Process development for the mass production of Ehrlichia ruminantium

This work describes the optimization of a cost-effective process for the production of an inactivated bacterial vaccine against heartwater and the first attempt to produce the causative agent of this disease, the rickettsia Ehrlichia ruminantium (ER), using stirred tanks. In vitro, it is possible to produce ER using cultures of ruminant endothelial cells. Herein, mass production of these cells was optimized for stirring conditions. The effect of inoculum size, microcarrier type, concentration of serum at inoculation time and agitation rate upon maximum cell concentration were evaluated. Several strategies for the scale-up of cell inoculum were also tested. Afterwards, using the optimized parameters for cell growth, ER production in stirred tanks was validated for two ER strains (Gardel and Welgevonden). Critical parameters related with the infection strategy such as serum concentration at infection time, multiplicity and time of infection, and medium refeed strategy were analyzed. The results indicate that it is possible to produce ER in stirred tank bioreactors, under serum-free culture conditions, reaching a 6.5-fold increase in ER production yields. The suitability of this process was validated up to a 2-l scale and a preliminary cost estimation has shown that the stirred tanks are the least expensive culture method. Overall, these results are crucial to define a scaleable and fully controlled process for the production of a heartwater vaccine and open "new avenues" for the production of vaccines against other ehrlichial species, with emerging impact in human and animal health.     

Protection against dengue type 2 virus induced in mice immunized with a DNA plasmid encoding the non-structural 1 (NS1) gene fused to the tissue plasminogen activator signal sequence

Dengue is one of the most important arboviral diseases in humans, and although efforts over the last decades have dealt with the development of a vaccine, this vaccine is not available yet. In order to evaluate the potential of a DNA vaccine based on the non-structural 1 (NS1) protein against dengue virus (DENV), we constructed the pcTPANS1 plasmid which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. Results indicate that pcTPANS1 promotes correct expression of NS1 in eukaryotic cells and drives secretion of the recombinant protein to the surrounding medium in a dimeric form. Balb/c mice, intramuscularly inoculated with this plasmid, presented high levels of antibodies, recognizing mainly surface-exposed conformational epitopes present in the NS1 protein expressed by insect cells. Long-term antibody response was observed in animals 56 weeks after the first plasmid inoculation, and a rapid, efficient secondary response was observed after a DNA boost. Vaccinated animals were challenged against DENV-2 in two murine models, based on intracerebral (i.c.) and intraperitoneal (i.p.) virus inoculations, and in both cases, pcTPANS1-immunized mice were protected. Overall, these results provide further support for the use of such a plasmid in a possible approach for the development of a vaccine against DENV.     

DNA methylation changes after 5-aza-2'-deoxycytidine therapy in patients with leukemia

5-Aza-2'-deoxycytidine (decitabine) is postulated to have clinical activity in myeloid leukemias via its ability to inhibit DNA methylation. To study this, we examined DNA methylation in patients with leukemia treated with decitabine. Five days after the treatment, total genomic 5-methylcytosine/cytosine decreased on average by 14% (from 4.3% to 3.7%), whereas methylation of repetitive DNA elements showed a mean decrease of 9% and 16% for Alu and long interspersed nucleotide elements, respectively. Methylation decreased linearly with increasing doses between 5 and 20 mg/m(2)/d (r = 0.88; P = 0.05) but showed a plateau above that. Hypomethylation correlated with response in patients with acute myelogenous leukemia treated with low doses (5-20 mg/m(2)/d), but patients with chronic myelogenous leukemia treated with high doses (100-180 mg/m(2)/d) showed no such correlation. Aberrant methylation of p15 (>10%) was found in 27% of patients, and 80% of these showed a decrease by at least one third, but this did not correlate with response. The imprinted gene H19 showed little change in methylation after decitabine. In conclusion, we show dose-dependent hypomethylation after decitabine at low doses. Increasing the dose, which has been shown previously to result in a reduced response rate, was not accompanied by further hypomethylation.     

Social capital and the research agenda in epidemiology

Social capital refers to those features of social organization that enable participants to act together and more effectively pursue common goals. A growing body of evidence suggests that societies with high levels of social capital have lower morbidity and mortality rates and higher life expectancy and are less violent. The main goal of this article is to review the relationship between social capital and health. First, the main concepts and criticisms of social capital theory are discussed. Next, commonly used assessment tools are elucidated. Then, the relationship between social capital and health is analyzed. Finally, the article comments on the theory's application to Brazilian reality. If scientific rigor is applied to social capital research, recognizing theoretical and methodological difficulties, it can expand the research agenda and contribute to a better understanding of how to effectively deal with health inequalities.     

The impact of sugar cane-burning emissions on the respiratory system of children and the elderly

We analyzed the influence of emissions from burning sugar cane on the respiratory system during almost 1 year in the city of Piracicaba in southeast Brazil. From April 1997 through March 1998, samples of inhalable particles were collected, separated into fine and coarse particulate mode, and analyzed for black carbon and tracer elements. At the same time, we examined daily records of children (<13 years of age) and elderly people (>64 years of age) admitted to the hospital because of respiratory diseases. Generalized linear models were adopted with natural cubic splines to control for season and linear terms to control for weather. Analyses were carried out for the entire period, as well as for burning and nonburning periods. Additional models were built using three factors obtained from factor analysis instead of particles or tracer elements. Increases of 10.2 microg/m3 in particles相似文献   

The nurse and the construction of professional autonomy in the care process

This article analyses some factors that interfere in the quality of nursing practice, focusing, particularly on the professional nursing practice. It brings about a critical approach of this acting, based on the assumption that the care process is the essence of the nursing work. The nurse's professional autonomy is considered as an important issue to be renewed by a new scientific paradigm, that takes into consideration the humanistic side of health care. Based on a qualitative approach, some important issues are considered, related to the labor process in an universitary public hospital. The article concludes by proposing a new attitude, which can be both more committed to the production of health and more connected to a social dimension.     

Genetic polymorphisms of CYP1A1, CYP2E1, GSTM1, and GSTT1 associated with head and neck cancer

BACKGROUND: Alcohol intake and tobacco smoke, in addition to other environmental and genetic factors, have been associated with head and neck cancer. We evaluated the role of metabolic enzyme polymorphisms on the risk of head and neck cancer in a hospital-based case-control study. METHODS: CYP1A1MspI, CYP2E1PstI, GSTM1, and GSTT1polymorphisms were evaluated in 103 histologically confirmed head and neck cancer cases and 102 controls by means of polymerase chain reaction-restriction fragment length polymorphism methods. RESULTS: GSTM1null increased the risk of head and neck cancer (odds ratio [OR], 2.2; 95% confidence interval [95% CI], 1.24-3.79), oral cancer (OR, 2.8; 95% CI, 1.28-5.98), and pharyngeal cancer (OR, 2.2; 95% CI, 1.08-4.63). CYP2E1PstI polymorphism indicated a risk for oral cancer (OR, 3.6; 95% CI, 1.29-11.56). The joint effect of GSTM1 null and CYP1A1 polymorphism increased the risk of head and neck cancer (OR, 2.4; 95% CI, 1.13-5.10). CONCLUSIONS: GSTM1 null alone or associated with CYP1A1 increased the risk of head and neck cancer; the CYP2E1PstI mutated allele increased the risk for only oral cancer.     

Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T cells in renal transplant recipients

BACKGROUND: Immunosuppression in renal transplantation, although manageable in the short-term, is a major hurdle for long-term graft survival. Recently, increased frequencies of CD4CD25 regulatory T cells (Tregs) have been described as an additional mechanism that induces alloimmune tolerance. METHODS: We assessed 64 renal transplant recipients with stable renal function for at least one year. Patients were divided into two groups according to the immunosuppression they were receiving at the moment of the study: one consisted of patients receiving rapamycin (Rapa) but not calcineurin inhibitors (CNI), and the other group received CNI but not Rapa. The Rapa group was further divided into three subgroups according to their previous experience with CNI: CNI-free, CNI withdrawal, and CNI conversion. Frequencies of blood Tregs were studied by flow cytometry after staining with monoclonal antibodies specific for different markers of Tregs. RESULTS: Frequencies of CD4 T cells with regulatory phenotype and function were significantly decreased in peripheral blood of renal transplant patients receiving CNI compared with those receiving Rapa. This effect was independent of an early exposure to CNI because the CNI-free patients in the Rapa group showed similar frequencies of Tregs to the CNI withdrawal and CNI conversion groups. CONCLUSIONS: CNI, but not Rapa, induce a decrease of circulating Tregs in stable renal transplant recipients. Thus, Rapa might be further explored in strategies using preservation of Tregs for transplant tolerance. Furthermore, quantification of blood Tregs may be a suitable tool to identify renal transplant recipients who may be candidates for reduced immunosuppression.     

Refinements in the technique for repair of the accessory nerve

Trapezius muscle palsy after accessory nerve injury leads to periscapular pain and shoulder motion deficit. The results of accessory nerve repair generally are good, but surgery is difficult. The difficulty consists of finding the nerve stumps that are embedded in fat and scar tissue from previous surgeries or injuries. Five patients with accessory nerve lesions had surgery and grafting of the accessory nerve. We dissected the proximal stump of the accessory nerve within the fibers of the sternocleidomastoid muscle and in the vicinity of the greater auricular nerve. To achieve dissection of the distal nerve stump, the deep cervical fascia was detached from the trapezius muscle 3 cm cephalad to the clavicle. The detached fascia and the trapezius muscle were flipped similar to book pages. The motor branches entering the trapezius muscle were visualized and followed toward the accessory nerve. A sural nerve graft with a mean length of 6.6 cm was used for grafting. Uncomplicated identification of the nerve stumps was possible in all patients. After accessory nerve grafting, pain and motion consistently improved in all patients. The technique proposed here ensures reliable and rapid identification of the divided stumps of the accessory nerve.