Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knock-out mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.

Ornithine decarboxylase expression in the small intestine of broilers submitted to feed restriction and glutamine supplementation

Six hundred and forty one-day-old Cobb male broilers were used to evaluate ornithine decarboxylase (ODC) expression in the mucosa of the small intestine. Birds were submitted to early feed restriction from 7 to 14 days of age. The provided feed was supplemented with glutamine. A completely randomized design with a 2 x 2 factorial arrangement was used (with or without glutamine, with or without feed restriction). Restricted-fed birds were fed at 30% the amount of the ad libitum fed group from 7 to 14 days of age. Glutamine was added at the level of 1% in the diet supplied from 1 to 28 days of age. Protein concentration in the small intestine mucosa was determined, and ODC expression at 7, 14, 21, and 28 days of age was evaluated by dot blotting. ODC was present in the mucosa of broilers, and the presence of glutamine in the diet increased ODC activation. Glutamine prevented mucosa atrophy by stimulating protein synthesis, and was effective against the effects of feed restriction. Dot blotting can be used to quantify ODC expression in the intestinal mucosa of broilers.

Antibody and blood leukocyte response in Rhipicephalus sanguineus (Latreille, 1806) tick-infested dogs and guinea pigs

The dog is considered to be the natural host of Rhipicephalus sanguineus and is unable to develop appreciable resistance even after repeated feedings. The guinea pig develops strong resistance after one infestation with adult ticks. Antibody (IgG) titres against tick salivary gland antigens (SGAs) and blood leukocyte numbers in dogs and guinea pigs undergoing experimental R. sanguineus tick infestations were measured to detect a possible correlation with susceptibility or resistance of hosts. Since infested dogs develop an immediate hypersensitivity reaction to R. sanguineus antigens, total and anti-R. sanguineus SGA IgE levels were also measured in this host species. IgG and IgE antibody levels were determined by an enzyme-linked immunosorbent assay (ELISA) along three consecutive infestations of both hosts. Most dogs and guinea pigs displayed low IgG levels against R. sanguineus SGAs, though marked differences in individual response were observed. Although dog's total serum IgE levels increased significantly after infestations, no change in the amount of anti-salivary gland IgE was detected. Total and differential blood cell counts were determined in dogs and guinea pigs during primary and secondary infestation. In dogs, a tertiary infestation and a subsequent higher infestation level were also evaluated. Infested dogs did not display any alteration in blood leukocyte counts throughout the experiment. Guinea pigs, on the other hand, developed a significant basophilia during primary infestation which increased further during secondary infestation. These data reveal similarities and differences in the reactions of resistant and non-resistant hosts to ticks. They contribute for the understanding of such host-parasite relationships and will hopefully aid in the development of immune control of ticks. (C) 2003 Elsevier B.V. All rights reserved.

Experimental infection of Newcastle disease virus in pigeons (Columba livia): Humoral antibody response, contact transmission and viral genome shedding

The aim of this study was to evaluate the humoral antibody response, the genome viral excretion and the contact transmission of pathogenic chicken origin Newcastle disease virus (NDV) from experimentally infected pigeons (Columba livia) to in-contact pigeon. The antibody response to infection was assessed by the hemagglutination inhibition (HI) test and the genome viral excretion was detected by RT-PCR. Viral strain induced high antibody levels, both in inoculated and in sentinel birds. The pathogenic viral strain for chickens was unable to produce clinical signs of the disease in experimentally infected pigeons, although it induced the Immoral antibody response and produced NDV genome shedding. NDV genome was detected intermittently throughout the experimental period, from 5 days post-infection (dpi) to 24 dpi. Therefore, viral genome shedding occurred for 20 days. The viral genome was detected in all birds, between I I and 13 dpi. Furthermore, the high infectivity of the virus was confirmed, as all non-inoculated sentinel pigeons showed antibody levels as high as those of inoculated birds. (C) 2007 Elsevier B.V. All rights reserved.

In vitro and in situ activity of carboxymethyl cellulase and glutamate dehydrogenase according to supplementation with different nitrogenous compounds

Two experiments were carried out to evaluate the effect of supplementation with different nitrogenous compounds on the activities of carboxymethil cellulase (CMCase) and glutamate dehydrogenase (GDH). In the first experiment, four treatments were evaluated in vitro: cellulose, cellulose with casein, cellulose with urea, and cellulose with casamino acids. After 6, 12 and 24 hours of incubation, CMCase and GDH activity, pH, and concentrations of ammonia nitrogen (AN) and microbial protein were measured. In the three incubation periods, the concentration of AN was higher when urea was used as a supplemental source of nitrogen. The activity of CMCase was higher with the addition of urea and casamino acids when compared with the control and the casein treatment. Supplementation with casamino acids provided higher GDH activity when compared with the control at 6 hours of incubation. At 12 hours of incubation, the GHD activity was also stimulated by casein. At 24 hours, there was no difference in GHD activity among treatments. In the second experiment, three rumen-fistulated bulls were used for in situ evaluation. Animals were fed Tifton hay (Cynodon sp.) ad libitum. The treatments consisted of control (no supplementation), supplementation with non-protein nitrogenous compounds (urea and ammonium sulphate, 9:1) and supplementation with protein (albumin). In treatments with nitrogenous compound supplementation, 1 g of crude protein/kg of body weight was supplied. The experiment was conducted in a 3 × 3 Latin square design. The measurements were performed at 6, 12 and 24 hours after supplementation. No difference in GDH activity was observed among treatments. The control treatment showed higher CMCase activity when compared with the treatments containing supplemental sources of nitrogen. However, urea supplementation provided higher CMCase activity compared to albumin.

Effect of mannanoligosaccharides and/or enzymes on antibody titers against infectious bursal and Newcastle disease viruses

O efeito da inclusão de mananoligossacarídeo (MOS) e/ou enzimas em dietas de frangos sobre os títulos de anticorpos contra os vírus das doenças de Gumboro (VDG) e de Newcastle (VDN). Setecentos e cinqüenta aves foram distribuídas em um delineamento experimental inteiramente ao acaso, em arranjo fatorial 2 x 2 + 1, com dois níveis de MOS (0 e 0,1% até 21 dias e 0,05% de 22 até 42 dias de idade), dois níveis de enzimas (0 e 0,05%) e uma dieta-controle-positivo contendo antibióticos, totalizando cinco tratamentos com cinco repetições. Para análise dos anticorpos, amostras de sangue foram colhidas semanalmente por punção da veia jugular em duas aves de cada repetição. A primeira e a última colheita foram realizadas aos sete e 42 dias de idade, respectivamente. A inclusão de MOS resultou em aumento dos títulos contra VDG na quarta (P<0,03) e quinta (P<0,02) semanas, e contra VDN na terceira (P<0,01), quarta (P<0,03) e quinta (P<0,03) semanas de idade. O MOS foi efetivo em estimular a resposta imune humoral contra VDG e VDN vacinais.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Investigation of the interaction between Tc85-11 protein and antibody anti-T cruzi by AFM and amperometric measurements

This present work reports on development of an amperometric immunosensor for the diagnosis of Chagas' disease using a specific glycoprotein of the trypomastigote surface, which belongs to the Tc85-11 protein family of Trypanosoma cruzi (T cruzi). An atomically flat gold surface on a silicon substrate and gold screen-printed electrodes were functionalized with cystatrine and later activated with glutaraldehyde (GA), which was used to form covalent bonds with the purified recombinant antigen (Tc85-11). The antigen reacts with the antibody from the serum, and the affinity reaction was monitored directly using atomic force microscopy or amperometry through a secondary antibody tagged to peroxidase (HRP). Surface imaging allowed to us to differentiate the modification steps and antigen-antibody interaction allowed to distinguish the affinity reactions. In the amperometric immunosensor, peroxidase catalyses the L-2 formation in the presence of hydrogen peroxide and potassium iodide, and the reduction current intensity was measured at a given potential with screen-printed electrodes. The immunosensor was applied to sera of chagasic patients and patients having different systemic diseases. (c) 2006 Elsevier Ltd. All rights reserved.

Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis

We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients' sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <.001) IgG4, but lower(P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-l or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form. (C) Elsevier, Paris.

IgE antibody response to the main antigenic component of Paracoccidioides brasiliensis in patients with paracoccidioidomycosis

Paracoccidioidomycosis (PCM) has two main clinical presentations, a chronic form (CF) and an acute, more severe form (AF). The AF is associated with a more marked dysfunction of the patient's immune response, and a distinct anti-Paracoccidioides brasiliensis immunoglobulin (Ig)A and IgG antibody subclass expression, compared with that seen in the CF. In this study we investigated the presence of IgE antibodies against the main P. brasiliensis antigen (a 43-kDa molecule) in the serum of PCM patients using an enzyme-linked immunosorbent assay. We found that 100% of the AF patients (n = 16) produced IgE antibodies, mostly at high levels, whereas only 9 (27%) out of 33 CF patients produced this isotype. Interestingly, these nine patients presented higher serological titers on the counter-immunoelectrophoresis assays than did those who did not produce IgE; a finding that suggests that they had a relatively more severe disease. As IgE is a characteristic feature of the AF patients, and switching to a positive IgE response is dependent on interleukin-4, our results support the notion that the relatively more severe impairment of cellular immunity in the AF is probably related to a Th-2 pattern of immune response.

Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cocaine-induced behavioral sensitization in adolescent rats endures until adulthood: Lack of association with GluR1 and NR1 glutamate receptor subunits and tyrosine hydroxylase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)