Plasmodium vivax Duffy binding protein: baseline antibody responses and parasite polymorphisms in a well-consolidated settlement of the Amazon Region

Objective To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) a leading malaria vaccine candidate in a well-consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBPII) within the local malaria parasite population. Methods Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBPII. Results The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti-PvDBP IgG antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) with subjects age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBPII diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement. Conclusion The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBPII diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.

Cardiovascular responses to glutamate microinjection in the dorsomedial periaqueductal gray of unanesthetized rats

The periaqueductal gray area (PAG) is a mesencephalic area involved in cardiovascular modulation. Glutamate (L-Glu) is an abundant excitatory amino acid in the central nervous system (CNS) and is present in the rat PAG. Moreover, data in the literature indicate its involvement in central blood pressure control. Here we report on the cardiovascular effects caused by microinjection of L-Glu into the dorsomedial PAG (dmPAG) of rats and the glutamatergic receptors as well as the peripheral mechanism involved in their mediation. The microinjection of L-Glu into the dmPAG of unanesthetized rats evoked dose-related pressor and bradycardiac responses. The cardiovascular response was significantly reduced by pretreatment of the dmPAG with a glutamatergic M-methyl-D-aspartate (NMDA) receptor antagonist (LY235959) and was not affected by pretreatment with a non-NMDA receptor antagonist (NBQX), suggesting a mediation of that response by the activation of NMDA receptors. Furthermore, the pressor response was blocked by pretreatment with the ganglion blocker pentolinium (5 mg/kg, intravenously), suggesting an involvement of the sympathetic nervous system in this response. Our results indicate that the microinjection of L-Glu into the dmPAG causes sympathetic-mediated pressor responses in unanesthetized rats, which are mediated by glutamatergic NMDA receptors in the dmPAG. (c) 2012 Wiley Periodicals, Inc.

Physiological effects of glyphosate over amino acid profile in conventional and transgenic soybean (Glycine max)

This paper compares the responses of conventional and transgenic soybean to glyphosate application in terms of the contents of 17 detectable soluble amino acids in leaves, analyzed by HPLC and fluorescence detection. Glutamate, histidine, asparagine, arginine + alanine, glycine + threonine and isoleucine increased in conventional soybean leaves when compared to transgenic soybean leaves, whereas for other amino acids, no significant differences were recorded. Univariate analysis allowed us to make an approximate differentiation between conventional and transgenic lines, observing the changes of some variables by glyphosate application. In addition, by means of the multivariate analysis, using principal components analysis (PCA), cluster analysis (CA) and linear discriminant analysis (LDA) it was possible to identify and discriminate different groups based on the soybean genetic origin. (C) 2011 Elsevier Inc. All rights reserved.

Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax

We evaluated the influence of allelic frequency of the human leukocyte antigen (HLA) -DRB1 on the acquisition of antibody response against malaria sporozoite and merozoite peptides in patients with Plasmodium vivax malaria acquired in endemic areas of Brazil. IgG antibodies were detected by enzyme-linked immunosorbent assay against four peptides of circumsporozoite protein (CSP) (amino, carboxyl, and VK210 and VK247 repeats) and peptides of merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and Duffy-binding protein (DBP). We found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to CSP amino terminal, as well as an association between HILA-DR3 and the highest antibody response to MSP1 (Pv200L). In conclusion, we suggest a potential regulatory role of the H1A-DRB1 alleles in the production of antibodies to a conserved region of P. vivax CSP and MSP1 in Brazilian population exposed to malaria. (C) 2011 Elsevier B.V. All rights reserved.

Fluctuation of Anti-Endothelial Cell Antibody Titers in Mixed Connective Tissue Disease

Background: Antibodies directed against endothelial cell surface antigens have been described in many disorders and have been associated with disease activity. Since the most prominent histopathologic feature in mixed connective tissue disease (MCTD) is the widespread and unique proliferative vascular lesion, our aim was to evaluate the frequency of anti-endothelial cell antibodies (AECA) in this condition. Objectives: To evaluate the frequency of AECA in this disease and assess its clinical and laboratory associations. Methods: Seventy-three sera from 35 patients with MCTD (Kasukawa's criteria), collected during a 7 year period, were tested for immunoglobulins G and M (IgG and IgM) AECA by cellular ELISA, using HUVEC (human umbilical vein endothelial cells). Sera from 37 patients with systemic lupus erythematosus (SLE), 22 with systemic sclerosis (SSc) and 36 sera from normal healthy individuals were used as controls. A cellular ELISA using HeLa cells was also performed as a laboratory control method. Results: IgG-AECA was detected in 77% of MCTD patients, 54% of SLE patients, 36% of SSc patients and 6% of normal controls. In MCTD, IgG-AECA was associated with vasculitic manifestations, disease activity and lymphopenia, and was also a predictor of constant disease activity. Immunosuppressive drugs were shown to reduce IgG-AECA titers. Since antibodies directed to HeLa cell surface were negative, AECA was apparently unrelated to common epitopes present on epithelial cell lines. Conclusions: AECA are present in a large proportion of patients with MCTD and these antibodies decrease after immunosuppressive treatment. IMAJ 2012; 14:84-87

Antinociceptive effect of stimulating the zona incerta with glutamate in rats

The zona incerta (ZI) is a subthalamic nucleus connected to several structures, some of them known to be involved with antinociception. The 21 itself may be involved with both antinociception and nociception. The antinociceptive effects of stimulating the ZI with glutamate using the rat tail-flick test and a rat model of incision pain were examined. The effects of intraperitoneal antagonists of acetylcholine, noradrenaline, serotonin, dopamine, or opioids on glutamate-induced antinociception from the ZI in the tail-flick test were also evaluated. The injection of glutamate (7 mu g/0.25 mu l) into the ZI increased tail-flick latency and inhibited post-incision pain, but did not change the animal performance in a Rota-rod test. The injection of glutamate into sites near the ZI was non effective. The glutamate-induced antinociception from the ZI did not occur in animals with bilateral lesion of the dorsolateral funiculus, or in rats treated intraperitoneally with naloxone (1 and 2 m/kg), methysergide (1 and 2 m/kg) or phenoxybenzamine (2 m/kg), but remained unchanged in rats treated with atropine, mecamylamine, or haloperidol (all given at doses of 1 and 2 m/kg). We conclude that the antinociceptive effect evoked from the ZI is not due to a reduced motor performance, is likely to result from the activation of a pain-inhibitory mechanism that descends to the spinal cord via the dorsolateral funiculus, and involves at least opioid, serotonergic and a-adrenergic mechanisms. This profile resembles the reported effects of these antagonists on the antinociception caused by stimulating the periaqueductal gray or the pedunculopontine tegmental nucleus. (C) 2012 Elsevier Inc. All rights reserved.

Ionotropic Glutamate Receptors in Hypothalamic Paraventricular and Supraoptic Nuclei Mediate Vasopressin and Oxytocin Release in Unanesthetized Rats

We report changes in plasma arginine vasopressin (AVP) and oxytocin (OT) concentrations evoked by the microinjection of L-glutamate (L-glu) into the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus(PVN) of unanesthetized rats, as well as which local mechanisms are involved in their mediation. L-Glu microinjection (10 nmol/100 nl) into the SON increased the circulating levels of both AVP and OT. The AVP increases were blocked by local pretreatment with the selective non-N-methyl-D-aspartate (NMDA) receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (2 nmol/100 nl), but it was not affected by pretreatment with the NMDA-receptor antagonist LY235959 (2 nmol/100 nl). The OT response to L-glu microinjection into the SON was blocked by local pretreatment with either NBQX or LY235959. Furthermore, the administration of either the non-NMDA receptor agonist (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) (5 nmol/100 nl) or NMDA receptor agonist NMDA (5 nmol/100 nl) into the SON had no effect on OT baseline plasma levels, but when both agonists were microinjected together these levels were increased. L-Glu microinjection into the PVN did not change circulating levels of either AVP or OT. However, after local pretreatment with LY235959, the L-glu microinjection increased plasma levels of the hormones. The L-glu microinjection into the PVN after the local treatment with NBQX did not affect the circulating AVP and OT levels. Therefore, results suggest the AVP release from the SON is mediated by activation of non-NMDA glutamate receptors, whereas the OT release from this nucleus is mediated by an interaction of NMDA and non-NMDA receptors. The present study also suggests an inhibitory role for NMDA receptors in the PVN on the release of AVP and OT. (Endocrinology 153: 2323-2331, 2012)

In Silico Analysis and Immunohistochemical Characterization of NaPi2b Protein Expression in Ovarian Carcinoma With Monoclonal Antibody Mx35

Introduction: Ovarian adenocarcinoma is frequently detected at the late stage, when therapy efficacy is limited and death occurs in up to 50% of the cases. A potential novel treatment for this disease is a monoclonal antibody that recognizes phosphate transporter sodium-dependent phosphate transporter protein 2b (NaPi2b). Materials and Methods: To better understand the expression of this protein in different histologic types of ovarian carcinomas, we immunostained 50 tumor samples with anti-NaPi2b monoclonal antibody MX35 and, in parallel, we assessed the expression of the gene encoding NaPi2b (SCL34A2) by in silico analysis of microarray data. Results: Both approaches detected higher expression of NaPi2b (SCL34A2) in ovarian carcinoma than in normal tissue. Moreover, a comprehensive analysis indicates that SCL34A2 is the only gene of the several phosphate transporters genes whose expression differentiates normal from carcinoma samples, suggesting it might exert a major role in ovarian carcinomas. Immunohistochemical and mRNA expression data have also shown that 2 histologic subtypes of ovarian carcinoma express particularly high levels of NaPi2b: serous and clear cell adenocarcinomas. Serous adenocarcinomas are the most frequent, contrasting with clear cell carcinomas, rare, and with worse prognosis. Conclusion: This identification of subgroups of patients expressing NaPi2b may be important in selecting cohorts who most likely should be included in future clinical trials, as a recently generated humanized version of MX35 has been developed.

Glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats

Background: Given the established fact that obesity interferes with male reproductive functions, the present study aimed to evaluate sperm production in the testis and storage in the epididymis in a glutamate-induced model of obesity. Methods: Male rats were treated neonatally with monosodium glutamate (MSG) at doses of 4 mg/kg subcutaneously, or with saline solution (control group), on postnatal days 2, 4, 6, 8 and 10. On day 120, obesity was confirmed by the Lee index in all MSG-treated rats. After this, all animals from the two experimental groups were anesthetized and killed to evaluate body and reproductive organ weights, sperm parameters, plasma hormone levels (FSH, LH and testosterone), testicular and epididymal histo-morphometry and histopathology. Results: Significant reductions in absolute and relative weights of testis, epididymis, prostate and seminal vesicle were noted in MSG-treated animals. In these same animals plasma testosterone and follicle-stimulating hormone (FSH) concentrations were decreased, as well as sperm counts in the testis and epididymis and seminiferous epithelium height and tubular diameter. The sperm transit time was accelerated in obese rats. However, the number of Sertoli cells per seminiferous tubule and stereological findings on the epididymis were not markedly changed by obesity. Conclusions: Neonatal MSG-administered model of obesity lowers sperm production and leads to a reduction in sperm storage in the epididymis of adult male rats. The acceleration of sperm transit time can have implications for the sperm quality of these rats.

Monosodium glutamate neonatal treatment induces cardiovascular autonomic function changes in rodents

OBJECTIVES: The aim of this study was to evaluate cardiovascular autonomic function in a rodent obesity model induced by monosodium glutamate injections during the first seven days of life. METHOD: The animals were assigned to control (control, n = 10) and monosodium glutamate (monosodium glutamate, n = 13) groups. Thirty-three weeks after birth, arterial and venous catheters were implanted for arterial pressure measurements, drug administration, and blood sampling. Baroreflex sensitivity was evaluated according to the tachycardic and bradycardic responses induced by sodium nitroprusside and phenylephrine infusion, respectively. Sympathetic and vagal effects were determined by administering methylatropine and propranolol. RESULTS: Body weight, Lee index, and epididymal white adipose tissue values were higher in the monosodium glutamate group in comparison to the control group. The monosodium glutamate-treated rats displayed insulin resistance, as shown by a reduced glucose/insulin index (-62.5%), an increased area under the curve of total insulin secretion during glucose overload (39.3%), and basal hyperinsulinemia. The mean arterial pressure values were higher in the monosodium glutamate rats, whereas heart rate variability (>7 times), bradycardic responses (>4 times), and vagal (similar to 38%) and sympathetic effects (similar to 36%) were reduced as compared to the control group. CONCLUSION: Our results suggest that obesity induced by neonatal monosodium glutamate treatment impairs cardiac autonomic function and most likely contributes to increased arterial pressure and insulin resistance.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome due to a mutation in the glutamate dehydrogenase gene

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a rare autosomal dominant disease manifested by hypoglycemic symptoms triggered by fasting or high-protein meals, and by elevated serum ammonia. HI/HA is the second most common cause of hyperinsulinemic hypoglycemia of infancy, and it is caused by activating mutations in GLUD1, the gene that encodes mitochondrial enzyme glutamate dehydrogenase (GDH). Biochemical evaluation, as well as direct sequencing of exons and exon-intron boundary regions of the GLUD1 gene, were performed in a 6-year old female patient presenting fasting hypoglycemia and hyperammonemia. The patient was found to be heterozygous for one de novo missense mutation (c.1491A>G; p.Il497Met) previously reported in a Japanese patient. Treatment with diazoxide 100 mg/day promoted complete resolution of the hypoglycemic episodes. Arq Bras Endocrinol Metab. 2012;56(8):485-9

THE NEGATIVE EFFECTS OF ALCOHOL HANGOVER ON HIGH-ANXIETY PHENOTYPE RATS ARE INFLUENCED BY THE GLUTAMATE RECEPTORS OF THE DORSAL MIDBRAIN

Alcoholism is a chronic disorder characterized by the appearance of a withdrawal syndrome following the abrupt cessation of alcohol intake that includes symptoms of physical and emotional disturbances, anxiety being the most prevalent symptom. In humans, it was shown that anxiety may increase the probability of relapse. In laboratory animals, however, the use of anxiety to predict alcohol preference has remained difficult. Excitatory amino acids as glutamate have been implicated in alcohol hangover and may be responsible for the seizures and anxiety observed during withdrawal. The dorsal periaqueductal gray (DPAG) is a midbrain region critical for the modulation/expression of anxiety- and fear-related behaviors and the propagation of seizures induced by alcohol withdrawal, the glutamate neurotransmission being one of the most affected. The present study was designed to evaluate whether low- (LA) and high-anxiety rats (HA), tested during the alcohol hangover phase, in which anxiety is the most prevalent symptom, are more sensitive to the reinforcing effects of alcohol when tested in a voluntary alcohol drinking procedure. Additionally, we were interested in investigating the main effects of reducing the excitatory tonus of the dorsal midbrain, after the blockade of the ionotropic glutamate receptors into the DPAG, on the voluntary alcohol intake of HA and LA motivated rats that were made previously experienced with the free operant response of alcohol drinking. For this purpose, we used local infusions of the N-metil D-Aspartato (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate receptors antagonist DL-2-Amino-7-phosphonoheptanoic acid - DL-AP7 (10 nmol/0.2 mu l) and L-glutamic acid diethyl ester - GDEE (160 nmol/0.2 mu l) respectively. Alcohol intoxication was produced by 10 daily bolus intraperitonial (IP) injections of alcohol (2.0 g/kg). Peak-blood alcohol levels were determined by gas-chromatography analysis in order to assess blood-alcohol content. Unconditioned and conditioned anxiety-like behavior was assessed by the use of the fear-potentiated startle procedure (FPS). Data collected showed that anxiety and alcohol drinking in HA animals are positively correlated in animals that were made previously familiarized with the anxiolytic effects of alcohol. In addition, anxiety-like behavior induced during alcohol hangover seems to be an effect of changes in glutamatergic neurotransmission into DPAG possibly involving AMPA/kainate and NMDA receptors, among others. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Production of Human Antibody Fragments Binding to Melittin and Phospholipase A2 in Africanised Bee Venom: Minimising Venom Toxicity

The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

Autoantibodies and High-Risk HLA Susceptibility Markers in First-Degree Relatives of Brazilian Patients with Type 1 Diabetes Mellitus: A Progression to Disease Based Study

The objective of this study was to determine the frequencies of autoantibodies to heterogeneous islet-cell cytoplasmic antigens (ICA), glutamic acid decarboxylase(65) (GAD(65)A), insulinoma-associated antigen-2 (IA-2A) and insulin (IAA)-and human leukocyte antigen (HLA) class II markers (HLA-DR and -DQ) in first degree relatives of heterogeneous Brazilian patients with type I diabetes(T1DM). A major focus of this study was to determine the influence of age, gender, proband characteristics and ancestry on the prevalence of autoantibodies and HLA-DR and -DQ alleles on disease progression and genetic predisposition to T1DM among the first-degree relatives. IAA, ICA, GAD(65)A, IA-2A and HLA- class II alleles were determined in 546 first-degree-relatives, 244 siblings, 55 offspring and 233 parents of 178 Brazilian patients with T1DM. Overall, 8.9% of the relatives were positive for one or more autoantibodies. IAA was the only antibody detected in parents. GAD(65) was the most prevalent antibody in offspring and siblings as compared to parents and it was the sole antibody detected in offspring. Five siblings were positive for the IA-2 antibody. A significant number (62.1%) of siblings had 1 or 2 high risk HLA haplotypes. During a 4-year follow-up study, 5 siblings (expressing HLA-DR3 or -DR4 alleles) and 1 offspring positive for GAD(65)A progressed to diabetes. The data indicated that the GAD(65) and IA-2 antibodies were the strongest predictors of T1DM in our study population. The high risk HLA haplotypes alone were not predictive of progression to overt diabetes.

Effects of feeding a multivalent polyclonal antibody preparation on feedlot performance, carcass characteristics, rumenitis, and blood gas profile in Bos indicus biotype yearling bulls

The objective of this study was to evaluate effects of feeding monensin (MON) or a multivalent polyclonal antibody preparation (PAP) against several rumen microorganisms on feedlot performance, carcass characteristics, blood gas profile, and rumenitis of Bos indicus biotype (BT) yearling bulls. The study was designed as a completely randomized design with a 3 x 2 factorial arrangement, replicated 4 times, in which 32 yearling bulls of each of 3 BT evaluated (3-way-cross, TC; Canchim, CC; and Nellore, NE) were fed diets containing either MON at 300 mg.d(-1) or PAP at 10 mL.d(-1) across 3 different periods. No significant (P > 0.10) feed additive (FA) main effects were observed for any of the feedlot performance variables and carcass characteristics with the exception of dressing percentage. Yearling bulls receiving PAP had a decreased (P = 0.047) dressing percentage when compared with yearling bulls receiving MON. Significant (P < 0.05) BT main effects were observed for all feedlot performance variables and carcass characteristics with the exception of kidney-pelvic fat expressed in kilograms (P = 0.49) and LM lipids content (P = 0.45). Crossbred yearling bulls (TC and CC) had greater (P < 0.001) ADG, DMI in kilograms, DMI as % of BW, and improved (P = 0.001) G: F when compared with NE yearling bulls. A tendency (P = 0.072) for a FA main effect was observed for rumenitis scores, in which yearling bulls receiving PAP had lesser rumenitis scores than those receiving MON. When the data were disposed as frequency percentage, 55.6% and 45.7% of the rumens from yearling bulls fed PAP and MON were scored between 0 and 1, respectively (0 = no lesions, 10 = severe lesions). Likewise, a significant BT main effect was observed (P = 0.008), where NE yearling bulls had greater rumenitis scores than those of crossbred yearling bulls (TC and CC). No signifi cant FA main effects were observed (P > 0.10) for any of the fatty acids measured in the subcutaneous adipose tissue, with the exception that yearling bulls receiving MON had greater (P < 0.05) concentrations of palmitic acid (16: 0), margaric acid (17: 0), docosapentaenoic acid (22: 5), and docosahexaenoic acid (22: 6) than those yearling bulls receiving PAP. Feeding PAP tended to decrease incidence of rumen lesions and led to similar feedlot performance compared with feeding MON. Thus, PAP is a new technology that presents a possible alternative for ionophores.