Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

Comparison of the effectiveness of initial combined antiretroviral therapy with nelfinavir or efavirenz at a university-based outpatient service in Brazil

Since there are some concerns about the effectiveness of highly active antiretroviral therapy in developing countries, we compared the initial combination antiretroviral therapy with zidovudine and lamivudine plus either nelfinavir or efavirenz at a university-based outpatient service in Brazil. This was a retrospective comparative cohort study carried out in a tertiary level hospital. A total of 194 patients receiving either nelfinavir or efavirenz were identified through our electronic database search, but only 126 patients met the inclusion criteria. Patients were included if they were older than 18 years old, naive for antiretroviral therapy, and had at least 1 follow-up visit after starting the antiretroviral regimen. Fifty-one of the included patients were receiving a nelfinavir-based regimen and 75 an efavirenz-based regimen as outpatients. Antiretroviral therapy was prescribed to all patients according to current guidelines. By intention-to-treat (missing/switch = failure), after a 12-month period, 65% of the patients in the efavirenz group reached a viral load <400 copies/mL compared to 41% of the patients in the nelfinavir group (P = 0.01). The mean CD4 cell count increase after a 12-month period was also greater in the efavirenz group (195 x 10(6) cells/L) than in the nelfinavir group (119 x 10(6) cells/L; P = 0.002). The efavirenz-based regimen was superior compared to the nelfinavir-based regimen. The low response rate in the nelfinavir group might be partially explained by the difficulty of using a regimen requiring a higher patient compliance (12 vs 3 pills a day) in a developing country.

Evaluation of genotype resistance testing for salvage antiretroviral therapy at AIDS care centers from Ribeirão Preto, São Paulo, Brazil

The availability of HIV-1 genotype resistance testing (GRT) to clinicians has been insufficiently studied outside randomized clinical trials. The present study evaluated the outcome of salvage antiretroviral therapy (ART) recommended by an expert physician based on GRT in a non-clinical trial setting in Ribeirão Preto, Brazil. A prospective, open, nonrandomized study evaluating easy access to GRT at six Brazilian AIDS Clinics was carried out. This cooperative study analyzed the efficacy of treatment recommended to patients whose salvage ART was guided by GRT with that of treatment with ART based only on previous ART history. A total of 112 patients with ART failure were included in the study, and 77 of them were submitted to GRT. The median CD4 cell count and viral load for these 77 patients at baseline were (mean ± SD) 252.1 ± 157.4 cells/µL and 4.60 ± 0.5 log10 HIV RNA copies/mL, respectively. The access time, i.e., the time elapsed between ordering the GRT and receiving the result was, on average, 71.9 ± 37.3 days. The study results demonstrated that access to GRT followed by expert recommendations did not improve the time to persistent treatment failure when compared to conventional salvage ART. Access to GRT in this Brazilian community health care setting did not improve the long-term virologic outcomes of HIV-infected patients experiencing treatment failure. This result is probably related to the long time required to implement ART guided by GRT.

Influence of renal denervation on blood pressure, sodium and water excretion in acute total obstructive apnea in rats

Obstructive apnea (OA) can exert significant effects on renal sympathetic nerve activity (RSNA) and hemodynamic parameters. The present study focuses on the modulatory actions of RSNA on OA-induced sodium and water retention. The experiments were performed in renal-denervated rats (D; N = 9), which were compared to sham (S; N = 9) rats. Mean arterial pressure (MAP) and heart rate (HR) were assessed via an intrafemoral catheter. A catheter was inserted into the bladder for urinary measurements. OA episodes were induced via occlusion of the catheter inserted into the trachea. After an equilibration period, OA was induced for 20 s every 2 min and the changes in urine, MAP, HR and RSNA were recorded. Renal denervation did not alter resting MAP (S: 113 ± 4 vs D: 115 ± 4 mmHg) or HR (S: 340 ± 12 vs D: 368 ± 11 bpm). An OA episode resulted in decreased HR and MAP in both groups, but D rats showed exacerbated hypotension and attenuated bradycardia (S: -12 ± 1 mmHg and -16 ± 2 bpm vs D: -16 ± 1 mmHg and 9 ± 2 bpm; P < 0.01). The basal urinary parameters did not change during or after OA in S rats. However, D rats showed significant increases both during and after OA. Renal sympathetic nerve activity in S rats increased (34 ± 9%) during apnea episodes. These results indicate that renal denervation induces elevations of sodium content and urine volume and alters bradycardia and hypotension patterns during total OA in unconscious rats.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Restoration of the rabbit corneal surface after total epithelial debridement and complete limbal excision

How is the corneal epithelium restored when all of it plus the limbus have been eliminated? This investigation explored the possibility that this may be achieved through the conjunctival epithelium. The corneal epithelium of the right eye of 12 rabbits (Oryctolagus cuniculus) was totally scraped followed by surgical excision of the limbus plus 1.0-1.5 mm of the adjacent conjunctiva. Antibiotics and corticosteroids were applied for 1 week after surgery. Histological and immunohistochemical techniques were used to monitor the events taking place on the eye surface 2 weeks and 1, 3 and 6 months thereafter. Initially, the corneal surface was covered by conjunctival-like epithelium. After 1 month and more prominently at 3 and 6 months an epithelium displaying the morphological features of the cornea and reacting with the AE5 antibody was covering the central region. It is likely that the corneal epithelium originated from undifferentiated cells of the conjunctiva interacting with the corneal stroma.

Comparison of the variability of the onset and recovery from neuromuscular blockade with cisatracurium versus rocuronium in elderly patients under total intravenous anesthesia

This study was designed to compare the variability of the onset and offset of the effect of two neuromuscular blocking drugs with different elimination pathways in adult and elderly patients during total intravenous anesthesia (TIVA). After Ethics Committee approval and patients’ informed consent, the drugs were compared in 40 adult and 40 elderly patients scheduled for elective surgery under TIVA with tracheal intubation who were randomized to receive a single bolus dose of 0.15 mg/kg cisatracurium or 0.9 mg/kg rocuronium. The time of onset of maximum depression, duration of action, and recovery index time were measured and recorded for each patient and variability is reported as means ± standard deviation. Time of onset was significantly shorter for rocuronium than cisatracurium for the adult and elderly groups (P = 0.000), but the variability of cisatracurium was significantly greater compared with rocuronium for the same age groups (93.25 vs 37.01 s in the adult group and 64.56 vs 33.75 s in the elderly group; P = 0.000). The duration of the effect in the elderly group receiving rocuronium was significantly longer than in the elderly group receiving cisatracurium, and the variability of the duration was significantly greater in the rocuronium group than in the cisatracurium group. Mean time of recovery was significantly longer for the elderly group receiving rocuronium than for the elderly group receiving cisatracurium (P = 0.022), and variability was also greater (P = 0.002). Both drugs favored good intubating conditions. In conclusion, cisatracurium showed less variability in these parameters than rocuronium, especially in the elderly, a fact that may be of particular clinical interest.

Acute effects of resistance exercise and intermittent intense aerobic exercise on blood cell count and oxidative stress in trained middle-aged women

The aim of this study was to compare the effect of an intermittent intense aerobic exercise session and a resistance exercise session on blood cell counts and oxidative stress parameters in middle-aged women. Thirty-four women were selected and divided into three groups: RE group (performing 60 min of resistance exercises, N = 12), spinning group (performing 60 min of spinning, N = 12), and control group (not exercising regularly, N = 10). In both exercise groups, lymphocytes and monocytes decreased after 1-h recuperation (post-exercise) compared to immediately after exercise (P < 0.05). Immediately after exercise, in both exercised groups, a significant increase in TBARS (from 16.5 ± 2 to 25 ± 2 for the spinning group and from 18.6 ± 1 to 28.2 ± 3 nmol MDA/mL serum for the RE group) and protein carbonyl (from 1.0 ± 0.3 to 1.6 ± 0.2 for the spinning group and from 0.9 ± 0.2 to 1.5 ± 0.2 nmol/mg protein for the RE group) was observed (P < 0.05). A decrease in antioxidant activities (non-protein sulfhydryl, superoxide dismutase, catalase) was also demonstrated with a negative correlation between damage markers and antioxidant body defenses (P < 0.05). These results indicate that an acute bout of intermittent or anaerobic exercise induces immune suppression and increases the production of reactive oxygen species, causing oxidative stress in middle-aged and trained women. Furthermore, we demonstrated that trained women show improved antioxidant capacity and lower oxidative damage than sedentary ones, demonstrating the benefits of chronic regular physical activity.

Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

Leukocytosis, muscle damage and increased lymphocyte proliferative response after an adventure sprint race

The effect of an adventure sprint race (ASR) on T-cell proliferation, leukocyte count and muscle damage was evaluated. Seven young male runners completed an ASR in the region of Serra do Espinhaço, Brazil. The race induced a strong leukocytosis (6.22±2.04×103 cells/mm3 beforevs 14.81±3.53×103 cells/mm3after the race), marked by a significant increase of neutrophils and monocytes (P<0.05), but not total lymphocytes, CD3+CD4+ or CD3+CD8+ cells. However, the T-cell proliferative response to mitogenic stimulation was increased (P=0.025) after the race, which contradicted our hypothesis that ASR, as a high-demand competition, would inhibit T-cell proliferation. A positive correlation (P=0.03, r=0.79) was observed between the proliferative response of lymphocytes after the race and the time to complete the race, suggesting that the proliferative response was dependent on exercise intensity. Muscle damage was evident after the race by increased serum levels of aspartate amino transferase (24.99±8.30 vs 50.61±15.76 U/L, P=0.003). The results suggest that humoral factors and substances released by damaged muscle may be responsible for lymphocyte activation, which may be involved in muscle recovery and repair.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs=0.317, P=0.006), hemoglobin levels (rs=0.210, P=0.049), and percentage of peripheral blood blasts (rs=0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs=–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.

Negative emotions affect postoperative scores for evaluating functional knee recovery and quality of life after total knee replacement

This study aimed to determine whether psychological factors affect health-related quality of life (HRQL) and recovery of knee function in total knee replacement (TKR) patients. A total of 119 TKR patients (male: 38; female: 81) completed the Beck Anxiety Inventory (BAI), Beck Depression Inventory (BDI), State Trait Anxiety Inventory (STAI), Eysenck Personality Questionnaire-revised (EPQR-S), Knee Society Score (KSS), and HRQL (SF-36). At 1 and 6 months after surgery, anxiety, depression, and KSS scores in TKR patients were significantly better compared with those preoperatively (P<0.05). SF-36 scores at the sixth month after surgery were significantly improved compared with preoperative scores (P<0.001). Preoperative Physical Component Summary Scale (PCS) and Mental Component Summary Scale (MCS) scores were negatively associated with extraversion (E score) (B=-0.986 and -0.967, respectively, both P<0.05). Postoperative PCS and State Anxiety Inventory (SAI) scores were negatively associated with neuroticism (N score; B=-0.137 and -0.991, respectively, both P<0.05). Postoperative MCS, SAI, Trait Anxiety Inventory (TAI), and BAI scores were also negatively associated with the N score (B=-0.367, -0.107, -0.281, and -0.851, respectively, all P<0.05). The KSS function score at the sixth month after surgery was negatively associated with TAI and N scores (B=-0.315 and -0.532, respectively, both P<0.05), but positively associated with the E score (B=0.215, P<0.05). The postoperative KSS joint score was positively associated with postoperative PCS (B=0.356, P<0.05). In conclusion, for TKR patients, the scores used for evaluating recovery of knee function and HRQL after 6 months are inversely associated with the presence of negative emotions.

Evaluation of total carotenoids, alpha- and beta-carotene in carrots (Daucus carota L.) during home processing

This study aims to analyze the influence of dehydration and different preparation methods during home processing related toalpha-carotene, beta-carotene and total carotenoids stability in carrots. Vitamin A values were evaluated after different treatments. Thus, carrots were submitted to steam cooking, water cooking with and without pressure, moist/dry cooking and conventional dehydration. Determination of alpha- and beta-carotenes was made by High-Performance Liquid Chromatography (HPLC) (conditions were developed by us) using spectrophotometric detection visible-UV at 470 nm; a RP-18 column and methanol: acetonitrile: ethyl acetate (80: 10: 10) as mobile phase. Total carotenoids quantification was made by 449 nm spectrophotometer. The retention of the analyzed carotenoids ranged from 60.13 to 85.64%. Water cooking without pressure promoted higher retention levels of alpha- and beta-carotene and vitamin A values, while water cooking with pressure promoted higher retention levels of total carotenoids. Dehydration promoted the highest carotenoid losses. The results showed that, among the routinely utilized methods under domestic condition, cooking without pressure, if performed under controlled time and temperature, is the best method as it reduces losses in the amount of alpha- and beta-carotene, the main carotenoids present in the carrots. Despite the significant carotenoid losses, carrots prepared through domestic methods, remain a rich source of provitamin A.

Avaliação da perda de mercúrio total em peixe antes e após os processos de fritura e cocção

A perda de mercúrio contido em músculo de peixes (Piraíba, Brachyplatystoma ssp; Traíra, Hoplias malabaricus) submetidos a dois distintos processos de cozimento foi investigada. Os resultados mostraram perdas variando entre 0 e 30% (base seca), indicando que a maior parte do mercúrio foi retida. Assim, tais processos não protegem as populações, que deles se utilizam, contra a exposição por ingestão ao mercúrio originariamente contido na amostra "in natura".