Immunoprophylactic strategies against enterotoxemia caused by Clostridium perfringens type D in goats

The serological response to an experimental vaccine against Clostridium perfringens type D enterotoxemia was evaluated in four groups of goats. Group 1 received colostrum from unvaccinated cows and no vaccine. Groups 2, 3 and 4 received colostrum from vaccinated cows. In addition, Groups 3 and 4 received a vaccine dose at 80 days of age, and Group 4 received a second vaccine dose at 120 days of age. Serum antibody levels were determined by ELISA in cows before and after calving, and in goats at 3, 80, 120 and 160 days of age. No significant difference in serum antibody levels was observed between vaccinated and unvaccinated cows, or between the four groups of goats evaluated at 3 days of life. Groups 3 and 4 presented mean antibody titers of 0.6 and 1.1 IU/ml, respectively, 40 days after first vaccination. The vaccine response of Group 4 was 1.8 IU/ml 40 days after the booster dose and was higher than that observed for Group 3 (0.2 IU/ml). Thus, in the proposed regimen the use of heterologous colostrum did not induce passive immunization in goat kids. However, first vaccination and a booster dose after 40 days triggered satisfactory antibody levels.

Interaction between repeated restraint stress and concomitant midazolam administration on sweet food ingestion in rats

Emotional changes can influence feeding behavior. Previous studies have shown that chronically stressed animals present increased ingestion of sweet food, an effect reversed by a single dose of diazepam administered before testing the animals. The aim of the present study was to evaluate the response of animals chronically treated with midazolam and/or submitted to repeated restraint stress upon the ingestion of sweet food. Male adult Wistar rats were divided into two groups: controls and exposed to restraint 1 h/day, 5 days/week for 40 days. Both groups were subdivided into two other groups treated or not with midazolam (0.06 mg/ml in their drinking water during the 40-day treatment). The animals were placed in a lighted area in the presence of 10 pellets of sweet food (Froot loops®). The number of ingested pellets was measured during a period of 3 min, in the presence or absence of fasting. The group chronically treated with midazolam alone presented increased ingestion when compared to control animals (control group: 2.0 ± 0.44 pellets and midazolam group: 3.60 ± 0.57 pellets). The group submitted to restraint stress presented an increased ingestion compared to controls (control group: 2.0 ± 0.44 pellets and stressed group: 4.18 ± 0.58 pellets). Chronically administered midazolam reduced the ingestion in stressed animals (stressed/water group: 4.18 ± 0.58 pellets; stressed/midazolam group: 3.2 ± 0.49 pellets). Thus, repeated stress increases appetite for sweet food independently of hunger and chronic administration of midazolam can decrease this behavioral effect.

Ethidium bromide-induced demyelination of the sciatic nerve of adult Wistar rats

Peripheral nerve ultrastructure was assessed after single or multiple local injections of the intercalating dye ethidium bromide. Thirty-four adult Wistar rats of both sexes were divided into five groups and maintained in a controlled environment with rat chow and water ad libitum throughout the experiment. The experimental animals were injected with 1 µl of 0.1% ethidium bromide in 0.9% saline into the central third of the left sciatic nerve 1 (group 1), 2 (group 2), 4 (group 3), 6 (group 4) or 8 (group 5) times. In groups 2 to 5 the injections were made at 28-day intervals. Control animals received the same amount of 0.9% saline. The animals were killed at different times after injection: group 1 at 7 days (2 rats) and 15 days (2 rats); for groups 2, 3, 4 and 5, all rats were killed 10 days after the last injection and the lesions were investigated by light and transmission electron microscopy. In the acute lesions, intoxicated Schwann cells showed a vacuolated cytoplasm and separation of the sheaths from the axon. Myelin sheaths underwent progressive vesiculation and subsequent segmental demyelination. Myelin debris were withdrawn by macrophages and remyelination by Schwann cells was prominent. With the increase in the number of injections collagen fibers also increased in number and progressively enveloped smaller numbers of remyelinated axons composing new fascicles. Wallerian degeneration of fibers apparently not affected by ethidium bromide was more intense in the nerves from groups 4 and 5. The peripheral nerve repairs itself after demyelinating challenges with a profusion of collagen fibers and new fasciculations. This experimental model is valid to mimic recurrent demyelinating neuropathies.

Comparison of morning and afternoon exercise training for asthmatic children

Fitness improvement was used to compare morning with afternoon exercise periods for asthmatic children. Children with persistent moderate asthma (according to GINA criteria), 8 to 11 years old, were divided into 3 groups: morning training group (N = 23), afternoon training group (N = 23), and non-training group (N = 23). The program was based on twice a week 90-min sessions for 4 months. We measured the 9-min running distance, resting heart rate and abdominal muscle strength (sit-up number) before and after the training. All children took budesonide, 400 µg/day, and an on demand inhaled ß-agonist. The distance covered in 9 min increased (mean ± SEM) from 1344 ± 30 m by 248 ± 30 m for the morning group, from 1327 ± 30 m by 162 ± 20 m for the afternoon group, and from 1310 ± 20 m by 2 ± 20 m for the control group (P < 0.05 for the comparison of morning and afternoon groups with the control group by ANOVA and P > 0.05 for morning with afternoon comparison). The reduction of resting heart rate from 83 ± 1, 85 ± 2 and 86 ± 1 bpm was 5.1 ± 0.8 bpm in the morning group, 4.4 ± 0.8 bpm in the afternoon group, and -0.2 ± 0.7 bpm in the control group (P > 0.05 for morning with afternoon comparison and P < 0.05 versus control). The number of sit-ups in the morning, afternoon and control groups increased from 22.0 ± 1.7, 24.3 ± 1.4 and 23 ± 1.1 sit-ups by 9.8 ± 0.9, 7.7 ± 1.4, and 1.9 ± 0.7 sit-ups, respectively (P > 0.05 for morning with afternoon comparison and P < 0.05 versus control). No statistically significant differences were detected between the morning and afternoon groups in terms of physical training of asthmatic children.

Somatic cytogenetic and azoospermia factor gene microdeletion studies in infertile men

The objective of the present study was to determine the frequency of somatic chromosomal anomalies and Y chromosomal microdeletions (azoospermia factor genes, AZF) in infertile males who seek assisted reproduction. These studies are very important because the assisted reproduction techniques (mainly intracytoplasmic sperm injection) bypass the natural selection process and some classical chromosomal abnormalities, microdeletions of AZF genes or some deleterious genic mutations could pass through generations. These genetic abnormalities can cause in the offspring of these patients male infertility, ambiguous external genitalia, mental retardation, and other birth defects. We studied 165 infertile men whose infertility was attributable to testicular problems (60 were azoospermic, 100 were oligospermic and 5 were asthenospermic). We studied 100 metaphases per patient with GTG banding obtained from temporary lymphocyte culture for chromosomal abnormality detection and performed a genomic DNA analysis using 28 Y chromosome-specific sequence-tagged sites for Y AZF microdeletion detection. Karyotyping revealed somatic anomalies in 16 subjects (16/165 = 9.6%). Of these 16, 12 were in the azoospermic group (12/60 = 20%) and 4 were in the oligospermic group (4/100 = 4%). The most common chromosomal anomaly was Klinefelter syndrome (10/165 = 6%). Microdeletions of AZF genes were detected in 12 subjects (12/160 = 7.5%). The frequencies detected are similar to those described previously. These results show the importance of genetic evaluation of infertile males prior to assisted reproduction. Such evaluation can lead to genetic counseling and, consequently, to primary and secondary prevention of mental retardation and birth defects.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Comparison of the effects of lactated Ringer solution with and without hydroxyethyl starch fluid resuscitation on gut edema during severe splanchnic ischemia

The type of fluid used during resuscitation may have an important impact on tissue edema. We evaluated the impact of two different regimens of fluid resuscitation on hemodynamics and on lung and intestinal edema during splanchnic hypoperfusion in rabbits. The study included 16 female New Zealand rabbits (2.9 to 3.3 kg body weight, aged 8 to 12 months) with splanchnic ischemia induced by ligation of the superior mesenteric artery. The animals were randomized into two experimental groups: group I (N = 9) received 12 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 6% hydroxyethyl starch solution; group II (N = 7) received 36 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 0.9% saline. A segment from the ileum was isolated to be perfused. A tonometric catheter was placed in a second gut segment. Superior mesenteric artery (Q SMA) and aortic (Qaorta) flows were measured using ultrasonic flow probes. After 4 h of fluid resuscitation, tissue specimens were immediately removed for estimations of gut and lung edema. There were no differences in global and regional perfusion variables, lung wet-to-dry weight ratios and oxygenation indices between groups. Gut wet-to-dry weight ratio was significantly lower in the crystalloid/colloid-treated group (4.9 ± 1.5) than in the crystalloid-treated group (7.3 ± 2.4) (P < 0.05). In this model of intestinal ischemia, fluid resuscitation with crystalloids caused more gut edema than a combination of crystalloids and colloids.

Effect of gallium-arsenide laser, gallium-aluminum-arsenide laser and healing ointment on cutaneous wound healing in Wistar rats

This study determined the effects of gallium-aluminum-arsenide laser (GaAlAs), gallium-arsenide laser (GaAs) and Dersani® healing ointment on skin wounds in Wistar rats. The parameters analyzed were: type I and III collagen fiber concentrations as well as the rate of wound closure. Five wounds, 12 mm in diameter, were made on the animals’ backs. The depth of the surgical incision was controlled by removing the epithelial tissue until the dorsal muscular fascia was exposed. The animals were anesthetized with ketamine and xylazine via intraperitoneal injection. The rats were randomly divided into five groups of 6 animals each, according to the treatment received. Group 1 (L4): GaAs laser (4 J/cm²); group 2 (L30): GaAlAs laser (30 J/cm²); group 3 (L60): GaAlAs laser (60 J/cm²); group 4 (D): Dersani® ointment; group 5 (control): 0.9% saline. The applications were made daily over a period of 20 days. Tissue fragments were stained with picrosirius to distinguish type I collagen from type III collagen. The collagen fibers were photo-documented and analyzed using the Quantum software based on the primary color spectrum (red, yellow and blue). Significant results for wound closing rate were obtained for group 1 (L4), 7.37 mm/day. The highest concentration of type III collagen fibers was observed in group 2 (L30; 37.80 ± 7.10%), which differed from control (29.86 ± 5.15%) on the 20th day of treatment. The type I collagen fibers of group 1 (L4; 2.67 ± 2.23%) and group 2 (L30; 2.87 ± 2.40%) differed significantly from control (1.77 ± 2.97%) on the 20th day of the experiment.

Possible in vivo mechanisms involved in photodynamic therapy using tetrapyrrolic macrocycles

Photodynamic therapy (PDT) mediated by oxidative stress causes direct tumor cell damage as well as microvascular injury. To improve this treatment new photosensitizers are being synthesized and tested. We evaluated the effects of PDT with 5,10,15,20-tetrakis(4-methoxyphenyl)-porphyrin (TMPP) and its zinc complex (ZnTMPP) on tumor levels of malondialdehyde (MDA), reduced glutathione (GSH) and cytokines, and on the activity of caspase-3 and metalloproteases (MMP-2 and -9) and attempted to correlate them with the histological alterations of tumors in 3-month-old male Wistar rats, 180 ± 20 g, bearing Walker 256 carcinosarcoma. Rats were randomly divided into five groups: group 1, ZnTMPP+irradiation (IR) 10 mg/kg body weight; group 2, TMPP+IR 10 mg/kg body weight; group 3, 5-aminolevulinic acid (5-ALA+IR) 250 mg/kg body weight; group 4, control, no treatment; group 5, only IR. The tumors were irradiated for 15 min with red light (100 J/cm², 10 kHz, 685 nm) 24 h after drug administration. Tumor tissue levels of MDA (1.1 ± 0.7 in ZnTMPP vs 0.1 ± 0.04 nmol/mg protein in control) and TNF-α (43.5 ± 31.2 in ZnTMPP vs 17.3 ± 1.2 pg/mg protein in control) were significantly higher in treated tumors than in controls. Higher caspase-3 activity (1.9 ± 0.9 in TMPP vs 1.1 ± 0.6 OD/mg protein in control) as well as the activation of MMP-2 (P < 0.05) were also observed in tumors. These parameters were correlated (Spearman correlation, P < 0.05) with the histological alterations. These results suggest that PDT activates the innate immune system and that the effects of PDT with TMPP and ZnTMPP are mediated by reactive oxygen species, which induce cell membrane damage and apoptosis.

Testosterone therapy delays cardiomyocyte aging via an androgen receptor-independent pathway

The testicular feminized (Tfm) mouse carries a nonfunctional androgen receptor (AR) and reduced circulating testosterone levels. We used Tfm and castrated mice to determine whether testosterone modulates markers of aging in cardiomyocytes via its classic AR-dependent pathway or conversion to estradiol. Male littermates and Tfm mice were divided into 6 experimental groups. Castrated littermates (group 1) and sham-operated Tfm mice (group 2, N = 8 each) received testosterone. Sham-operated Tfm mice received testosterone in combination with the aromatase inhibitor anastrazole (group 3, N = 7). Castrated littermates (group 4) and sham-operated untreated Tfm mice (group 5) were used as controls (N = 8 and 7, respectively). An additional control group (group 6) consisted of age-matched non-castrated littermates (N = 8). Cardiomyocytes were isolated from the left ventricle, telomere length was measured by quantitative PCR and expression of p16INK4α, retinoblastoma (Rb) and p53 proteins was detected by Western blot 3 months after treatment. Compared with group 6, telomere length was short (P < 0.01) and expression of p16INK4α, Rb and p53 proteins was significantly (P < 0.05) up-regulated in groups 4 and 5. These changes were improved to nearly normal levels in groups 1 and 2 (telomere length = 0.78 ± 0.05 and 0.80 ± 0.08; p16INK4α = 0.13 ± 0.03 and 0.15 ± 0.04; Rb = 0.45 ± 0.05 and 0.39 ± 0.06; p53 = 0.16 ± 0.04 and 0.13 ± 0.03), but did not differ between these two groups. These improvements were partly inhibited in group 3 compared with group 2 (telomere length = 0.65 ± 0.08 vs 0.80 ± 0.08, P = 0.021; p16INK4α = 0.28 ± 0.05 vs 0.15 ± 0.04, P = 0.047; Rb = 0.60 ± 0.06 vs 0.39 ± 0.06, P < 0.01; p53 = 0.34 ± 0.06 vs 0.13 ± 0.03, P = 0.004). In conclusion, testosterone deficiency contributes to cardiomyocyte aging. Physiological testosterone can delay cardiomyocyte aging via an AR-independent pathway and in part by conversion to estradiol.

Percent body fat is a better predictor of cardiovascular risk factors than body mass index

The objective of the present study was to evaluate the predictive values of percent body fat (PBF) and body mass index (BMI) for cardiovascular risk factors, especially when PBF and BMI are conflicting. BMI was calculated by the standard formula and PBF was determined by bioelectrical impedance analysis. A total of 3859 ambulatory adult Han Chinese subjects (2173 males and 1686 females, age range: 18-85 years) without a history of cardiovascular diseases were recruited from February to September 2009. Based on BMI and PBF, they were classified into group 1 (normal BMI and PBF, N = 1961), group 2 (normal BMI, but abnormal PBF, N = 381), group 3 (abnormal BMI, but normal PBF, N = 681), and group 4 (abnormal BMI and PBF, N = 836). When age, gender, lifestyle, and family history of obesity were adjusted, PBF, but not BMI, was correlated with blood glucose and lipid levels. The odds ratio (OR) and 95% confidence interval (CI) for cardiovascular risk factors in groups 2 and 4 were 1.88 (1.45-2.45) and 2.06 (1.26-3.35) times those in group 1, respectively, but remained unchanged in group 3 (OR = 1.32, 95%CI = 0.92-1.89). Logistic regression models also demonstrated that PBF, rather than BMI, was independently associated with cardiovascular risk factors. In conclusion, PBF, and not BMI, is independently associated with cardiovascular risk factors, indicating that PBF is a better predictor.

Effect of vitamin D therapy in addition to amitriptyline on migraine attacks in pediatric patients

The purpose of this study was to investigate the effect of supplementary vitamin D therapy in addition to amitriptyline on the frequency of migraine attacks in pediatric migraine patients. Fifty-three children 8-16 years of age and diagnosed with migraine following the International Headache Society 2005 definition, which includes childhood criteria, were enrolled. Patients were classified into four groups on the basis of their 25-hydroxyvitamin D [25(OH)D] levels. Group 1 had normal 25(OH)D levels and received amitriptyline therapy alone; group 2 had normal 25(OH)D levels and received vitamin D supplementation (400 IU/day) plus amitriptyline; group 3 had mildly deficient 25(OH)D levels and received amitriptyline plus vitamin D (800 IU/day); and group 4 had severely deficient 25(OH)D levels and was given amitriptyline plus vitamin D (5000 IU/day). All groups were monitored for 6 months, and the number of migraine attacks before and during treatment was determined. Calcium, phosphorus alkaline phosphatase, parathormone, and 25(OH)D levels were also determined before and during treatment. Results were compared between the groups. Data obtained from the groups were analyzed using one-way analysis of variance. The number of pretreatment attacks in groups 1 to 4 was 7±0.12, 6.8±0.2, 7.3±0.4, and 7.2±0.3 for 6 months, respectively (all P>0.05). The number of attacks during treatment was 3±0.25, 1.76±0.37 (P<0.05), 2.14±0.29 (P<0.05), and 1.15±0.15 (P<0.05), respectively. No statistically significant differences in calcium, phosphorus, alkaline phosphatase, or parathormone levels were observed (P>0.05). Vitamin D given in addition to anti-migraine treatment reduced the number of migraine attacks.

Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in São Paulo, Brazil, from 1994 and 2006 to 2010

Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.

Spectrophotometric and spectrofluorimetric determination of some drugs containing secondary amino group in bulk drug and dosage forms via derivatization with 7-Chloro-4-Nitrobenzofurazon

Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for determination of some drugs such as Pramipexole, Nebivolol, Carvedilol, and Eletriptan, which commonly contain secondary amino group. The subject methods were developed via derivatization of the secondary amino groups with 7-Chloro-4-Nitrobenzofurazon in borate buffer where a yellow colored reaction product was obtained and measured spectrophotometrically or spectrofluorimetrically. Concentration ranges were found as 2.0 to 250 μg mL-1 and 0.1 to 3.0 μg mL-1, for spectrophotometric and spectrofluorimetric study, respectively. The described methods can be easily applied by the quality control laboratories in routine analyses of these drugs in pharmaceutical preparations.

Epidemiology and antimicrobial resistance of B. fragilis group organisms isolated from clinical specimen and human intestinal microbiota

Epidemiological aspects and the antimicrobial susceptibility profile of the Bacteroides fragilis group isolated from clinical and human intestinal specimens were examined in this study. B. fragilis group strains were isolated from 46 (37%) of 124 clinical specimens and the source of the samples was: Blood culture (3), intraabdominal infection (27), brain abscess (2), soft tissue infection (17), respiratory sinus (3), pleural aspirate (9), breast abscess (3), surgical infected wound (22), pelvic inflammatory disease (22), chronic otitis media (9) and miscellaneous (7). Intraabdominal and soft tissue infections were responsible for more than half of the clinical isolates. Susceptibility to penicillin, cefoxitin, tetracycline, metronidazole, chloramphenicol and clindamycin was examined. All isolates were susceptible to metronidazole and chloramphenicol. For clindamycin and cefoxitin the resistance rates observed were 21.7% and 10.9% respectively. Susceptibility profiles varied among the different species tested. A total of 37 species of B. fragilis group isolated from intestinal microbiota of individuals who had no antimicrobial therapy for at least 1 month before the sampling was also examined. All strains were also susceptible to chloramphenicol and motronidazole and the resistance rates to clindamycin and cefoxitin were 19.4% and 5.4% respectively. A few institutions, in Brazil, have monitored the antimicrobial susceptibility of B. fragilis group strains isolated from anaerobic infections. The resistance rates to cefoxitin and clindamycin and the variation in susceptibility patterns among the species isolated in this study emphasize the need for monitoring of susceptibility patterns of B. fragilis group organisms isolated, especially at our University Hospitals.