While a decrease of wild felid population has led to disruption of conservation programme, recent studies have shown the importance of immune regulation for determining health outcomes and co-infection. Immunoglobulin G is important for detecting and evaluating responses to infectious diseases and vaccination. But, there is limited information on felid immunoglobulins and their role for functional immunity. This study aimed at isolating and characterizing lion’s immunoglobulin G. Lions’ sera (n = 68) were processed using the MagReSyn® magnetic beads and the final protein concentration was determined using the Xpose™ Trinean Spectrophotometer. The cross-reactivity of goat anti-cat immunoglobulin with sera of lions and other species was analysed using ELISA. High cross-reactivity was observed in lions ranging from 87.7 to 100%, and low reactivity with rhino (22.4%) followed by chicken (0.01%). The protein concentration from purified sera yielded 39.09 mg/ml. Molecular weight of lion IgG 150-160 kDa was detected with both chains at 54-56 kDa and 24-26 kDa on SDS PAGE. These results indicate a potential aid in developing serological tools to monitor exposure to micro-organisms of lions. / Agriculture and  Animal Health / M. Sc. (Agriculture)

O objetivo do presente estudo foi avaliar a transferência de imunidade passiva, saúde e o desempenho de bezerros colostrados com diferentes doses de imunoglobulinas provenientes do substituto de colostro SCCL&#174; ou colostro materno. Os animais foram separados das mães imediatamente após o nascimento. Cinquenta bezerros da raça holandesa (machos e fêmeas) foram alocados em blocos casualizados de acordo com sexo, peso ao nascer e data de nascimento e distribuídos em diferentes protocolos de colostragem: 1) 2L de colostro materno (2CM); 2) 4L de colostro materno (4CM); 3) 2L de colostro materno + uma dose do substituto de colostro (2MC1SC); 4) 2 doses substituto de colostro ao nascer (2SC); e 5) 2 doses substituto de colostro ao nascer + uma dose substituto de colostro entre 6 a 8 horas após nascimento (3SC). Foram colhidas amostras de sangue ao nascimento, antes do fornecimento do colostro (0h) e com 48h após a ingestão do colostro para avaliar a transferência de imunidade passiva. Durante o período de aleitamento os bezerros receberam 6L/d de sucedâneo, tiveram acesso ad libitum à água e concentrado e foram alojados em abrigos individuais, tipo tropical. O consumo de concentrado foi avaliado diariamente, e semanalmente avaliou-se o peso e medidas corporais. A saúde dos animais foi monitorada diariamente e a incidência de diarreia foi avaliada através do escore fecal. Durante o aleitamento as amostras de sangue foram colhidas a cada 14 dias, para determinação de proteína total, albumina, glicose, BHBA, AGNE e insulina. A transferência de imunidade passiva não foi afetada pelos protocolos de colostragem (P>0,05). No entanto, doses mais elevadas de colostro resultaram em maior ingestão de imunoglobulinas, porém reduz a eficiência aparente de absorção (P <0,0001). Da mesma forma, os protocolos de colostragem não afetaram os parâmetros de saúde como escore fecal (P=0,5873), dias com diarreia (P=0,3685), dias com febre (P=0,7072); número de tratamentos para diarreia (P=0,8499) e o número de tratamentos para pneumonia (P>0,005). No entanto, afetou o número de tratamentos contra tristeza parasitária bovina (P=0,0042). O peso médio e o GMD, assim como os parâmetros sanguíneos avaliados durante o aleitamento, não foram afetados pelos protocolos de colostragem (P> 0,05). O substituto de colostro avaliado no presente estudo pode ser uma alternativa ao colostro materno, uma vez que, tanto o colostro materno como o substituto de colostro foram eficientes na transferência de imunidade passiva, resultando em desempenho similar mesmo em condições de alto desafio sanitário. / The objective of the present study was to evaluate the passive transfer of immunity (PTI), health and performance of calves fed different doses of immunoglobulins from the SCCL&#174; colostrum replacer or maternal colostrum. The animals were separated from the mothers immediately after birth. Fifty holstein calves (male and female) were allocated in randomized blocks according to sex, birth weight and date of birth and were distributed among different colostrum feeding protocols: 1) 2L of maternal colostrum (2MC); 2) 4L of maternal colostrum (4MC); 3) 2L of maternal colostrum + one dose of colostrum replacer (1MC+1CR); 4) 2 doses of colostrum replacer (2CR) and 5) 2 doses of colostrum replacer given at birth + one dose of colostrum replacer given between 6 and 8 hours after birth (3CR). Blood samples were taken at birth, before colostrum fed (0h) and at 48h after colostrum intake to assess passive immunity transfer. Calves were individually housed and bucket fed 6L/d of milk replacer, and had ad libitum access to water and starter. Starter intake was measured daily, and weekly assessed weight and body measurements. Animal health was monitored daily and the incidence of diarrhea was assessed by fecal score. During the pre-weaning, blood samples were harvested every 14 days to for determination of total protein, albumin, glucose, BHBA, NEFA and insulin. The passive transfer of immunity was not affected by colostrum feeding protocols (P>0.05). However, higher doses of colostrum resulted in higher immunoglobulins intake, but lower apparent efficiency absorption (P<0.0001). Similarly, the colostrum feeding protocols did not affect health parameters (P>0.05), such as fecal score (P=0,5873), days with diarrhea (P=0,3685), days with fever (P=0,7072), number of treatments for diarrhea (P=0,8499) and number of treatments for pneumonia (P>0,005). However, affected the number of treatments for cattle tick fever (P=0.0042). The mean weight and ADG, as well as the blood parameters evaluated during pre-weaning were not affected by the colostrum feeding protocols (P> 0.05).The colostrum replacer evaluated in this study may be an alternative to maternal colostrum, once the, both maternal colostrum and colostrum replacer were efficient in the transfer of passive immunity, resulting in similar performance even under conditions of high sanitary challenge.

Neste trabalho, anticorpos anti-IgGh foram conjugados às nanopartículas de prata (NPAg) para detectar imunoglobulina G humana (IgGh). Um imunoensaio colorimétrico baseado na diminuição da agregação devido ao aumento da repulsão eletrostática após a interação ligante-alvo. A agregação é induzida pela variação da força iônica e uma mudança da coloração da suspensão coloidal de amarelo para vermelho pode ser observada. Na presença de IgGh, a agregação é inibida e a coloração da suspensão coloidal não se altera. As nanopartículas foram obtidas por meio de cinco procedimentos diferentes e caracterizadas por espectroscopia UV-Vis, espalhamento dinâmico de luz, difração de raios-X e microscopia eletrônica. Glicose e borohidreto de sódio foram utilizados como agentes redutores, enquanto CTAB e &#946;-ciclodextrina foram utilizados como estabilizantes. Citrato de sódio foi utilizado como agente redutor e/ou estabilizante. Nanoesferas de carbono foram obtidas por tratamento hidrotérmico de uma solução aquosa de glicose e também foram utilizadas no preparo das nanopartículas. As nanopartículas foram funcionalizadas com ácido mercaptossuccínico e a conjugação ocorreu devido à interação entre grupos aminas e grupos carboxílicos ionizados, presentes no anticorpo e agente de acoplamento, respectivamente. A estabilidade dos conjugados e o efeito da adição de IgGh foram avaliados para todos os sistemas preparados. As nanopartículas de prata preparadas com borohidreto de sódio e citrato de sódio foram selecionadas para serem aplicadas no desenvolvimento do imunoensaio e as condições experimentais foram avaliadas. Em condições ótimas, observou-se uma correlação linear entre a diminuição da agregação do sistema (NPAg-anti-IgGh) e a concentração de IgGh (0 a 200 ng mL-1). O limite de detecção foi estimado em 25 ng mL-1. O método colorimétrico apresentou boa seletividade para a detecção de IgGh. Além disso, foi obtido um resultado satisfatório ao aplicar o método para determinação do fator IX de coagulação. Foi desenvolvido também um método para determinação de ATP baseado na agregação de nanopartículas de ouro. Aptâmeros foram utilizados como elemento de reconhecimento. Em princípio, o método pode ser aplicável à determinação de outros analitos, por meio da substituição do aptâmero utilizado neste trabalho pelo oligonucleotídeo específico para o alvo de interesse. / In this work, antibodies to human immunoglobulin G (anti-IgGh) were used in combination with silver nanoparticle (NPAg) to detect IgGh. A colorimetric immunoassay based on the decrease of aggregation due to increased electrostatic repulsion upon ligand-target interaction. Aggregation was induced by varying the ionic strength and the solution of NPAg-anti-IgGh shows obvious visible color change from yellow to red. In the presence of IgGh aggregation the nanoparticle is inhibited and coloration of the colloidal solution does not change. The nanoparticles were obtained using five different procedures and they were characterized by UV-Vis spectroscopy, dynamic light scattering, X-ray diffraction and electron microscopy. Glucose and sodium borohydride were used as reducing agent, as CTAB and &#946;-cyclodextrin reagents were used as stabilizers. Sodium citrate was used as reducing and stabilizing agent. Carbon nanospheres were prepared by hydrothermal treatment of glucose and used in the preparation of NPAg. The nanoparticles were functionalized with dimercaptosuccinic acid and their conjugation occurred due to the interaction of positively charged amine groups and anionic groups (-COO-) present on the antibody and coupling agent. The stability of conjugates and the variation of aggregation in the presence of IgGh were evaluated for all systems. The NPAg prepared by sodium borohydride were selected for use in the immunoassay and the optimum conditions of the assay were investigated. Under the optimal conditions, the ration between the absorbance at 396 nm and 564 nm was linearly proportional to the IgGh concentration on a range from 0 to 200 ng mL-1, with a detection limit of 25 ng mL-1. The colorimetric method showed good selectivity for IgGh detection. It was possible to adapt the method to the determination of other proteins, such as factor IX. In another approach, anti-aptamer ATP was used to develop a colorimetric method for the determination of ATP based on stabilization of gold nanoparticles provided by strands of DNA.The strategy was based on stabilization of nanoparticles due to interaction with single strands of DNA, and the change of the stability of the nanoparticles provided by the conformational change of the aptamer following recognition. This method could in principle be used to detect analytes by substituting the aptamer used in this study by the specific aptamer for the target of interest

by Shiu Kar Chi. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 61-67). / Abstract also in Chinese. / ACKNOWLEDGEMENTS / ABSTRACT / LIST OF TABLES / LIST OF FIGURES / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- Historical perspective --- p.2 / Chapter 1.3 --- Biochemistry of the IgG subclasses --- p.4 / Chapter 1.4 --- IgG subclasses and human diseases --- p.7 / Chapter 1.4.1 --- Glomerulonephritis --- p.7 / Chapter 1.4.2 --- Blistering skin lesions --- p.7 / Chapter 1.4.3 --- Insulin dependent diabetes mellitus (IDDM) --- p.8 / Chapter 1.5 --- Primary antibody deficiencies --- p.8 / Chapter 1.5.1 --- CVID --- p.8 / Chapter 1.5.2 --- X-linked antibody deficiency --- p.8 / Chapter 1.5.3 --- IgG subclass deficiency --- p.10 / Chapter 1.5.4 --- Specific antibody deficiencies --- p.10 / Chapter 1.5.5 --- Selective IgA deficiency --- p.10 / Chapter 1.6 --- IgG subclasses deficiency --- p.10 / Chapter 1.7 --- Clinical manifestation of IgG subclass deficiency --- p.11 / Chapter 1.8 --- Restriction of IgG subclass responses to exogenous antigens --- p.12 / Chapter 1.9 --- Expression of IgG subclasses --- p.14 / Chapter 1.10 --- Mechanisms of IgG subclass deficiency --- p.14 / Chapter 1.10.1 --- Gene deletion --- p.14 / Chapter 1.10.2 --- Immune dysregulation --- p.17 / Chapter 1.10.2.1 --- T-cell receptor defects --- p.18 / Chapter 1.10.2.2 --- Interferon gamma (IFN-y) --- p.18 / Chapter 1.10.2.3 --- Interleukin-4 (IL-4) --- p.19 / Chapter 1.10.2.4 --- Interleukin-6 (IL-6) --- p.19 / Chapter 1.11 --- Prevalence of IgG subclass deficiency --- p.19 / Chapter 1.12 --- Reference intervals for IgG subclass --- p.20 / Chapter 1.13 --- Methods for investigation of IgG subclass deficiency --- p.20 / Chapter 1.13.1 --- Radial-immunodiffusion --- p.21 / Chapter 1.13.2 --- Enzyme linked immunsorbent assay --- p.21 / Chapter 1.13.3 --- Nephelometry/turbidmetry --- p.21 / Chapter 1.14 --- Aim of study --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHOD I The Binding Site IgG Subclass Assay --- p.23 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- IgG subclass assay --- p.24 / Chapter 2.1.2 --- Evaluation of patients immune status --- p.24 / Chapter 2.1.3 --- Apparatus and equipment --- p.24 / Chapter 2.2 --- Evaluation of The Binding Site human IgG subclass assay on Beckman Array 360 protein system --- p.25 / Chapter 2.2.1 --- Principle of the Beckman Array Protein System --- p.25 / Chapter 2.2.2 --- Assay preparation and procedure --- p.25 / Chapter 2.2.3 --- Performance characteristic of the IgG subclasses assay --- p.28 / Chapter 2.2.3.1 --- Gain setting --- p.28 / Chapter 2.2.3.2 --- Within batch precision --- p.28 / Chapter 2.2.3.3 --- Interassay precision --- p.28 / Chapter 2.2.3.4 --- Linearity of the assay --- p.29 / Chapter 2.2.3.5 --- Interference of the IgG subclass assay --- p.29 / Chapter 2.2.3.6 --- Recovery experiment --- p.30 / Chapter CHAPTER 3 --- MATERIALS AND METHOD II IgG Subclass Deficiency in Hong Kong --- p.32 / Chapter 3.1 --- Patients and controls --- p.33 / Chapter 3.2 --- Blood samples --- p.33 / Chapter 3.3 --- Serum total haemolytic complement and alternative pathway haemolytic complement assay --- p.34 / Chapter 3.3.1 --- Total haemolytic complement --- p.34 / Chapter 3.3.2 --- Alternative pathway haemolytic complement --- p.34 / Chapter 3.4 --- Statistical analysis --- p.35 / Chapter CHAPTER 4 --- RESULTS I: Evaluation of The Binding Site IgG Subclass Array Kit --- p.36 / Chapter 4.1 --- Gain setting --- p.37 / Chapter 4.2 --- Within batch precision --- p.37 / Chapter 4.3 --- Inter-assay precision --- p.37 / Chapter 4.4 --- Linearity and lowest limit of detection --- p.37 / Chapter 4.5 --- Interference experiments --- p.37 / Chapter 4.6 --- Recovery experiment --- p.37 / Chapter CHAPTER 5 --- RESULTS II: IgG Subclass Deficiency in Hong Kong --- p.45 / Chapter 5.1 --- IgG subclass concentrations and humoral immune status evaluation results of patients and control subjects --- p.46 / Chapter 5.2 --- Statistical tests --- p.45 / Chapter CHAPTER 6 --- DISCUSSION I: The Binding Site IgG Subclass Array Kit --- p.52 / Chapter 6.1 --- IgG subclass assays --- p.53 / Chapter 6.2 --- Within batch and inter-assay precision --- p.53 / Chapter 6.3 --- Lowest limit of detection --- p.53 / Chapter 6.4 --- Interference --- p.54 / Chapter 6.5 --- Recovery of IgG --- p.54 / Chapter 6.6 --- Overall performance of the nephelometric assay --- p.55 / Chapter CHAPTER 7 --- DISCUSSION II: IgG Subclass Deficiency in Hong Kong --- p.56 / Chapter 7.1 --- IgG subclass deficiency in adults --- p.57 / Chapter 7.2 --- Paetiatric patients --- p.59 / Chapter 7.3 --- Recurrent infections and IgG subclass deficiency --- p.59 / Chapter 7.4 --- Summary --- p.60 / REFERENCES --- p.61

Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE(2) receptors coupled to adenylate cyclase via a G(i) protein (EP(3) receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/ L) increased PGE(2), PGF(2 alpha), and PGD(2) synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP(3)) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glyco gen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mu mol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes.

Cellular adhesion research has recently focused on the small scale at the level of individual receptor-ligand bonds. This trend in research is primarily due to experimental advances which allow such individual bond force measurements. Here, one of these techniques, micromanipulation, has been extended to not only determine the bond force of individual receptor-ligand pairs, but also the intrinsic kinetic rates of the interaction. Using transmembrane (TM ) Fc gamma receptor III (CD16a-TM) and human IgG (hIgG), the dependence of adhesion probability on receptor-ligand expression densities, contract duration and contact area was quantitated. A probabilistic based theoretical formulation was developed and validated that relates the intrinsic molecular kinetic rates of the receptorVligand interaction to the experimentally determined adhesion probability. This theoretical formulation describing individual receptor-ligand kinetics has also allowed direct evaluation of existing biophysical bond strength/kinetics paradigms at the extreme condition of single bonds. A force-displacement model was also developed to quantitate the force exerted on the RBC membrane transducer during the micropipette retraction process and found to be in agreement with previous work. In addition to CD16a-TM, the kinetic rates of CD16a anchored via a glycosyl phosphatidylinositol (GPI) moiety (CD16a-GPI) and the two alleles of CD16b (NA1 and NA2) were determined for human, rabbit, and mouse IgG species. The binding affinity of these CD16 interactions to soluble IgG was also measured by traditional bulk chemistry approaches and compared to those measured via the micromanipulation protocol in which the IgG ligand is membrane bound in the solid phase. These data suggest that the membrane anchor itself can alter CD16 binding properties. This represents the first reported effect of the anchor on an intrinsic receptor property, its kinetic rates and binding affinity. This thesis presents two specific aims or goals. These goals were achieved and reported in this thesis. During the course of this research, I also explored other directions and gathered initial data. These directions were further explored by other researchers but the initial data is also presented here.

A suppressed immune system is a complicating health factor in cancer patients that keeps them from achieving the highest quality of life possible. Moderate exercise is thought to boost the immune system in cancer patients. The aim of this project was to determine the effects of an eight week aerobic exercise program on the mucosal immune system of cancer survivors compared to non-cancer patients. Our hypothesis was that the immune system of the cancer patients would positively respond to a moderate exercise program, specifically increasing antibody production. To examine our hypothesis, five cancer and six non-cancer patients undertook a supervised moderate aerobic exercise program at the University of Northern Colorado. The subjects performed an incremental peak treadmill test to exhaustion at the start of the program and after 8 weeks of training. Saliva samples were taken at specific times for each peak exercise test: prior to testing, immediately after testing, and 30 minutes post-test. Enzyme-Linked ImmunoSorbent Assays (ELISA) were performed at Ball State University to analyze the levels of immunoglobulins (IgA, IgG, IgM) in saliva samples of cancer and non-cancer patients. Our findings demonstrated there was a significant increase in IgG after 8 weeks of moderate exercise in non-cancer patients 30 minutes after the treadmill test. A significant increase was also seen in salivary IgA levels after 8 weeks of moderate exercise in cancer patients 30 minutes after the treadmill test was administered, supporting our hypothesis that exercise enhances immune function. Eight weeks of moderate exercise has been shown to enhance immune function demonstrated by the increase of IgA and IgG levels in saliva. / Department of Biology

Group A streptococcal disorders can result from autoantibodies generated against M proteins. These autoantibodies cross react with the basal ganglia resulting in movement disorders. Previously, we demonstrated binding of streptococcal mAb10F5, with CPu and phospholipids. To determine if mAb10F5 binding to basal ganglia and phospholipids is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hours. Brains were harvested and immunofluorescence was used to analyze brain slices. Control IgG2 rats showed significantly less fluorescence in the CPu than mAb10F5 injected rats at every time point. These findings reaffirm 10F5 is an anti-basal ganglia antibody. To evaluate mechanism of antibody entry, mAb10F5 was examined for anti-phospholipid activity. MAb10F5 displayed greater affinity to phospholipids when compared to IgG2 controls. Our findings support mAb10F5 is an anti-basal ganglia and anti-phospholipid antibody due to its own virulence. / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

Previous data in our lab demonstrated an inability to induce oral tolerance when using a feeding needle gavage for 14 days. Given that the upper gastrointestinal (GI) tract is the site of antigen introduction, and the interplay between immune cells of the mucosal tissues, we questioned if inflammation in this tissue, induced by physical trauma, would affect oral tolerance induction. We performed studies on Balb/c mice using a needle gavage or syringe feeding method followed by doses of the immunogenic protein ovalbumin (OVA) to induce tolerance. Immunohistochemistry was used to assess inflammation in esophageal tissues and to correlate with an ability or inability to induce tolerance. Non-cellular alterations within the tissue were also assessed using a pathology grading score. Although fluctuations in cell populations were observed in both the syringe and gavage treated mice, the needle gavage caused significant noncellular damage to esophageal mucosal tissue, which is the most likely cause of failed tolerance induction to the OVA. / Department of Biology

Autoantibodies generated against M proteins can cause post-streptococcal disorders such as Rheumatic Fever. A severe complication of rheumatic fever is rheumatic heart disease which may involve both cardiomyopathy and valvulitis. Rheumatic fever has been associated with the class I M protein epitope of Group A streptococcus (GAS). This epitope can be recognized by monoclonal antibodies (mAbs) 10B6 and 10F5. Previously, we demonstrated binding of streptococcal mAb10F5 in the heart tissue (apex, atria, and valves) of Lewis rats as compared to anti-myosin binding. To determine if mAb10F5 binding in the heart is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hrs. Hearts were harvested and immunofluorescence was used to analyze the hearts. The immunofluorescence intensities for IgG2b were compared to mAb10F5 using previously acquired data. Control IgG2b rats showed significantly less immunofluorescence intensities in the heart regions than mAb10F5 injected rats at the 48 and 72 hr time points. These findings reaffirm mAb10F5 as an anti-cardiac antibody thatbinds heart tissue due its own virulence. To differentiate between the two IgG subtypes, binding intensities of IgG2a were compared to the binding intensities of IgG2b. The binding intensities of IgG2a increased with time. This finding was supported by previous work in our laboratory suggesting IgG2a remained in the bloodstream longer than the IgG2b. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science