Radioimmunoassay of insulin and glucagon with studies on the obese hyperglycaemic syndrome in mice

Sensitive and precise radioimmunoassays for insulin and glucagon have been established. Although it was possible to employ similar precepts to the development of both hormone assays, the establishment of a reliable glucagon radioimmunoassay was complicated by the poor immunogenicity and instability of the peptide. Thus, unlike insulin antisera which were prepared by monthly injection of guinea pigs with crystalline insulin emulsified in adjuvant, the successful production of glucagon antisera was accomplished by immunisation of rabbits and guinea pigs with glucagon covalently linked to bovine plasma albumin. The conventional chloramine-T iodination with purification by gel chromatography was only suitable for the production of labelled insulin. Quality tracer for use in the glucagon radioimmunoassay was prepared by trace iodination, with subsequent purification of monoiodinated glucagon by anion exchange chromatography. Separation of free and antibody bound moieties by coated charcoal was applicable to both hormone assays, and a computerised data processing system, relying on logit-log transformation, was used to analyse all assay results. The assays were employed to evaluate the regulation of endocrine pancreatic function and the role of insulin and glucagon in the pathogenesis of the obese hyperglycaemic syndrome in mice. In the homozygous (ob/ob) condition, mice of the Birmingham strain were characterised by numerous abnormalities of glucose homeostasis, several of which were detected in heterozygous (ob/+) mice. Obese mice exhibited pancreatic alpha cell dysfunction and hyperglucagonaemia. Investigation of this defect revealed a marked insensitivity of an insulin dependent glucose sensing mechanism that inhibited glucagon secretion. Although circulating glucagon was of minor importance in the maintenance of hyperinsulinaemia, lack of suppression of alpha cell function by glucose and insulin contributed significantly to both the insulin insensitivity and the hyperglycaemia of obese mice.

Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

Influence of microbial antigen formulation and delivery route on the immune response

Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccharide vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH aroA mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.

Assessment of different formulations of oral Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine in rodent models for immunogenicity and protection against aerosol challenge with M-bovis

Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economic loss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to control bTB in the UK are hampered by the infection in Eurasian badgers (Metes metes) that represent a wildlife reservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TB vaccine, M. bovis Bacille Calmette-Guerin (BCG), in oral bait represents a possible disease control tool and holds the best prospect for reaching badger populations over a wide geographical area. Using mouse and guinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidate badger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsular hybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation for their protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significant immune responses (based on lymphocyte proliferation and expression of IFN-gamma) were only seen with the lipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viable BCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferred protection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen with delivery doses in the range 10(6)-10(7) CFU, although this was more consistent in the spleen at the higher dose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid in alginate microcapsules, although 10(7) in the latter formulation conferred protection in terms of increasing body weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlight the potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery vehicles for an oral BCG vaccine in badgers.

Efecto de la extirpación de las espículas del glande del cuy como técnia de esterilización reproductiva y su influencia en agresividad y ganancia de peso en comparación con un método químico alcohol yodado al 2%

La presente investigación evaluó el efecto de la extirpación de las espículas del glande en cobayos como método de esterilización reproductiva y su influencia en agresividad y ganancia de peso en comparación con un método químico (alcohol yodado 2%) y un testigo. Se contemplaron dos etapas de estudio, la primera valoró agresividad y ganancia de peso en 90 cuyes machos de 45 días de edad con peso promedio de 658,3±8,54gr los cuales fueron distribuidos en grupos de 5 animales por jaula; para la segunda etapa se consideró fertilidad y prolificidad en 90 cuyes hembras sexualmente maduras de 3 meses de edad, las cuales fueron empadradas con 18 machos obtenidos al azar de una sub-muestra de la primera etapa, la relación de empadre fue cinco hembras un macho. Los animales en estudio fueron distribuidos en los siguientes tratamientos: T1 animales enteros (n=30), T2 animales extirpados las espículas (n=30) y T3 animales castrados con alcohol yodado 2% (n=30). Los datos obtenidos fueron procesados y analizados en el programa SPSS® versión 22.0 determinándose que la ganancia total de peso fue para T1 836,4±33,89gr, T2 860,5±24,54gr y T3 725,5±30,45 con significancia estadística (P<0,05) para T2 y T1 con relación a T3. En referencia a agresividad medida a través del daño de la canal a nivel de la zona dorso posterior se obtuvieron resultados porcentuales estadísticos no significativos (p>0,05) entre tratamientos. En cuanto a fertilidad se obtuvo: para T1 un valor de 66,7%, T2 86,7% y T3 12% existiendo significancia estadística (p<0,05) entre tratamientos. Para la variable prolificidad se obtuvo valores medios de: T1 2,47±0,34; T2 3,43±0,32 y T3 0,40±0.22, con diferencias significativas (p<0,05) únicamente para el T3 que fue el que menos crías obtuvo. En conclusión la extirpación de las espículas del glande del cuy no esteriliza reproductivamente por el contrario aumenta el porcentaje de fertilidad, no disminuye agresividad, obtiene pesos no distintos al testigo pero alcanza mejores pesos que el método químico.

Influencia de las espículas peneanas del cobayo sobre el comportamiento sexual, valoración espermática y fertilidad del macho

Determinar la influencia de las espículas peneanas en los cobayos machos sobre el comportamiento sexual, fertilidad y valores espermáticos, fue el objetivo principal de este proyecto de investigación, para ello se incluyeron 10 cobayos machos de cinco meses de edad, peso promedio 988,3±11,40 g y 40 hembras de cuatro meses de edad, peso promedio 815,3±11,80 g, bajo las mismas condiciones de alimentación y mantenimiento. Cinco machos seleccionados al azar fueron extirpados quirúrgicamente las espículas peneanas. Un cobayo entero y un intervenido fueron mantenidos, como reemplazo en el caso de muerte de una unidad experimental y se los excluyó del procedimiento inicial. Se realizaron tres ensayos: en el primero se dividieron en dos tratamientos T1= 4 cobayos machos con espículas peneanas + 20 hembras en jaulas separadas, T2= 4 cobayos machos extirpados las espículas peneanas + 20 hembras en jaulas separadas. Se analizó durante ocho días consecutivos el comportamiento sexual por observación directa de los cobayos en cada jaula. Para el segundo ensayo los mismos tratamientos T1 y T2, permanecieron por treinta días en empadre, para evaluación de la fertilidad. Finalmente en el último ensayo se analizaron parámetros espermáticos, por medio de la extirpación quirúrgica de los testículos y disección del epidídimo de los cobayos en estudio, para este ensayo se incluyeron los cobayos de reemplazo. El diseño experimental que se utilizó en la investigación fue un diseño completamente al azar, las pruebas de significación fueron, T de Student, prueba de Shapiro Wilk, para el análisis de homogeneidad de varianza se utilizó la técnica de Levene y se realizó el análisis de medias repetidas, todo esto con el programa SPSS para Windows versión 22®. Los parámetros de comportamiento sexual, olfateos, mordiscos y montas fueron similares (P>0,05), el número promedio de acicalamientos fue mayor en el grupo de machos enteros en relación al grupo sin espículas (P<0,05). El grupo de hembras que fueron copuladas por cobayos enteros quedaron preñadas un 65% más en comparación con las hembras que fueron cubiertas por los machos extirpados las espículas (P<0,01). No se encontraron diferencias significativas en los análisis de parámetros seminales de los machos en estudio. Por lo tanto se concluye que la remoción de las espículas peneanas influye en la fertilidad, pero no en el comportamiento sexual y los valores espermáticos

Evaluación cuali-cuantitativa de espermatozoides de la cola del epedídimo de cuyes cavia porcellus criollos y mejorados en dos edades reproductivas

El objetivo de esta investigación fue evaluar las características cuali-cuantitativas de espermatozoides de cuyes extraídos de la cola del epidídimo según su fenotipo y edad reproductiva. Se realizó en la granja Irquis de la U. de Cuenca en 20 reproductores identificados por sus características fenotípicas y dispuestos en cuatro grupos: 5 criollos jóvenes (CJ), 5 criollos adultos (CA), 5 mejorados jóvenes (MJ), y 5 mejorados adultos (MA). Los cuyes fueron hemicastrados y de los epidídimos fueron disectados la cola sobre una caja petri. Se recuperó los espermatozoides por Swim up, diluidos en 1ml de medio (18% rafinosa y 3% leche descremada), procesados con Triladyl®, refrigerados a 5oC/1 hora, y equilibrados por 0, 2, 24, 48, 96, 192, y 360 horass para su análisis de viabilidad espermática. Se congelaron únicamente los espermatozoides de 2 hs de equilibrio en vapores de nitrógeno. Se usó un DCA de 2x2: fenotipo y edad, y se usó un ANOVA para comprobar significancia. Se obtuvo interacción (P<0,05) entre factores con eficiencia atribuida a MJ a las 0 hs: en Concentración (C) y Anormalidades de cola (AC), a las 24 hs: en motilidad individual (MIP) y 48 hs: en Vitalidad (VE). En MIP no se encontró diferencias (P>0,05) en ningún tiempo de medición. En VE sólo encontró diferencias (P<0,05) a las 96 hs (CJ:18,0;MJ:10,2;MA:8,6;CA:6,0%). En anormalidades totales (AT) sólo se encontró diferencias (P<0,05) a las 0 hs (MJ:26,3;CJ:32,6;MA:36,2;CA:38,5%); y en AC se encontró diferencias (P<0,05) a las 0 hs (MJ:4,6; CJ:9,5; CA:11,5; MA:16,4%), y a las 48 hs (CA:5,7;CJ:7,3;MJ:16,0;MA:18,1%). En Integridad de la membrana (HOS-Test) se obtuvo (P<0,05) diferencias a las 2 hs (MJ:20,0; MA:13,1;CA:10,7;CJ:9,0%) y a las 96 hs (CA:25,4;CJ:15,3;MJ:9,7; MA:8,8%). A la congelabilidad no se obtuvo sobrevivencia de espermatozoides en ninguno de los tratamientos. En conclusión, la cantidad y calidad de espermatozoides epididimarios de cuyes identificados fenotípicamente varía según su edad; sin embargo, no se pudo comprobar su variación en la congelabilidad mostrándose absolutamente inviables a la crío conservación

Cardiovascular actions of the venom from the Irukandji (Carukia barnesi) jellyfish: Effects in human, rat and guinea-pig tissues in vitro and in pigs in vivo

1. We have investigated the cardiovascular pharmacology of the crude venom extract (CVE) from the potentially lethal, very small carybdeid jellyfish Carukia barnesi, in rat, guinea-pig and human isolated tissues and anaesthetized piglets. 2. In rat and guinea-pig isolated right atria, CVE (0.1-10 mu g/mL) caused tachycardia in the presence of atropine (I mu mol/L), a response almost completely abolished by pretreatment with tetrodotoxin (TTX; 0.1 mu mol/L). In paced left atria from guinea-pig or rat, CVE (0.1-3 mu g/mL) caused a positive inotropic response in the presence of atropine (1 mu mol/L). 3. In rat mesenteric small arteries, CVE (0.1-30 mu g/mL) caused concentration-dependent contractions that were unaffected by 0.1 mu mol/L TTX, 0.3 mu mol/L prazosin or 0.1 mu mol/L co-conotoxin GVIA. 4. Neither the rat right atria tachycardic response nor the contraction of rat mesenteric arteries to CVE were affected by the presence of box jellyfish (Chironex fleckeri) antivenom (92.6 units/mL). 5. In human isolated driven right atrial trabeculae muscle strips, CVE (10 mu g/mL) tended to cause an initial fall, followed by a more sustained increase, in contractile force. In the presence of atropine (I mu mol/L), CVE only caused a positive inotropic response. In separate experiments in the, presence of propranolol (0.2 mu mol/L), the negative inotropic effect of CVE was enhanced, whereas the positive inotropic response was markedly decreased. 6. In anaesthetized piglets, CVE (67 mu g/kg, i.v.) caused sustained tachycardia and systemic and pulmonary hypertension. Venous blood samples demonstrated a marked elevation in circulating levels of noradrenaline and adrenaline. 7. We conclude that C. barnesi venom may contain a neural sodium channel activator (blocked by TTX) that, in isolated atrial tissue (and in vivo), causes the release of transmitter (and circulating) catecholamines. The venom may also contain a 'direct' vasoconstrictor component. These observations explain, at least in part, the clinical features of the potentially deadly Irukandji syndrome.

Virulence factors in Escherichia coli strains isolated from pigs in the Ribeirao Preto region, State of Sao Paulo, Brazil.

Three-hundred faecal swabs were obtained from pigs with diarrhoea in farms located in different areas of the Ribeirao Preto region in the State of Sao Paulo. One-hundred Escherichia coli strains were isolated and tested for production of thermolabile (TL) and thermostable (STRa and STb) enterotoxins, and for the presence of colonization factors F4, F5 and F6. The strains were also tested for sensitivity to 14 antibiotics and chemotherapeutic agents. Twenty-four Escherichia coli strains produced enterotoxin STb, 5 produced LT and 3 produced STa. In the mannose-resistant haemagglutination reaction, one strain reacted positively with sheep, chicken, horse and human red blood cells and another reacted positively with guinea pig, sheep, chicken, horse and human red cells. However, both strains were negative for colonization factors F4, F5 and F6 when submitted to the slide agglutination test. All Escherichia coli strains were resistant to at least one antibiotic, the highest percentages being obtained for resistance to penicillin, tetracycline and cephalotin. In addition to the importance of the virulence factors normally encountered in enterotoxigenic Escherichia coli strains from pigs, the present results show the possible existence of new colonization factors other than F4, F5 and F6 participating in E. coli-induced pigs colibacillosis in the Ribeirao Preto region.

Disorderly categories in picture postcards from colonial Papua and New Guinea

At the turn of the century in Melbourne, a notice typed on the verso of a postcard stated that the South Yarra Baptist Young Men's class was meeting on the following Sunday at 2.45 p.m. The card, published in the United Kingdom, was numbered 51828 in the Valentine series of Papuan postcards.1 The image, a photograph of Hanuabada village taken in the early 1880s, and the text, written early in 1900, are contradictory and constitute separate realms of evidence that invite a renegotiation of meaning, analysis, and interpretation of the relationships between images, tourism, colonial rule, and ethnographic knowing. The visual evidence suggests the postcard may have played an ethnographic, educative role in the public understanding of Papua, which had just become an Australian Territory and was not yet well known. It is also suggestive of educative roles related to mission endeavours, subimperialist ambitions and the new tourist traffic through the ports of Port Moresby, Samarai, and Rabaul.

Market access of Papua New Guinea bananas (Musa spp.) with particular respect to banana fly (Bactrocera musae (Tryon)) (Diptera: Tephritidae)

International market access for fresh commodities is regulated by international accepted phytosanitary guidelines, the objectives of which are to reduce the biosecurity risk of plant pest and disease movement. Papua New Guinea (PNG) has identified banana as a potential export crop and to help meet international market access requirements, this thesis provides information for the development of a pest risk analysis (PRA) for PNG banana fruit. The PRA is a three step process which first identifies the pests associated with a particular commodity or pathway, then assesses the risk associated with those pests, and finally identifies risk management options for those pests if required. As the first step of the PRA process, I collated a definitive list on the organisms associated with the banana plant in PNG using formal literature, structured interviews with local experts, grey literature and unpublished file material held in PNG field research stations. I identified 112 organisms (invertebrates, vertebrate, pathogens and weeds) associated with banana in PNG, but only 14 of these were reported as commonly requiring management. For these 14 I present detailed information summaries on their known biology and pest impact. A major finding of the review was that of the 14 identified key pests, some research information occurs for 13. The single exception for which information was found to be lacking was Bactrocera musae (Tryon), the banana fly. The lack of information for this widely reported ‘major pest on PNG bananas’ would hinder the development of a PNG banana fruit PRA. For this reason the remainder of the thesis focused on this organism, particularly with respect to generation of information required by the PRA process. Utilising an existing, but previously unanalysed fruit fly trapping database for PNG, I carried out a Geographic Information System analysis of the distribution and abundance of banana in four major regions of PNG. This information is required for a PRA to determine if banana fruit grown in different parts of the country are at different risks from the fly. Results showed that the fly was widespread in all cropping regions and that temperature and rainfall were not significantly correlated with banana fly abundance. Abundance of the fly was significantly correlated (albeit weakly) with host availability. The same analysis was done with four other PNG pest fruit flies and their responses to the environmental factors differed to banana fly and each other. This implies that subsequent PRA analyses for other PNG fresh commodities will need to investigate the risk of each of these flies independently. To quantify the damage to banana fruit caused by banana fly in PNG, local surveys and one national survey of banana fruit infestation were carried out. Contrary to expectations, infestation was found to be very low, particularly in the widely grown commercial cultivar, Cavendish. Infestation of Cavendish fingers was only 0.41% in a structured, national survey of over 2 700 banana fingers. Follow up laboratory studies showed that fingers of Cavendish, and another commercial variety Lady-finger, are very poor hosts for B. musae, with very low host selection rates by female flies and very poor immature survival. An analysis of a recent (within last decade) incursion of B. musae into the Gazelle Peninsula of East New Britain Province, PNG, provided the final set of B. musae data. Surveys of the fly on the peninsular showed that establishment and spread of the fly in the novel environment was very rapid and thus the fly should be regarded as being of high biosecurity concern, at least in tropical areas. Supporting the earlier impact studies, however, banana fly has not become a significant banana fruit problem on the Gazelle, despite bananas being the primary starch staple of the region. The results of the research chapters are combined in the final Discussion in the form of a B. musae focused PRA for PNG banana fruit. Putting the thesis in a broader context, the Discussion also deals with the apparent discrepancy between high local abundance of banana fly and very low infestation rates. This discussion focuses on host utilisation patterns of specialist herbivores and suggests that local pest abundance, as determined by trapping or monitoring, need not be good surrogate for crop damage, despite this linkage being implicit in a number of international phytosanitary protocols.