Avaliação do perfil eletrocardiográfico pré e trans-anestesia em lobos-guará / Evaluation of electrocardiographic profile pre and trans-anesthesic in maned wolves

O presente estudo tem como objetivo avaliar o efeito da administração do Zolazepam/Tiletamina nas funções cardiorrespiratórias e eletrocardiográficas em lobos-guará (Chrysocyon brachyurus) mantidos em cativeiro. Foram utilizados dez lobos-guará clinicamente saudáveis (seis machos e quatro fêmeas), com média de peso 23,5±3,5kg, e idade de 6,5±2,8 anos. Os lobos eram mantidos em cativeiro e foram capturados pelos tratadores, proporcionando o mínimo de estresse possível para avaliação dos parâmetros pré-anestésicos. Foram avaliadas frequência cardíaca e respiratória, temperatura retal, pressão arterial média e eletrocardiografia. Após coleta dos parâmetros fisiológicos e eletrocardiográficos pré-anestesia, foi administrada a dose de 5,1±0,7mg/kg de Zolazepam/Tiletamina intramuscular. Depois da anestesia, colocaram-se os eletrodos do eletrocardiograma nos membros torácicos e pélvicos. Os animais eram monitorados durante uma hora, sendo que, a cada 10 minutos, era realizado o registro dos valores eletrocardiográficos, assim como os valores dos parâmetros fisiológicos e cardiorrespiratórios. Os resultados mostraram alteração significativa na amplitude da onda P entre 10 a 50 minutos pós-anestesia. Frequência cardíaca (153±20bmp), frequência respiratória (29±6mpm), temperatura corporal (38,4±1oC), pressão arterial média (114±20mmHg) e as outras variáveis eletrocardiográficas não apresentaram alterações. O aumento da amplitude da onda P nos animais deste trabalho sugeriu um aumento atrial, oriundo de doenças cardíacas ou simplesmente pelo aumento da frequência cardíaca durante a contenção.

The aim of this study was to assess the effects of the anesthetic combination of Tiletamine/Zolazepam on the cardiorespiratory function and electrocardiographic profile in captive maned wolves (Chrysocyon brachyurus). Ten maned wolves were used in this study (6 males and 4 females). All animals were healthy, with an average body weight of 23.5±3.5kg, and age of 6.5±2.8years. The wolves were conditioned to be physically restrained by their keepers in order to minimize stress during assessment of pre-anesthetic parameters. Data on heart and respiratory rates, rectal temperature, mean arterial blood pressure and electrocardiography were collected. Pre-anesthetic physiological an eletrocardiographic parameters were collected before the administration of 5.1±0.73mg/kg Tiletamine/Zolazepam intramuscularly. Under anesthesia, electrocardiogram electrodes were placed on thoracic and pelvic limbs and eletrocardiographic data was recorded every 10 minutes for approximately one hour, totaling 6 electrocardiograms. Heart rate 153±20bmp, respiratory rate 29±6mpm, rectal temperature 38,4±1oC, mean arterial blood pressure 114±20mmHg, and the other electrocardiographic parameters did not change; however, the P wave amplitude changed from 10 to 50 minutes after anesthesia. The increase in the P wave on the animals in this study suggested an atrial increase, probably due to cardiac disease or just by increasing the heart rate during the capture.

Expression of schistosomal cathepsin L1 in Escherichia coli and evaluation of its protective capacity in an animal challenge

Schistosomes use proteinases to accomplish some tasks such as tissue penetration, tissue digestion for nutrition and evasion of host immune responses. The Cathepsin L is a cysteine proteinase of the papain superfamily detected in the gut lumen indicating that this enzyme contributes to the proteolysis of ingested hemoglobin. Due to these roles they play in the schistosome biology, proteolytic enzymes are considered potential targets to develop and direct anti-schistosomal therapies. In this work, the cDNA coding Cathepsin L1 of Schistosoma mansoni was cloned into the pAE vector that provides high-level expression of heterologous proteins in Escherichia coli. The recombinant protein was expressed as inclusion bodies, purified under denaturing conditions through nickel charged chromatography and used for experimental animal vaccination. ELISA was performed with the pooled sera. Although this protein showed to be immunogenic, mice immunized with three doses of recombinant protein plus aluminum hydroxide as adjuvant did not protect against S. mansoni infection.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Improved method to calculate the antibody avidity index.

The methods currently used to determine the immunoglobulin avidity index (AI) require the choice of a reference point in the ELISA titration curve. Since both curves, with and without denaturating reagents, seldom run in parallel, the AI value becomes highly dependent on this reference. The new method for AI calculation presented here takes into account the whole data of the ELISA titration curve in which the final numerical AI is the average of each point.

PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A--pspA') gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-gamma) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.

r-Sm14 - pRSETA efficacy in experimental animals.

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.

BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.

Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice.

The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M. fortuitum beta-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B. pertussis infection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.

A Bordetella pertussis acellular vaccine candidate: antigenic characterization and antibody induction.

A single-step chromatography on Matrex-Gel Blue A has been employed to obtain soluble extracts containing some of the most important antigens of Bordetella pertussis, pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (69-kDa outer membrane protein), fimbriae (FIM2 and FIM3) and adenylate cyclase (AC). Two supernatants, P19 (48.8 mg PT, 6.8 mg FHA, 17.3 mg AC, 13 mg FIM2 and 4.9 mg FIM3 per liter) and P21 (0.1 mg PT, 0.07 mg FHA, 0.46 mg FIM2 and 0.94 mg FIM3 per liter), resulting from bacteria grown in Stainer-Scholte medium, were submitted to chromatography. Fractions with the antigens were obtained after stepwise elution with 60 mM sodium phosphate buffer, pH 6.0; 50 mM Tris-HCl, pH 7.4; 50 mM Tris-HCl, pH 7.4/0.75 M MgCl2; 50 mM Tris-HCl, pH 7.4/4 M MgCl2 and 4 M urea. Preparations from P19 (containing 4.05 micrograms PT, 8.14 micrograms FHA, 6.3 micrograms AC, 3.37 micrograms 69-kDa, 9.54 micrograms FIM2 and 2.23 micrograms FIM3) and from P21 (with 0.175 micrograms PT, 0.28 micrograms FHA, 0.002 micrograms 69-kDa, 0.005 micrograms FIM2 and 0.122 micrograms FIM3) were detoxified with glutaraldehyde and tested as an acellular pertussis vaccine. These products were non-toxic for mice and induced high levels of antibodies against purified pertussis antigens, as judged by ELISA.

A Bordetella pertussis acellular vaccine candidate: antigenic characterization and antibody induction

A single-step chromatography on Matrex-Gel Blue A has been employed to obtain soluble extracts containing some of the most important antigens of Bordetella pertussis, pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (69-kDa outer membrane protein), fimbriae (FIM2 and FIM3) and adenylate cyclase (AC). Two supernatants, P19 (48.8 mg PT, 6.8 mg FHA, 17.3 mg AC, 13 mg FIM2 and 4.9 mg FIM33 per liter) and P21 (0.1 mg PT, 0.07 mg FHA, 0.46 mg FIM2 and 0.94 mg FIM3 per liter), resulting from bacteria grown in Stainer-Scholte medium, were submitted to chromatography. Fractions with the antigens were obtained after stepwise elution with 60 mM sodium phosphate buffer, pH 6.0; 50 mM Tris-HC1, pH 7.4; 50 mM Tris-HC1, pH 7.4/0.75 M MgCl2; 50mM Tris-HCl, pH 7.4/4 M MgCl2 and 4 M urea. Preparations from P19 (containing 4.05 µg PT, 8.14 µg FHA, 6.3 µg AC, 3.37 µg 69-kDA, 9.54 µg FIM2 and 2.23 µg FIM3) and P21 (with 0.175 µg PT, 0.28 µg PT, 0.28 µg FHA, 0.002 µg69-kDa, 0.005 µg FIM2 and 0.122 µg FIM3) were detoxified with glutaraldehyde and tested as an acellular pertussis vaccine. These products were non-toxic for mice and induced high levels of antibodies against purified pertussis antigens, as judged by ELISA