The Brazilian legal framework on the scientific use of animals

Brazil has an exceptionally dynamic research sector in Latin America in health, biotechnology, and pharmacology, backed by defined government policies on science and technology and a health research agenda focusing on important neglected diseases: malaria, leishmaniasis, Chagas disease, turberculosis, leprosy, and dengue. The Brazilian health research policy promotes partnerships and networks among scientists in academic institutions in both wealthy industrialized and disease-endemic countries, and in these efforts the government's guidelines for animal use in biomedical research are considered fundamental to guarantee both animal welfare and the quality of research. Given international discussions of animal experimentation regulations and guidelines, in this article we describe current Brazilian legislation governing the use of animals in scientific investigations. We conclude that, despite advances in the implementation of the 3Rs (reduction, refinement, replacement), the new regulatory framework does not sufficiently incorporate ethical considerations, lacking explicit reference to the 3Rs as well as measures for their full application. The more humane use of animals in research will depend on the approach adopted by Brazil's National Council for the Control of Animal Experimentation to promote the 3Rs and to improve internal regulations as well as data collection and analysis in research institutions. In Brazil as elsewhere, one of the greatest challenges to policymakers is to harmonize the myriad and intertwined legal provisions without hindering biomedical research.     

A multicentre randomized controlled trial of the efficacy and safety of single-dose praziquantel at 40 mg/kg vs. 60 mg/kg for treating intestinal schistosomiasis in the Philippines, Mauritania, Tanzania and Brazil

Background

Praziquantel at 40 mg/kg in a single dose is the WHO recommended treatment for all forms of schistosomiasis, but 60 mg/kg is also deployed nationally.

Methodology/Principal Findings

Four trial sites in the Philippines, Mauritania, Tanzania and Brazil enrolled 856 patients using a common protocol, who were randomised to receive praziquantel 40 mg/kg (n = 428) or 60 mg/kg (n = 428). While the sites differed for transmission and infection intensities (highest in Tanzania and lowest in Mauritania), no bias or heterogeneity across sites was detected for the main efficacy outcomes. The primary efficacy analysis was the comparison of cure rates on Day 21 in the intent-to-treat population for the pooled data using a logistic model to calculate Odd Ratios allowing for baseline characteristics and study site. Both doses were highly effective: the Day 21 cure rates were 91.7% (86.6%–98% at individual sites) with 40 mg/kg and 92.8% (88%–97%) with 60 mg/kg. Secondary parameters were eggs reduction rates (ERR), change in intensity of infection and reinfection rates at 6 and 12 months. On Day 21 the pooled estimate of the ERR was 91% in both arms. The Hazard Ratio for reinfections was only significant in Brazil, and in favour of 60 mg/kg on the pooled estimate (40 mg/kg: 34.3%, 60 mg/kg: 23.9%, HR = 0.78, 95%CI = [0.63;0.96]). Analysis of safety could not distinguish between disease- and drug-related events. 666 patients (78%) reported 1327 adverse events (AE) 4 h post-dosing. The risk of having at least one AE was higher in the 60 than in the 40 mg/kg group (83% vs. 73%, p<0.001). At 24 h post-dosing, 456 patients (54%) had 918 AEs with no difference between arms. The most frequent AE was abdominal pain at both 4 h and 24 h (40% and 24%).

Conclusion

A higher dose of 60 mg/kg of praziquantel offers no significant efficacy advantage over standard 40 mg/kg for treating intestinal schistosomiasis caused by either S. mansoni or S. japonicum. The results of this study support WHO recommendation and should be used to inform policy decisions in the countries.

Trial Registration

Controlled-Trials.com ISRCTN29273316 ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00403611","term_id":"NCT00403611"}}NCT00403611     

Abnormal glucose homeostasis and pancreatic islet function in mice with inactivation of the Fem1b gene

Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both beta cells and non-beta cells, and is highly expressed in INS-1E cells, a pancreatic beta-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.     

MamMiBase: a mitochondrial genome database for mammalian phylogenetic studies

MamMiBase, the mammalian mitochondrial genome database, is a relational database of complete mitochondrial genome sequences of mammalian species. The database is useful for phylogenetic analysis, since it allows a ready retrieval of nucleotide and aminoacid individual alignments, in three different formats (NEXUS for PAUP program, for MEGA program and for PHYLIP program) of the 13 protein coding mitochondrial genes. The user may download the sequences that are useful for him/her based on their parameters values, such as sequence length, p-distances, base content, transition transversion ratio, gamma, which are also given by MamMiBase. A simple phylogenetic tree (neighbor-joining tree with Jukes Cantor distance) is also available for download, useful for parameter calculations and other simple tasks. AVAILABILITY: MamMiBase is available at http://www.mammibase.lncc.br     

Cytokine gene expression in Walker 256: a comparison of variants A (aggressive) and AR (regressive)

Two variants of this Walker 256 tumor have been previously reported as Walker 256 A and variant AR. The variant A has more aggressive property than variant AR and can induce systemic effects such as anorexia, sodium and water retention, followed by weight loss and death. The mechanisms involved in enhancing tumor regression and progression in this model are still incompletely understood. In the present study, serum and spleen mononuclear cells and tumor cells from animals inoculated with variants A and AR, were isolated to investigate the TGF-beta, IL-12, IFN-gamma and TNF-alpha and relationship with anemia, weight of animals, weight of spleen, volume of tumor and osmotic fragility compared with controls inoculated with Ringer Lactate. Results demonstrate that the group inoculated with variant A, compared to variant AR, shows high levels of TGF-beta gene expression in both tumor tissue and spleen cells, no expression of IFN-gamma and a progressive and higher levels of IL-12 in tumor tissue without inflammatory infiltrate visualized by optical microscopy. These results suggest that the aggressively of variant A is relate to cytokine modulation, facilitating the growth and escape of tumor cells. Furthermore, IL-12 seems to be constitutively expressed in both tumor lineage A and AR.     

RP-HPLC/MS-APCI analysis of odd-chain TAGs from Rhodococcus erythropolis including some regioisomers

We used reversed phase liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) for direct analysis of triacylglycerols (TAGs) from four different cultivations of Rhodococcus erythropolis CCM 2595. This technique enabled us to identify and quantify the specific molecular species of TAGs directly from lipid extracts of the bacterium, including the determination of their basic characteristics such as retention time and mass spectra. A total of 17 TAGs having at least one odd-numbered-chain FA (fatty acid) were found. This is the first time when odd-chain TAGs, predominantly with pentadecanoic, margaric, and margaroleic acids, were identified. Multiple linear regression analyses were used for predicting retention times of molecular species, predominantly of odd-chain TAGs. Cultivations on two different substrates at two different temperatures (20 and 30 °C) showed that only temperature had any effect on the content of TAGs. In cultivations with succinate as a carbon source the content of these TAGs increased by half (i.e., from 23.5 to 34.2%) when the cultivation temperature was lowered from 30 to 20 °C. The content of the PoPoMo, PoMoO and PPoMo (see Table 1) TAGs containing the odd-numbered-chain margaroleic acid rose from 0.0 to 7.1% while in cultivation on phenol as a carbon source the lowering of cultivation temperature caused an increase of these TAGs from 0.5 to 6.7%.     

Novel role for surfactant protein A in gastrointestinal graft-versus-host disease

Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A-deficient mice on a C57BL/6 background (H2b; SP-A(-/-)) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A-deficient recipient mice that have undergone an allogeneic BMT [SP-A(-/-)alloBMT] or SP-A-sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A-deficient recipient mice that have undergone a syngeneic BMT or SP-A-sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A(-/-) alloBMT or SP-A-sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A(-/-)alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A(-/-)alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.