Assessment of the acid phosphatase CP01850 from Corynebacterium pseudotuberculosis in DNA and subunit vaccine formulations against caseous lymphadenitis / Avaliação da fosfatase ácida recombinante CP01850 de Corynebacterium pseudotuberculosis em formulações vacinais de subunidade e de DNA contra a linfadenite caseosa

The target cp1002_RS01850 from Corynebacterium pseudotuberculosis was used to construct a DNA and recombinant subunit vaccine against caseous lymphadenitis. Recombinant protein rCP01850 was expressed in Escherichia coli using pAE vector, and DNA vaccine was engineered with pTARGET vector. BALB/c mice were divided in five groups containing eight animals each, inoculated with: pTARGET/cp01850 as DNA vaccine (G1); rCP01850 plus Al (OH)3 as recombinant subunit vaccine (G2); pTARGET/cp01850 and a boost with rCP01850 plus Al (OH)3 (G3); pTARGET (G4); or Al (OH)3 (G5). Mice were inoculated and blood samples were collected on days 0, 21, and 42 for the analysis of total IgG, IgG1 and IgG2a by ELISA. In each group, five animals were challenged with Mic-6 C. pseudotuberculosis strain, and three were used for cytokine quantification by qPCR. Although no group has been protected by vaccines against lethal challenge, G2 showed an increase in the survival rate after challenge. Significantly higher levels of IL-4, IL-12, IFN-γ, total IgG, IgG1 and IgG2a were also detected for G2, evidencing a mixed Th1/Th2 immunological profile. In conclusion, despite no protection level provided by different vaccinal strategies using cp1002_RS01850 from C. pseudotuberculosis, G2 developed a Th1/Th2 immune response with an increase in survival rate.(AU)

O alvo cp1002_RS01850 de Corynebacterium pseudotuberculosis foi utilizado para construir uma vacina recombinante de subunidade e de DNA contra a linfadenite caseosa. A proteína recombinante rCP01850 foi expressa em Escherichia coli usando o vetor pAE, e a vacina de DNA foi construída com o vetor pTARGET. Camundongos BALB/c foram divididos em grupos de oito animais, inoculados com: pTARGET/cp01850 como vacina de DNA (G1); rCP01850 e Al (OH)3 como vacina recombinante de subunidade (G2); pTARGET/cp01850 e um boost com rCP01850 e Al (OH)3 (G3); pTARGET (G4); ou Al (OH)3 (G5). Os animais foram inoculados e amostras de sangue foram coletadas nos dias 0, 21, e 42 do experimento para a análise de IgG total, IgG1 e IgG2a por ELISA. De cada grupo, cinco animais foram desafiados com a cepa Mic-6 de C. pseudotuberculosis, e três foram usados para a quantificação de citocinas por qPCR. Apesar de nenhum grupo ter sido protegido pelas vacinas testadas contra o desafio letal, G2 apresentou taxa de sobrevida e níveis de IL-4, IL-12, IFN-γ, IgG total, IgG1 e IgG2a significativamente mais altos, evidenciando um perfil imunológico misto Th1/Th2. Conclui-se que apesar das diferentes estratégias vacinais utilizando cp1002_RS01850 de C. pseudotuberculosis não terem sido capazes de gerar proteção, G2 desenvolveu uma resposta Th1/Th2 e elevou a taxa de sobrevida.(AU)

Exercise training restores oxidative stress and nitric oxide synthases in the rostral ventrolateral medulla of renovascular hypertensive rats.

We hypothesize that exercise training (EX) reverses the level of nitric oxide (NO) and oxidative stress into rostral ventrolateral medulla (RVLM) of renovascular hypertensive rats (two kidneys, one clip - 2K1C). Microinjections of L-arginine (5 nmol), L-NAME (10 nmol), or saline (100 nl) were made into RVLM of 2K1C and normotensive (SHAM) rats sedentary (SED) or subjected to swimming for 4 weeks. mRNA expression (by qRT-PCR) of nitric oxide synthases isoforms (nNOS, eNOS, and iNOS), manganese superoxide dismutase (MnSOD), copper and zinc superoxide (Cu/ZnSOD), catalase (CAT), NADPH oxidase subunit p22(phox), concentration of thiobarbituric acid-reactive substances (TBARS), and CAT activity into RVLM were evaluated. The mean arterial pressure was reduced in 2K1C EX compared with that in 2K1C SED rats. L-arginine into RVLM induced hypertensive effect in 2K1C and SHAM SED rats, while L-NAME prevented hypertensive effect only in SHAM-SED. EX reduced hypertensive effect of L-arginine in SHAM and 2K1C rats. mRNA expression of NOS isoforms, p22(phox), and concentration of TBARS were increased while CAT and Cu/ZnSOD expression and CAT activity decreased into RVLM of 2K1C-SED compared with SHAM-SED rats. Additionally, EX reversed mRNA expression of CAT and NOS isoforms, concentration of TBARS, and CAT activity into RVLM of 2K1C-EX rats. These data suggest that the levels of NOS and oxidative stress into RVLM are important to determine the level of hypertension. Furthermore, EX can restore the blood pressure by reversing the levels of NOS and CAT expression, and reducing TBARS concentration into RVLM for the physiological state.

Morphometric and quantitative analysis of theafferent renal artery variation

The kidney is a retroperitoneal organ that weight from 125 to 170 g in the adult men and 115to 155 g in adult women. Irrigation kidney is characterized by the presence of large anatomical variability thatmay be influenced by ethnic and to a lesser extent by gender. Among the variations, there may be the presenceof an accessory renal artery that is projected into the upper or lower end of the kidney. This research aimsto observe the incidence of anatomical variations of the afferent renal artery and quantify both right and leftkidney weight.Materials and Methods:We analyzed kidney weights and accessory renal artery variations in48 adult kidneys of both genders obtained from Institute of Anatomy of the University of Severino Sombra.Subsequently, we compared the mean weights of kidneys in order to ascertain whether there was significantdiscrepancy between the left and the right kidney. For this, we performed the Student t test considering aP < 0.05.Results:The mean weight of the right kidney was 140.4 ± 22.6 g and the left was 148.8 ± 20.5 g.In 40% of right kidneys was observed anatomic variation with the presence of accessory renal artery. To the leftkidney was observed a variation of 35%.Conclusion:We found that the accessory renal artery, when present,was more closely related to the end of the kidney especially in the right kidney.

Interactions between straw size and thawing rates on the cryopreservation of agouti (Dasyprocta aguti) epididymal sperm.

This study verifies the interactions between straw size and thawing rates and their impact on the epididymal sperm from this species. Caudae epididymidum from 10 agoutis were subjected to retrograde washing using a coconut water extender (ACP-109c(®) ). Epididymal sperm were evaluated and extended in ACP-109c(®) plus egg yolk (20%) and glycerol (6%). The samples were packaged in 0.25- or 0.50-ml straws, frozen in liquid nitrogen and thawed at 37°C/1 min or 70°C/8 s, followed by a re-evaluation. The use of 0.25-ml straws thawed at 37°C/1 min provided a value of 26.6% for sperm motility. No interactions between straw size and thawing rates were verified on agouti sperm (p > 0.05), but when 0.5-ml straws were thawed at 70°C/8 s, sperm vigour decreased significantly (p < 0.05). It is recommended that the agouti epididymal sperm cryopreserved in ACP-109c(®) extender should be packaged in 0.25- or 0.50-ml straws and thawed at 37°C/60 s.

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu).

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen-thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen-thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.

Influence of the thawing rate on the cryopreservation of semen from collared peccaries (Tayassu tajacu) using Tris-based extenders.

The objective was to evaluate the influence of the thawing rate on the quality of frozen-thawed (cryopreserved in Tris-based extenders) semen obtained from collared peccaries (Tayassu tajacu). Semen from 13 sexually mature collared peccaries males were collected by electroejaculation, and evaluated for motility, vigor, sperm viability, membrane integrity, and sperm morphology. Semen was divided in two equal portions: the first was diluted in Tris-fructose and the other in Tris-glucose, with egg yolk (20%) and glycerol (3%) added to each portion. Extended semen was frozen in liquid nitrogen and thawed using two thawing protocols (37 degrees C for 1 min or 55 degrees C for 7 s, followed by an additional 30 s at 37 degrees C). There were no significant differences between the two extenders after extension, chilling, or glycerol addition. After thawing at 37 degrees C, there were 37.9 +/- 4.2% and 28.5 +/- 5.1% motile spermatozoa for samples extended in Tris-fructose and Tris-glucose, respectively, with 33.8 +/- 3.7% and 28.2 +/- 3.5% motile spermatozoa after thawing at 55 degrees C (no significant differences). Furthermore, there were no significant interactions between extenders and thawing protocols for any semen end point. In conclusion, semen from collared peccaries was successfully cryopreserved in Tris-based extenders and thawed with two protocols (37 degrees C for 1 min or 55 degrees C for 7 s).

Pisosterol induces interphase arrest in HL60 cells with c-MYC amplification.

The leukaemia cell line HL60 is widely used in studies of the cell cycle, apoptosis and adhesion mechanisms in cancer cells. One marked characteristic of HL60 cells is the c-MYC proto-oncogene amplification, resulting in the formation of homogeneously staining regions (HSRs) at 8p24. We conducted a fluorescence in situ hybridization study in an HL60 cell line, using a locus-specific probe for c-MYC, before and after treatment with pisosterol (at 0.5, 1.0 and 1.8 microg/mL), a triterpene isolated from the fungus Pisolithus tinctorius. Before treatment, 87.5% of the cells showed HSRs. After treatment, no effects were detected at lower concentrations of pisosterol (0.5 and 1.0 microg/mL). However, at 1.8 microg/mL only 15% of the cells presented HSRs, and 39.5% presented few fluorescent signals (3 or 4 alleles), suggesting that pisosterol probably blocks the cells with HSRs at interphase. This result is particularly interesting because cells that do not show a high degree of c-MYC gene amplification have a less aggressive and invasive behaviour and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

Cell cycle arrest induced by pisosterol in HL60 cells with gene amplification.

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 microg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 microg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 microg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 microg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

Prevalência de teníase no município de Pedra Branca Estado do Ceará, Brasil / Prevalence of taeniasis in municipal district Pedra Branca of Ceará State, Brazil

A teníase é reconhecida como importante problema de saúde pública em regiões onde as condições sanitárias são precárias e em países industrializados que recebem imigrantes de regiões onde a infecção é endêmica. O homem, responsável pela manutenção do ciclo parasitário, é o único hospedeiro da forma adulta dos parasitos Taenia solium e Taenia saginata. Apesar da importância do porco no ciclo de vida da Taenia solium, pouca atenção é dada em relação à infecção suína e humana. Estudo epidemiológico realizado no município de Pedra Branca no período de 1997 a 1998 demonstrou alto índice de carne de porco com cisticercose. O objetivo do presente estudo foi investigar a prevalência de teníase na população de Pedra Branca a fim de identificar os casos de teníase. Para isso empregou-se o método de Kato modificado por Katz por ser de fácil execução e de nos fornecer preparações quase permanentes. Nesse estudo, foram analisadas 831 amostras, de indivíduos de ambos os sexos, na faixa etária de 02 a 70 anos, com 54 (6,5%) de positivas. Os resultados mostraram a prevalência de diferentes espécies de helmintos, onde a maior freqüência foi de Ascaris lumbricoides 19 (2,30%), seguido de ancilostomideos 12 (1,44%) e em terceiro de Taenia sp 10 (1,20%). A prevalência de teníase foi bastante significativa levando-se em consideração que o método utilizado não é específico para o diagnóstico de teníase.

Taeniasis are recognized as important public health problem in many developed countries for regions with bad sanitary conditions as well as for industrial countries in the cases of emigration from the regions with endemic infection. Human intestin is responsible for realization of the parasite cycle and is unique accommodation for adult parasite form: Taenia solium and Taenia saginata In spite of importance of pigs in the life cycle of Taenia solium, it was small attention to relation between human and pig infection. Epidemiological study accomplished in Pedra Branca in the period 1997–1998 demonstrated the high index of cysticercosis contamination of pork. The purpose of the present study was for measuring the prevalence of human taeniasis on the Pedra Branca population. For this it was used the Kato method, modificated by Katz in order to be easy in use and to supply the preparations quasi permanent. In this study were analyzed 831 samples of stool of persons of both sexes between 2 and 70 year, with 54 (6.5%) of positives. The results show prevalent of different types of helminths, where Ascaris lumbricoides 19 (2.30%) has the biggest index, second was ancilostomideos 12 (1.44%) and third Taenia sp 10 (1.20%). The prevalence of taeniasis was significant considering that the used method was not specific for it diagnostic.

Quantitative and descriptive analysis of the meniscotibial ligament in human corpses

The meniscotibial ligaments (LMT) of humans are capsular fibers with proximal origin in the lateral border of the menisci and distal insertion in the lateral border of the tibial condyle, also called coronary ligament of the knee. However, few studies describe the presence, course and anatomical variations of this ligament. Our objective was to evaluate the LMT through quantitative and descriptive methods. Eighty-five knees were examined(42 right knees [RK] and 43 left knees [LK]), dissected, evaluated, catalogued and photographed by four examiners. Twenty knees (23.53%) presented LMT, 6 (30%) in RK and 14 (70%) in LK. In two RKs, the LMT presented proximal insertion in the medial menisci and distal insertion in medial tibial condyle; in the other ones, the LMT presented proximal insertion in the lateral menisci and distal insertion in the lateral tibial condyle. In the LK, four LMT presented proximal insertion in the medial menisci and distal insertion in the medial tibial condyle, while the other ones presented proximal insertion in the lateral menisci and distal insertionin the lateral tibial condyle. One LMT in the LK presented proximal and distal insertion in both menisci and tibial condyle. The analysis of human corpses in our study suggests that the presence of the LMT is much more common in the LK than in the RK, with more frequent insertion in the proximal region of the lateral menisci and distal insertion in the lateral tibial condyle.

Comparative study on the effect of cyclic nucleotides related to the function of Biomphalaria glabrata hemocytes and human granulocytes.

The biochemical processes linked up to cyclic nucleotides related to the phenomenon of phagocytosis in Biomphalaria glabrata hemocytes were studied. In hemocytes the results suggest the presence of a phagocytic capacity similar to that observed in human cells related to regulation by cyclic adenylate monophosphate (cAMP), but not by cyclic guanylate monophosphate (cGMP). This similarity and differences in the metabolic process of phagocytary capacity between human phagocytary cells and hemocytes could represent an interesting model aimed at studying the phylogenetic evolution of enzymatic complexes.

Quantification of the population and phagocytary activity of hemocytes of resistant and susceptible strains of Biomphalaria glabrata and Biomphalaria tenagophila infected with Schistosoma mansoni

Entre os fatores determinantes na resistencia e suscetibilidade da Biomphalaria ao Schistosoma mansoni, os hemocitos desempenharam importante papel. Com o objetivo de estudar as interacoes S. mansoni/Biomphalaria relativas aos hemocitos o primeiro passo e certamente relacionado a padronizacao desta populacao de celulas em Biomphalaria nao infectada. Desta forma a quantificacao desta populacao de celulas na hemolinfa bem como sua capacidade fagocitaria foi determinada pela primeira vez. Alem disso, usando cepas de B. glabrata e B. tenagophila suscetiveis e resistentes, o hemocitograma e a capacidade fagocitaria dos hemocitos, apos infeccao com S. mansoni, foram tambem determinados. Cepas de B. glabrata (BA e BH respectivamente) resistentes e suscetiveis bem como cepas de B. tenagophila (TAIM e CF respectivamente) foram infectadas com 10 miracidios das cepas LE e SJ de S. mansoni, respectivamente. Estes caramujos infectados e respectivos controles nao infectados foram avaliados em relacao ao numero de hemocitos circulantes e alteracao da capacidade fagocitaria, usando Zimozan e MTT...

Quantification of the population and phagocytary activity of hemocytes of resistant and susceptible strains of Biomphalaria glabrata and Biomphalaria tenagophila infected with Schistosoma mansoni.

Among the determinant factors in the resistance and susceptibility of Biomphalaria to Schistosoma mansoni, hemocytes play an important role. Aiming at studying S. mansoni/Biomphalaria interactions related to hemocytes, the first step is certainly connected with the standardization of this cell population in uninfected Biomphalaria. In this way, quantification of this cell population in hemolymph, as well as its phagocitary capacity, have been determined for the first time. Furthermore, using susceptible and resistant strains of B. glabrata and B. tenagophila, the hemocytegram and phagocytary capacity of hemocytes after infection with S. mansoni were determined too. Resistant and susceptible strains of B. glabrata (BA and BH, respectively), as well as resistant and susceptible strains of B. tenagophila (TAIM and CF, respectively) were infected with 10 miracidia of the LE and SJ strains of S. mansoni, respectively. These infected snails and respective uninfected controls were assessed in relation to the number of circulating hemocytes and alteration in the phagocytary capacity, by using Zymozan and MTT. Reading was taken by means of a spectrophotometer at 5 hours and 1, 2, 5, 10, 20 and 30 days after infection. The results showed a decrease in population of the circulating phagocytary cells, 5 hours after infection. One day post-infection, the circulating cells of the susceptible snails showed an increased metabolic activity, but the same event could not be observed in the resistant strains. In the subsequent observation periods, significant differences among the strains studied could not be observed until the end of the experiment.

Schistosoma mansoni: protective immunity in mice cured by chemotherapy at the chronic phase of the disease.

Aiming at demonstrating a decrease of acquired immunity after chemotherapeutic cure, a group of mice was infected with 25 Schistosoma mansoni cercariae (LE strain). A part of these animals was treated with 400 mg/kg oxamniquine, at 120 days after infection. Challenge infections were carried out at 45, 90 and 170-day-intervals after treatment (185, 210 and 290 days after primo-infection, respectively). Recovery of worms at 20 days after reinfections showed that a residual immunity remains up to 90 days after treatment, and disappears at 170 days after cure. Using the ELISA method, it was possible to detect a decrease of antibody levels (total IgG) in the treated group, when antigens from different evolutive stages of S. mansoni were used. The epidemiological implications of the present results, and the possible mechanisms involved in the decrease of acquired immunity after treatment are discussed.