Killing of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> yeast cells by IFN-γ and TNF-α activated murine peritoneal macrophages: evidence of H<Subscript>2</Subscript>O<Subscript>2</Subscript> and NO effector mechanisms

Paracoccidioidomycosis is a deep mycosis, endemic in Latin America, caused by Paracoccidioides brasiliensis. Macrophage activation by cytokines is the major effector mechanism against this fungus. This work aimed at a better understanding of the interaction between yeast cells-murine peritoneal macrophages and the cytokine signals required for the effective killing of high virulence yeast-form of P. brasiliensis. In addition, the killing effector mechanisms dependent on the generation of reactive oxygen or nitrogen intermediates were investigated. Cell preincubation with IFN-gamma or TNF-alpha, at adequate doses, resulted in effective yeast killing as demonstrated in short-term (4-h) assays. Both, IFN-gamma and TNF-alpha activation were associated with higher levels of H(2)O(2) and NO when compared to nonactivation. Treatment with catalase (CAT), a H(2)O(2 )scavenger, and N(G)-monomethyl-L: -arginine (L: -NMMA), a nitric oxide synthase inhibitor, reverted the killing effect of activated cells. Taken together, these results suggest that both oxygen and L: -arginine-nitric oxide pathways play a role in the killing of highly virulent P. brasiliensis.     

Metabolomic analysis of cancer cachexia reveals distinct lipid and glucose alterations

Cancer cachexia remains a challenging clinical problem with complex pathophysiology and unreliable diagnostic tools. A blood test to detect this metabolic derangement would aid in early treatment of these patients. A ¹H NMR-based metabolomics approach was used to determine the unique metabolic fingerprint of cachexia and to search for biomarkers in serum samples taken from an established murine model of cancer cachexia. Male CD2F1 mice received a subcutaneous flank injection of C26 adenocarcinoma cells to induce experimental cancer-related cachexia. Two molecular markers of muscle atrophy, upregulation of the E3 ubiquitin ligase Muscle Ring Finger 1 (MuRF1) and aberrant glycosylation of β-dystroglycan (β-DG), were used to confirm muscle wasting in the tumor-bearing mice. Serum samples were collected for metabolomic analysis during the development of the cachexia: at baseline, when the tumor was palpable, and when the mice demonstrated cachexia. The unsupervised statistical analysis demonstrated a distinct metabolic profile with the onset of cachexia. The critical metabolic changes associated with cachexia included increased levels of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), with decreased serum glucose levels. Regression analysis demonstrated a very high correlation of the presence of aberrant glycosylation of β-DG with the unique metabolic profile of cachexia. This study demonstrates for the first time that metabolomics has potential as a diagnostic tool in cancer cachexia, and in further elucidating simultaneous metabolic pathway alterations due to this syndrome. In addition, variations in VLDL and LDL deserve more investigation as surrogate serum biomarkers for cancer cachexia.     

Ventricular arrhythmogenesis: Insights from murine models

Ventricular arrhythmias are the key underlying cause of sudden cardiac death, a common cause of mortality and a significant public health burden. Insights into the electrophysiological basis of such phenomena have been obtained using a wide range of recording techniques and a diversity of experimental models. As in other fields of biology, the murine system presents both a wealth of opportunities and important challenges when employed to model the human case. This article begins by reviewing the extent to which the murine heart is representative of that of the human. It then presents a novel physiological classification of mechanisms of arrhythmogenesis, critically assessing the extent to which the study of murine hearts has offered worthwhile insights.     

Inhibition of the metastatic progression of breast and colorectal cancer in vitro and in vivo in murine model by the oxidovanadium(IV) complex with luteolin

The anticancer and antimetastatic behavior of the flavonoid luteolin and its oxidovanadium(IV) complex [VO(lut)(H₂O)₂]Na·3H₂O (VOlut) has been investigated. Considering that the complex displayed strong anticancer activity on MDAMB231 human breast cancer cell line we herein determined through in vitro assays that the complex would probably reduce breast cancer cell metastasis in a higher extent than the natural antioxidant. In the CT26 colon cancer cell line a stronger anticancer effect has also been determined for the complex (IC₅₀ 0.9 μM) and in addition it did not exert toxic effects on normal colon epithelial cells at concentrations up to 10 μM. Working with a murine model of highly aggressive, orthotopic colon cancer model (CT26 cancer cell lines) it has been determined that the complex might prevent metastatic dissemination of the colon cancer cells to the liver. The flavonoid luteolin also exerted anticancer effects (at a low degree, IC₅₀ 5.9 μM) on CT26 cell line and produced a 24% reduction of colon cancer liver metastasis.     

An improved protocol for primary culture of cardiomyocyte from neonatal mice

The primary culture of neonatal mice cardiomyocyte model enables researchers to study and understand the morphological, biochemical, and electrophysiological characteristics of the heart, besides being a valuable tool for pharmacological and toxicological studies. Because cardiomyocytes do not proliferate after birth, primary myocardial culture is recalcitrant. The present study describes an improved method for rapid isolation of cardiomyocytes from neonatal mice, as well as the maintenance and propagation of such cultures for the long term. Immunocytochemical and gene expression data also confirmed the presence of several cardiac markers in the beating cells during the long-term culture condition used in this protocol. The whole culture process can be effectively shortened by reducing the enzyme digestion period and the cardiomyocyte enrichment step. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Knockdown of Atg7 suppresses Tumorigenesis in a murine model of liver cancer

Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer in adults and a leading cause of cancer-related deaths worldwide. Studies have shown that autophagy is significantly involved in carcinogenesis, in particular, driven by activated RAS signaling. Autophagy related 7 (Atg7) is a critical component for the formation of autophagosome and required for autophagy processes. We investigated the role of autophagy in RAS-driven tumorigenesis in the liver, via the knockdown of Atg7 in the model. Transposon vectors encoding short hairpin RNAs targeting Atg7 (Atg7 shRNA) were constructed. Inhibition of autophagy via Atg7 knockdown was tested in Hep3B cells cultured in nutrient-starved medium. Formation of autophagosome was suppressed in nutrient-starved Hep3B cells expressing Atg7 shRNA, demonstrating that it efficiently inhibited autophagy in HCC cells. Transposons encoding Atg7 shRNA were mixed with those expressing HRAS^G12V and p53 shRNA, and subsequently used for hydrodynamic injection to 5-week-old C57BL/6 mice. Tumorigenesis in livers induced by HRAS^G12V and p53 shRNA was significantly suppressed by Atg7 knockdown. The inhibition of autophagy led to a decreased proliferation of cancer cells, as determined by Ki-67 staining. Our data indicate that knockdown of Atg7 led to a significant decrease in tumorigenesis in a murine HCC model induced by activated RAS. Inhibition of autophagosome formation is expected to be a therapeutic option for liver cancer.     

Comparison of cholera toxin A2/B and murine interleukin-12 as adjuvants of Toxoplasma multi-antigenic SAG1-ROP2 DNA vaccine

Toxoplasmosis can lead to severe pathology in both humans and animals. However, an effective vaccine for humans has not been successfully developed. In this study, we used multi-antigenic SAG1-ROP2 as a DNA vaccine and cholera toxin A2/B subunit and murine interleukin-12 to compare their effectiveness as genetic adjuvants. Bagg albino/c (BAL/c) mice were immunized intramuscularly with pcDNA3.1-SAG1-ROP2 alone (control group), or pcDNA3.1-SAG1-ROP2 with co-administration of pCTA2/B or pIL-12, respectively. After immunization, the effectiveness of these two adjuvants were compared using lymphocyte proliferation assay, cytokine and antibody measurements. The group co-administered pIL-12 elicited stronger humoral and Th1-type cellular immune responses than those immunized with pcDNA3.1-SAG1-ROP2 alone, while in the group co-administered pCTA2/B there was no obvious enhancement of immunity. When challenged with Toxoplasma gondii RH strain, mice immunized with pIL-12 co-administration had significantly higher survival rates, whereas there was no notable augmentation of immunity in pCTA2/B group. Therefore, since pIL-12 significantly enhanced the antigenicity of multi-antigenic DNA vaccine, this suggests that IL-12 is a better and more effective adjuvant than CTA2/B in this situation.     

Protective Capacity of L-Form Salmonella typhimurium against Murine Typhoid in C3H/HeJ Mice

L forms of Salmonella typhimurium LT2 conferred strong protection to a lethal challenge with its parental bacterium on innately hypersusceptible C3H/HeJ mice, and its minimal protective dose was approximately 150 L-forming units. Although L-form S. typhimurium was avirulent for C3H/HeJ mice, it multiplied slowly in both the liver and spleen with the maximal growth 2–3 weeks after immunization and thereafter it persisted in the liver until 24 weeks. Protective immunity began to work between 4 and 6 weeks after immunization, and it remained active as long as the L forms colonized the liver (until 24 weeks after immunization). Vaccination with the L form induced a population of T cells responding to L-form whole-cell lysate (WCL), while delayed-type hypersensitivity (DTH) to the extract of S. typhimurium was induced after the establishment of solid immunity. Moreover, neither T-cell responses nor DTH to heat-killed S. typhimurium was generated. In addition, antibody responses were elicited to WCL but not to heat-killed S. typhimurium. These results indicate that protection conferred by the L forms is attributable to the persistent colonization of the L forms rather than the presence of DTH, and also that Salmonella cytoplasmic antigens are involved in induction of immunological responses by vaccination with the L forms.     

Evaluation of the usefulness of anti-glyceraldehyde-3-phosphate dehydrogenase antibodies as a treatment for invasive candidiasis in a murine model

We have evaluated the effect of antibodies against the Candida albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a potential immunotherapeutic treatment for acute invasive candidiasis in a murine model of infection. Three different approaches were assayed: (i) active immunization of mice using recombinant His-tagged GAPDH, (ii) treatment of fungal yeast cells with anti-GAPDH antibodies prior to infection, and (iii) passive transfer of polyclonal anti-GAPDH antibodies. Results showed that all three approaches, although tending to show a slight beneficial effect in some instances, fail to have a relevant and statistically significant effect on the infection course, determined by survival curves and fungal burden in kidneys. This suggests that the cell wall-associated GAPDH of C. albicans, despite its potential role in virulence, does not appear to be a suitable target protein for the development of immunotherapeutic strategies against candidiasis, although further studies may be required to confirm this observation.     

Contribution of Catalase and Superoxide Dismutase to the Intracellular Survival of Clinical Isolates of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Murine Macrophages

The present study was performed in order to carefully investigate the interaction of Staphylococcus aureus with murine macrophages and the contribution of catalase and superoxide dismutase in intracellular persistence of Staphylococcus aureus within murine macrophages during in vitro infection. We have reported that Staphylococcus aureus internalized by murine macrophages did not appear to be rapidly killed. Data indicating the contribution of a single catalase and superoxide dismutase in intracellular survival of Staphylococcus aureus were provided using established biochemical assays. The results of the present experiment suggest that the survival of Staphylococcus aureus within phagocytic cells is facilitated by its ability to resist oxidative products. Organisms in the log phase of growth clearly demonstrate a resistance to oxidative products.