Adaptation and validation into Portuguese language of the HIV Antibody Testing Attitude Scale

Objective. To culturally adapt and validate a version in European Portuguese language of the HIV Antibody Testing Attitude Scale. Methods. Study conducting a methodological investigation for the adaptation and validation of an attitude measurement instrument. The instrument translation and back-translation were performed. Then, a pre-test was conducted. The study used a sample of 317 subjects from the academic community - students, professors and other professionals - who were contacted in the campus. Ethical principles were observed. Results. Three analyses were conducted using the method of principal component analysis (PCA) with five, four and three factors. A three-factor solution was achieved, which presents 50.82% variance. In the analysis of inter-item correlation, values between -0.018 and 0.749 were observed. Internal consistency shows Cronbach’s alpha coefficients of 0.860 overall and between 0.865 and 0.659 in the three factors. Conclusion. The instrument version shows psychometric properties that allow its use in Portuguese-speaking countries.

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.

Effects of supplemental osteopontin on intestinal development and serum antibody responses to rotavirus vaccination in piglets

Milk contains numerous bioactive substances including immunoglobulins, cytokines, growth factors and components that exert antibiotic and prebiotic activity (Field, 2005). Little is known about the biological effects of individual milk bioactives, despite the fact that natural milk improves intestinal development and immune system functions in neonates (Donovan et al., 1994; Field, 2005) relative to milk formula. Characterization of the biological effects of such components is important for optimal production of infant milk formulas to be used when mother’s milk is not available. Milk components with preliminary evidence of positive effects on the intestinal growth and mucosal immunity include osteopontin (OPN). Osteopontin is a phosphorylated acidic glycoprotein expressed by a number of different immune and non-immune cells and tissues (Sodek et al., 2000). It is also present in body fluids including blood, bile and milk (Sodek et al., 2000). Osteopontin is a multifunctional protein that is implicated in a wide number of biological processes including cell survival, bone remodeling, and immune modulatory functions (Sodek et al., 2000). Furthermore, Schack and colleagues (2009) demonstrated that the concentration of OPN in human milk is considerably higher than in bovine milk and infant formulas. Taken together, it is likely that OPN plays a role in the early development of gastrointestinal tract and mucosal immune responses in infants. Since the neonatal pig shares anatomical, physiological, immunological, and metabolic similarities with the human infants (Moughan, et al., 1992), they were selected as the animal model in our studies. Our first aim was to investigate the effects of OPN on piglet intestinal development. Newborn, colostrum-deprived piglets (n=27) were randomized to receive three treatments: formula with bovine OPN (OPN; 140 mg/L); formula alone (FF); or sow reared (SR) for 21 days. Body weight, intestinal weight and length, mucosal protein and DNA content, disaccharidase activity, villus morphology, and crypt cell proliferation were measured. Statistical significance was assigned at P<0.05. No significant effects of OPN were observed for body weight, intestinal weight and length. Mucosal protein content of SR piglets was lower than FF and OPN piglets in the duodenum, but higher than FF and OPN piglets in the ileum. No significant effects of diet in mucosal DNA content were detected for the three regions of the small intestine. Lactase and sucrase activities of SR piglets were higher than the two formula-fed groups in the duodenum, lower in the ileum. No significant effects of diet on lactase and sucrase activities were noted between two formula-fed groups in the duodenum and ileum. Jejunal lactase activity of FF piglets was higher than SR piglets, whereas no significant effect of diet was observed in jejunal sucrase activity among the three groups. Duodenal and ileal villus height and villus area of SR piglets were lower than two formula-fed groups, while OPN piglets did not differ from FF piglets. There was a significant effect of diet (P<0.0001) on jejunal crypt cell proliferation, with proliferation in OPN piglets being intermediate between that of FF and SR. In summary, supplemental OPN increased jejunal crypt cell proliferation, independent of evident morphological growth, and had a minor impact on disaccharidase activity in the small intestine of neonatal piglets. Rotavirus (RV) is the most common viral cause of severe gastroenteritis in infants and young children worldwide (Parashar et al., 2006). Maeno et al. (2009) reported that OPN knockout (OPN-KO) suckling mice were more susceptible to RV infection compared to wild-type (WT) suckling mice. To detect the role of OPN in intestinal immune responses of neonates, the goal of the second study was to evaluate whether supplemental OPN influenced the serum antibody responses to RV vaccination in neonatal piglets. Newborn, colostrum-deprived piglets were randomized into two dietary groups: formula with bovine OPN (OPN; 140 mg/L) and formula alone (FF) for 35 days. On d7, piglets in each dietary group were further randomized to receive rotavirus (RV) vaccination (Rotarix®) (FF+RV and OPN+RV) or remained non-vaccinated (FF+NV and OPN+NV). Booster vaccination was provided on d14. Blood samples were collected on d7, 14, 21, 28 and 35. RV-specific serum immunoglobulin (Ig) G, IgA, IgM and total serum IgG, IgA, IgM were measured by ELISA. Statistical significance was assigned at P<0.05, with trends reported as P<0.10. Body weight gain was unaffected by diet and/or vaccination. No significant effect of oral OPN supplementation was observed for RV-specific antibody responses and total Igs levels. After the combination of dietary groups, RV piglets had significantly higher RV-specific IgM concentrations compared to NV piglets. Although there were higher means of RV-specific IgG and RV-specific IgA concentrations in RV group than their counterparts in NV group, the difference did not reach statistical significance. RV-specific IgM reached a peak at d7 post booster vaccination (PBV), whereas the RV-specific IgG and IgA peaked later at PBV 14 or 21. Total Igs were unaffected by RV vaccination but were significantly increased over time, following similar pattern as RV-specific Igs. In summary, neonatal piglets generated weak antibody responses to RV vaccination. Supplemental OPN did not enhance RV-specific serum antibody responses and total serum Igs levels in neonatal piglets with or without RV vaccination. In conclusion, we observed normal developmental changes in the small intestine and serum Igs levels in neonatal piglets over time. Oral OPN supplementation showed minimal impacts on intestinal development and no effect on serum Igs levels. The role of supplemental OPN on the growth and development of infants is still inconclusive. Future studies should measure other physiological and immunological parameters by using different models of vaccination or infection.

An investigation into the folding and assembly of engineered antibodies and novel antibody formats

Monoclonal antibodies and novel antibody formats are currently one of the principal therapeutic in the biopharmaceutical industry worldwide and are widely used in the treatment of autoimmune diseases and cancer. It is for this reason that the productivity and quality of antibody production requires improvement; specifically investigations into the engineering of antibodies and any issues that may arise from the production of these therapeutics. The work presented in this thesis describes an investigation into the folding and assembly of seven antibodies plus the novel antibody format FabFv. IgG is comprised of two identical HCs and two identical LCs. The folding process of immunoglobulin is controlled by the CH1 domain within the HC. The CH1 domain remains in a disordered state and is sequestered by BiP in the endoplasmic reticulum. Upon the addition of a folded CL domain, BiP is displaced, the CH1 domain is able to fold and the complete IgG protein can then be secreted from the cell. The results presented in this thesis however, have outlined an additional mechanism for the folding of the CH1 domain. We have shown that the CH1 domain is able to fold in the absence of LC resulting in the secretion of HC dimers in a VH dependent manner. The proposed mechanism for the secretion of HC dimers suggests that some VH domains can interact with each other in order to bring the CH1 domains in close proximity to enable folding to occur. As HC dimer secretion is a hindrance in antibody production, this result has highlighted an engineering target to improve antibody yield. Examination of the folding of IgG4 with the variable region A33 has revealed the inability to secrete LC dimers, cleavage of the HC during expression and secretion of HC dimers in the Fab, FabFv and full length forms. The attributes described have also been shown to be variable region dependent. This has introduced a new concept that the variable domain is important in determining the expression and secretion of antibodies and their individual chains. Pulse chase and 2D gel electrophoresis analysis of the novel antibody format FabFv has revealed that the folding and expression of the LC and HC causes multimeric species of FabFv to be secreted, as opposed to the monomeric form which is the desired therapeutic. Our hypothesis is that this process occurs via a LC dependent mechanism. The proposed hypothesis suggests that further engineering to the LC could diminish the formation and secretion of FabFv multimers. The results from these investigations can be applied to increase the productivity of therapeutics and increase the biological understanding of the domain interactions of IgG during folding, assembly and secretion.

Comparative assessment of seller’s staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies.

Background: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller’s stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. Objective: This study was designed to assess the sensitivity and specificity of the SST in relation to the ‘gold standard’ dFAT in diagnosis of rabies in Nigeria. Methods: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. Results: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). Conclusion: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.

The Myosin Heavy Chain specific A4.1025 antibody discriminates different cardiac segments in ancient groups of gnathostomes: Morphological and evolutionary implications

The pan-Myosin Heavy Chain (pan-MyHC) marker MF20 have been reported to show similar, homogeneous signal in the myocardial segments of the heart of teleosts and tetrapods. However, in an ongoing study of the myocardial structure of the dogfish (Scyliorhinus canicula; Chondrichthyes), we observed differential immunostaining of the cardiac segments using another pan-MyHC, the A4.1025 antibody. In order to investigate the relevance of this finding for better understanding of the morphology and evolution of the vertebrate heart, we performed immunohistochemistry, slot blot and western blot in several species of chondrichthyans, actinopterygians and mammals using the above mentioned antibodies. In the dogfish heart, A4.1025 and MF20 specifically recognized MyHC isoforms, although with different degree of affinity. MF20 reactivity was homogeneous and high in all the myocardial segments. However, A4.1025 reactivity was heterogeneous. It was high in the sinus venosus (external layer), atrium and atrioventricular region, low in the ventricle and conus arteriosus, and null in the internal layer of the sinus venosus. A heterogeneous pattern of A4.1025 immunoreactivity was also detected in two other elasmobranchs, a holocephalan, a polypteryform and an acipenseriform. In all of these species, MF20 immunoreactivity was homogeneous. In addition, both markers showed a homogeneous immunoreactivity pattern in teleosts and mammals. Our results indicate that in the hearts of ancient gnathostomes, in all of which a conspicuous conus arteriosus exists, one or more MyHC isoforms with low affinity for A4.1025 show segment-specific distributions. Thus, A4.1025 appears to be an appropriated marker to identify the cardiac segments and their boundaries. We propose that the segmentspecific distribution of MyHC isoforms may generate a particular type of myocardial contractility associated with the presence of a conus arteriosus.

Modulation of GABA and glutamate release by brain-derived neurotrophic factor : role of adenosine A2A receptors

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Development of paclitaxel-loaded liposomal systems with anti-her2 antibody for targeted therapy

Purpose: To develop liposome formulations containing monoclonal antibody anti-HER2 (MabHer2), and Paclitaxel (PTX). Methods: Seven different liposomal systems containing PTX, or MabHer2 or a combination of PTX and MabHer2 were made using lipid film hydration technique and sonication. The effects of liposome preparation conditions and extraction methods on antibody structure were investigated by polyacrylamide gel electrophoresis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The characteristics of the liposomes were determined by a zetasizer, while drug-loading efficiency was evaluated by high-performance liquid chromatography. The cytotoxic effect of the liposome formulations was evaluated on MDA-MB-453 (HER2+) and MCF-7 (HER2-) breast cancer cell lines by MTT assay. Results: The antibody was not significantly affected by the stress conditions and the method of extraction. The particle size of liposomes was < 200 nm while the amount of incorporated PTX was 97.6 % for liposome without cationic agent and 98.2 % for those with cationic agent. Recovery of MabHer2 was 94.38 % after extraction. Combined PTX/MabHer2 liposome was more toxic on HER2 overexpressing positive MDA-MB-453 cell line than PTX-loaded liposomes and MabHer2. MabHer2 and combined PTX/MabHer2 liposomes showed no toxic effects on HER2 overexpressing negative MCF-7 cells relative to cationic PTX-loaded liposomes. Conclusions: This results obtained show that PTX can be encapsulated successfully into liposoma systems and that owing to Her2 specific antibody, these systems can be delivered directly to the target cell.

Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column. Results: There were 12 hybridoma strains selected into the polyol-responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA. Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

Anticonvulsant and sedative effect of Fufang Changniu pills and probable mechanism of action in mice

Purpose: To investigate the anticonvulsant and sedative effects of Fufang Changniu Pills (FCP) and its probable mechanism of action in mice. Methods: The water decoction of FCP was prepared and the main constituents were determined by high performance liquid chromatography (HPLC). The anticonvulsant activities of FCP were evaluated by maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizures in mice. Pentobarbital sodium-induced sleeping time and locomotor activity measurements were performed to evaluate the sedative effects of FCP in mice. Finally, PTZ-induced chronic seizures were established, and expressions of gamma-aminobutyric acid A receptor (GABA-A) and glutamic acid decarboxylase 65 (GAD65) in the brains of the mice were assayed by western blot in order to explore the probable mechanisms of action of the drug. Results: Gallic acid, liquiritin, cinnamyl alcohol, cinnamic acid and glycyrrhizic acid were detected in FCP decoction. FCP (50, 100 and 200 mg/kg) showed significant anticonvulsant and sedative effects on epileptic mice induced by MES (p < 0.05) and PTZ (p < 0.05). Moreover, pentobarbital sodium-induced sleeping time and locomotor activity tests showed that FCP possesses sedative effect (p < 0.05). Western blot data indicate that FCP significantly up-regulated GABA-A and GAD 65 in the brains of chronic epileptic rats (p < 0.05). Conclusion: FCP has significant anticonvulsant and sedative effects, and the mechanism of its action may be related to the up-regulation of GABA-A and GAD 65 in mice brain.

Structural determination and gynecological tumor diagnosis using antibody chip captured proteins

Purpose: To identify markers for gynecological tumor diagnosis using antibody chip capture. Methods: Marker proteins, including cancer antigen 153 (CA153), CA125, and carcinoembryonic antigen (CEA), were analyzed using antibody chip capture of serum samples. Fifteen agglutinin types that specifically recognized five common glycans (fucose, sialic acid, mannose, N - acetylgalactosamine, and N-acetylglucosamine) were used to detect marker protein glycan levels. The levels of CA153, CA125, and CEA from 49 healthy control samples, 31 breast cancer samples, 24 cervical cancer samples, and 19 ovarian cancer samples were used to measure the glycan levels of these marker proteins. Results: In breast cancer samples, CA153 and CA125 were down-regulated (p < 0.01), while differences in ovarian cancer samples were not statistically significant (p > 0.01). The total accuracy was 85.1 %, with 96.8 % accuracy for breast cancer, 75 % in cervical cancer, and 78.9 % in ovarian cancer. Cross-validation analyses showed that breast cancer had 93.5 % accuracy, cervical cancer was 66.7 %, and ovarian cancer was 68.4 %, leading to 78.4 % total accuracy (58/74). Conclusions: The results indicate that better clinical diagnosis of gynecological tumors can be obtained by monitoring changes in glycan levels of serum proteins and types of proteoglycan changes.

Oral glutamate intake reduces acute and chronic effects of ethanol in rodents

Purpose: To assess the effects of oral glutamate intake on acute motor effects and chronic intake of ethanol in rodents. Methods: The acute effects of ethanol on motor function were studied in ICR mice by giving 2 or 6 g/kg of ethanol 2 h after distilled water or 2.5 g/kg glutamate per os. Thirty minutes after ethanol treatment, behavioral assays, including rotarod tests and foot print analysis were monitored. In chronic ethanol treatment, male Wistar rats were trained to consume ethanol-sucrose solution during a 2-h period daily, starting with 2 % ethanol/10 % sucrose and gradually increasing to 10 % ethanol/5 % sucrose solution over 56 days. After training session, the drug treatment phase was done for 10 days. The animals were force-fed 50 mg/kg/day topiramate or 2.5 g/kg/day glutamate 2 h before ethanol treatment sessions. Each day, ethanol intake, water intake, food intake and body weight were recorded. Results: Mice that received 2 or 6 g/kg of ethanol orally, showed a significant reduction in time on the rod in the rotarod test and a significant increase in both forelimb and hindlimb stride lengths when compared to control. Oral treatment with 2.5 g/kg of glutamate reversed the acute motor effects of ethanol. In chronic ethanol treatment, the intake of 10 % ethanol/5 % sucrose, accessible for 2 h, was significantly decreased in rats treated with either topiramate or glutamate. Conclusion: These results provide evidence that oral glutamate administration help to reduce the acute motor effects of ethanol in mice and ethanol intake in the chronic ethanol drinking rats.

Development of metabotropic glutamate receptors from trigeminal nuclei to barrel cortex in postnatal mouse

Expression patterns of group I (mGluR1α and mGluR5)and group II (mGluR2/3) metabotropic glutamate receptor subtypes were examined immunocytochemically in the trigeminal system of mice during the first 3 weeks of postnatal development, when somatotopic whisker representations are sequentially established from brainstem through thalamus to cerebral cortex. Immunostaining for all three epitopes formed whisker-related patterns in the trigeminal nuclei from postnatal day (P) 0, in the ventral posterior thalamic nucleus from P2, and in the posteromedial barrel subfield of somatosensory cortex (SI) from P4. The appearance of whisker-related patterns was preceded by increased levels of immunostaining of the neuropil, which subsequently declined from the trigeminal nuclei upward. In SI, mGluR1α-positive neurons were observed in all cortical layers from P2. mGluR5 was localized in neurons, glial cells, and neuropil from P2. mGluR2/3 immunostaining was distributed only in the neuropil at all ages. The three receptor subtypes showed moderate to high expression in deep layer V throughout development. Transient expression peaked in the hollows of layer IV barrels from P4 to P9, and then fell off as expression increased in supragranular layers from P14 to P21. The deep aspect of the cortical subplate (layer VIb) showed dense mGluR5 and less dense mGluR1α immunostaining throughout development. Up-regulation of expression of group I and II mGluRs is correlated with the growth and refinement of connectivity and the establishment of somatotopic patterns in the three main relay stations of the trigeminal system. This finding suggests roles for mGluRs in the early processing of sensory information and in developmental plasticity.