetric staining) in bronchial epithelial cells both in vitro and in ex vivo experiment in mice. NPs decreased the toxicity of RES and increased its ability to counteract CSC cytotoxicity. NPs significantly increased the ability of phenethyl isothiocyanate to attenuate CSC-induced DNA fragmentation at the highest tested dose. In contrast, this potentiating effect was observed at all tested doses of RES, NPs dramatically increasing RES-induced apoptosis in CSC-treated cells. These results provide evidence that NPs are highly effective at increasing the efficacy of lipophilic drugs (RES) but are not effective towards hydrophilic agents (N-acetyl-L-cysteine), which already possess remarkable bioavailability. Intermediate effects were observed for phenethyl isothiocyanate. These findings are relevant to the identification of cancer chemopreventive agents that would benefit from lipid NP delivery.

Modeling spatial distribution of cancer incidence in Cordoba, Argentina

Background: Cancer is the second leading cause of death in Argentina, and there is little knowledge about its incidence. The first study based on population-based cancer registry described spatial incidence and indicated that there existed at least county-level aggregation. The aim of the present work is to model the incidence patterns for the most incidence cancer in Córdoba Province, Argentina, using information from the Córdoba Cancer Registry by performing multilevel mixed model approach to deal with dependence and unobserved heterogeneity coming from the geo-reference cancer occurrence. Methods: Standardized incidence rates (world standard population) (SIR) by sex based on 5-year age groups were calculated for 109 districts nested on 26 counties for the most incidence cancers in Cordoba using 2004 database. A Poisson twolevel random effect model representing unobserved heterogeneity between first level-districts and second level-counties was fitted to assess the spatial distribution of the overall and site specific cancer incidence rates. Results: SIR cancer at Córdoba province shown an average of 263.53±138.34 and 200.45±98.30 for men and women, respectively. Considering the ratio site specific mean SIR to the total mean, breast cancer ratio was 0.25±0.19, prostate cancer ratio was 0.12±0.10 and lower values for lung and colon cancer for both sexes. The Poisson two-level random intercepts model fitted for SIR data distributed with overdispersion shown significant hierarchical structure for the cancer incidence distribution. Conclusions: a strong spatial-nested effect for the cancer incidence in Córdoba was observed and will help to begin the study of the factors associated with it.

Heterogeneous time-dependent response of adipose tissue during the development of cancer cachexia

Cancer cachexia induces loss of fat mass that accounts for a large part of the dramatic weight loss observed both in humans and in animal models; however, the literature does not provide consistent information regarding the set point of weight loss and how the different visceral adipose tissue depots contribute to this symptom. To evaluate that, 8-week-old male Wistar rats were subcutaneously inoculated with 1 ml (2 x 10(7)) of tumour cells (Walker 256). Samples of different visceral white adipose tissue (WAT) depots were collected at days 0, 4, 7 and 14 and stored at -80 degrees C (seven to ten animals/each day per group). Mesenteric and retroperitoneal depot mass was decreased to the greatest extent on day 14 compared with day 0. Gene and protein expression of PPAR gamma(2) (PPARG) fell significantly following tumour implantation in all three adipose tissue depots while C/EBP alpha (CEBPA) and SREBP-1c (SREBF1) expression decreased over time only in epididymal and retroperitoneal depots. Decreased adipogenic gene expression and morphological disruption of visceral WAT are further supported by the dramatic reduction in mRNA and protein levels of perilipin. Classical markers of inflammation and macrophage infiltration (f4/80, CD68 and MIF-1 alpha) in WAT were significantly increased in the later stage of cachexia (although showing a incremental pattern along the course of cachexia) and presented a depot-specific regulation. These results indicate that impairment in the lipid-storing function of adipose tissue occurs at different times and that the mesenteric adipose tissue is more resistant to the 'fat-reducing effect' than the other visceral depots during cancer cachexia progression. Journal of Endocrinology (2012) 215, 363-373

Preclinical development of cancer vaccines

Triplex cell vaccine is a cancer immunopreventive cell vaccine that can prevent almost completely mammary tumor onset in HER-2/neu transgenic mice. A future translation of cancer immunoprevention from preclinical to clinical studies should take into account several aspects. The work reported in this thesis deals with the study of three of these aspects: vaccine schedule, activity in a therapeutic set-up and second-generation DNA vaccines. An important element in determining human acceptance and compliance of a treatment protocol is the number of vaccinations. In order to improve the vaccination schedule a minimal protocol was searched, i.e. a schedule consisting of a lower number of administrations than standard protocol but with a similar efficacy. A candidate optimal protocol was identified by the use of an in silico model, SimTriplex simulator. The in vivo test of this schedule in HER-2/neu transgenic mice only partially confirmed in silico predictions. This result shows that in silico models have the potential ability to aid in searching of optimal treatment protocols, provided that they will be further tuned on experimental data. As a further result this preclinical study highlighted that kinetic of antibody response plays a major role in determining cancer prevention, leading to the hypothesis of a threshold that must be reached rapidly and maintained lifetime. Early clinical trials would be performed in a therapeutic, rather than preventive, setting. Thus, the activity of Triplex vaccine was investigated against experimental lung metastases in HER-2/neu transgenic mice in order to evaluate if the immunopreventive Triplex vaccine could be effective also against a pre-existing tumor mass. This preclinical model of aggressive metastatic development showed that the vaccine was an efficient treatment also 4 for the cure of micrometastases. However the immune mechanisms activated against tumor mass were not antibody dependent, i.e. different from those preventing the onset of primary mammary carcinoma. DNA vaccines could be more easily used than cellular ones. A second generation of Triplex vaccine based on DNA plasmids was evaluated in an aggressive preclinical model (BALBp53neu female mice) and compared with the preventive ability of cellular Triplex vaccine. It was observed that Triplex DNA vaccine was as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Improving adoptive T cell therapies of cancer by ectopic expression of miR181a to repress inhibitory phosphatases

Adoptive T cell therapy using antigen-specific T lymphocytes is a powerful immunotherapeutic approach against cancer. Nevertheless, many T cells against tumor-antigens exhibit only weak anti-tumoral response. To overcome this barrier it is necessary to improve the potency and anti-tumoral efficacy of these T cells. Activation and activity of T cells are tightly controlled to inhibit unwanted T cell responses and to reduce the risk of autoimmunity. Both are regulated by extrinsic signals and intrinsic mechanisms which suppress T cell activation. The intrinsic mechanisms include the expression of phosphatases that counteract the activation-inducing kinases. Modifying the expression of these phosphatases allows the targeted modulation of T cell reactivity. MicroRNAs (miRNAs) are regulatory small noncoding RNA molecules that control gene expression by targeting messenger RNAs in a sequence specific manner. Gene-specific silencing plays a key role in diverse biological processes, such as development, differentiation, and functionality. miR181a has been shown to be highly expressed in immature T cells that recognize low-affinity antigens.rnThe present study successfully shows that ectopic expression of miR181a is able to enhance the sensitivity of both murine and human T cells. In CD4+ T helper cells as well as in CD8+ cytotoxic T cells the overexpression of miR181a leads to downregulation of multiple phosphatases involved in the T cell receptor signaling pathway. Overexpression of miR181a in human T cells achieves a co-stimulatory independent activation and has an anti-apoptotic effect on CD4+ T helper cells. Additionally, increasing the amount of miR181a enhances the cytolytic activity of murine CD8+ TCRtg T cells in an antigen-specific manner.rnTo test miR181a overexpressing T cells in vivo, a mouse tumor model using a B cell lymphoma cell line (A20-HA) expressing the Influenza hemagglutinin (Infl.-HA) antigen was established. The expression of model antigens in tumor cell lines enables targeted elimination of tumors using TCRtg T cells. The transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ T cells alone has no positive effect neither on tumor control nor on survival of A20-HA tumor-bearing mice. In contrast, the co-transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ and CD4+ T cells leads to improved tumor control and prolongs survival of A20-HA tumor-bearing mice. This effect is characterized by higher amounts of effector T cells and the expansion of Infl.-HA TCRtg CD8+ T cells.rnAll effects were achieved by changes in expression of several genes including molecules involved in T cell differentiation, activation, and regulation, cytotoxic effector molecules, and receptors important for the homing process of T cells in miR181a overexpressing T cells. The present study demonstrates that miR181a is able to enhance the anti-tumoral response of antigen-specific T cells and is a promising candidate for improving adoptive cell therapy.

Changing patterns of cancer incidence in the early- and late-HAART periods: the Swiss HIV Cohort Study

The advent of highly active antiretroviral therapy (HAART) in 1996 led to a decrease in the incidence of Kaposi's sarcoma (KS) and non-Hodgkin's lymphoma (NHL), but not of other cancers, among people with HIV or AIDS (PWHA). It also led to marked increases in their life expectancy.