The importance of gut associated lymphoid tissues has been extensively reported in higher vertebrates, but less is known in lower vertebrates. In mammals immunoglobulin (Ig)A is the primary Ig of mucosal immunity. But no IgA has been identified in cold-blooded animals. In higher vertebrates, antigen must stimulate the lymphoid tissues in the intestines to elicit an IgA response, and cytokines from CD4 positive helper T cells are required for B cell switch. It is not known if this is the case in lower vertebrates, or if T cell help evolved before or after class switch recombination between functional antibody isotypes. My study will fill in these gaps in our knowledge by comparing oral antigen inoculation relative to intraperitoneal antigen inoculation in frogs (Xenopus sp.). Oral immunization is a novel approach to eliciting immune responses in Xenopus. I propose that IgX will increase with oral inoculation compared to intraperitoneal injection. This would be the first demonstration of class switch upon oral immunization to a mucosal isotype in the first vertebrates that employs higher vertebrate Ig heavy chain switch mechanism, which would shed light on the most fundamental aspects of our humoral adaptive immune system. Using a total Ig ELISA protocol, measuring total relative levels of IgM, there was no difference between the first three groups of orally immunized frogs compared to intraperitoneally immunized frogs. However, a response to serum IgX was seen in the first group. On the other hand, the refined Ag-specific ELISA protocol did present a significant increase in serum IgM response in frogs immunized systemically over orally immunized animals, but not an overall IgX response. Phylogenetic analysis suggests that, contrary to initial reports, IgA evolved from IgX. With consideration of entire constant region and individual constant domain analyses as well as synteny and function, we suggest new hypotheses of vertebrate antibody evolution to be tested as immunogenetic coverage of more species continues to expand.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 113-131) Also available in print.

Made available in DSpace on 2014-06-12T18:31:49Z (GMT). No. of bitstreams: 2 arquivo8339_1.pdf: 1343346 bytes, checksum: 9a728290de0229465d8e8c3920952b44 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Toxoplasmose é a principal causa de uveíte posterior em nosso meio. A determinação de anticorpos no soro contribui para o esclarecimento etiológico da doença. Dentre as imunoglobulinas na toxoplasmose a mais relevante é a IgG, considerada marcador de contato prévio, seguido pela IgM, cuja presença denota atividade da doença e pela IgA e IgE. Determinações também podem ser realizadas no humor aquoso. A IgA secretora específica da lágrima tem sido observada com níveis de reatividade maior, em indivíduos com toxoplasmose ocular, quando comparados com pacientes normais. O presente estudo objetivou validar o teste de IgA secretora anti T. gondii na lágrima. Foram analisados 197 indivíduos portadores de uveíte posterior ativa, sendo 82 deles caracterizados como provavelmente toxoplásmica (padrão ouro), 74 pacientes portadores de uveítes posteriores de outras etiologias e 41 pacientes de possível toxoplasmose, porém sem padrão ouro. Considerou-se como padrão de seleção (padrão ouro), lesões de uveíte posterior ativa, satélite de lesão cicatrizada. Foi determinada a IgG e IgM sérica em 125 pacientes, por método de imunofluorescência. Para pesquisa de IgA secretora anti T. gondii na lágrima foi utilizado antígeno bruto do mesmo e técnica de imnunoensaio (ELISA). A determinação de dosagens de anticorpos IgG e IgM, mostrou que 106 pacientes (84,8%) foram positivos para IgG específica. Um paciente teve IgM positiva. A pesquisa de IgA secretora específica na lágrima mostrou sensibilidade de 65,9% (IC = 54,5-75,4) e especificidade de 71,6% (IC= 59,8 - 81,2) quando trabalhados o grupo de uveítes posterior ativa padrão ouro e o grupo de uveítes posteriores de outras etiologias.Valor preditivo (+): 72%, (IC= 60,3-81,5) e negativo:65,4% (IC=54,0-75,4). Razão de verossimilhança (+)=2,33. Probabilidades de doença pré-teste:52,5% e pós-teste 72,0% Probabilidades de não doença pré-teste: 52,5% e pós-teste:34,6%. Esses valores diminuíram quando feitas simulações com o grupo não padrão ouro. Os índices de reatividade da IgA secretora específica da lágrima foram maiores no grupo de uveítes posteriores padrão ouro que no grupo de uveítes de outras etiologias (p=0,004), o que não aconteceu com o grupo de pacientes não padrões ouro. Houve reatividade também do olho sadio (p=0,874). Conclui-se que o teste para determinação de IgA secretora anti-Toxoplasma gondii é um teste válido para diferenciar pacientes portadores de uveíte posterior ativa por toxoplasmose ocular de pacientes portadores de uveítes causadas por outras etiologias. Acreditamos que aprimoramentos precisam ser realizados á nível laboratorial que venham a melhorar o desempenho do teste

Cigarette smoke is the leading cause of preventable mortality worldwide. This excess death is attributable to an increased risk of acquiring a variety of conditions, including chronic respiratory/cardiovascular diseases and various types of cancer. Smokers are additionally predisposed to develop infectious diseases, notably including pneumonia caused by the influenza virus, one of the most prevalent and burdensome pathogens in existence today. Although cigarette smoke is well known to modulate many aspects of the immune system, the specific mechanisms by which this predisposition is mediated are incompletely understood. Also unclear is the effect of cigarette smoke on responses to intranasal immunization strategies aimed at eliciting immunity against pathogens such as influenza in the upper airways, where protection may substantially contribute to sterilizing immunity. This PhD thesis focused primarily on addressing these knowledge gaps. In the first study, we assessed the effect of cigarette smoke on antibody induction following intranasal immunization in the upper airways of mice, finding that smoke exposure attenuated antigen-specific IgA induction in the upper respiratory tract, reproductive tract, and systemic circulation. In addition, we found that these nasal IgA demonstrated a reduced antigen-binding avidity in the acute post-immunization period. Mechanistically, deficits in nasal IgA were associated with a reduced accumulation of antigen-specific IgA antibody-secreting cells (ASCs) in the nasal mucosa, induction of these cells in nasal-draining lymphoid tissues, and upregulation of molecules critical to ASC homing (vascular cell adhesion molecule-1; VCAM-1) and IgA transepithelial transport (polymeric immunoglobulin receptor; pIgR) in the nasal mucosa. Ultimately, in tandem with recent clinical work published by others, our study strongly suggests that cigarette smoke can attenuate IgA induction in the upper airways, which may have implications for aspects of intranasal vaccine efficacy. Thus, smoking status should be more consistently considered in the design of clinical trials for IgA-oriented intranasal vaccines. The second study did not assess smoking and host defense directly, but rather served to optimize protocols for assessing immunoglobulins in human mucoid respiratory samples as a precursor to future studies in smoking-related disease. In this regard we found that, relative to phosphate-buffered saline (PBS), dithiothreitol (DTT)-based processing of human sputum samples increased total IgA yields, decreased IgE yield, and improved the detection of a specific IgG autoantibody. These findings suggest that processing choices for human mucoid respiratory samples should be made with specific goals in mind as they pertain to antibody isotype(s) of interest. Finally, in the third study we investigated potential mechanisms by which cigarette smoke exposure promotes influenza, given that smokers are at increased risk of acquiring the pathogen, progressing to severe disease, and being admitted to hospital/ICU following infection. In doing so, we found that concurrent smoke exposure increased morbidity, hypoxemia, pulmonary edema, neutrophilia, and ultimately mortality in a mouse model of H1N1 infection. These changes were associated with an increased accumulation of viral (v)RNA in cells independent of any change in the shedding of replication-competent viral particles. Using a novel dysregulation score approach, we found that interleukin (IL)-6 and colony-stimulating factor (CSF)3 expression was highly exacerbated in the lungs and circulation of smoke-exposed, infected mice relative to controls. Supplementation of recombinant (r)CSF3 increased morbidity, hypothermia and edema, while blockade of the cognate receptor (CSF3R) improved alveolar-capillary barrier function. On the cellular level, single cell RNA-sequencing revealed a shift in the distribution of Csf3+ cells towards neutrophils. Finally, deep transcriptional analysis of neutrophils revealed a gene signature that was largely indicative of an exacerbated form of typical disease with select unique regulatory elements. Ultimately, this work identifies potential therapeutic targets (CSF3R signaling, excess vRNA accumulation) for the treatment of cigarette smoke-augmented influenza, and warns against clinical rCSF3 therapy to treat neutropenia during viral infectious disease. In conclusion, the work presented in this PhD dissertation expands our understanding of the relationship between cigarette smoke and antimicrobial host defense as it pertains to both IgA immunity in the upper airways, and the pathogenesis of cigarette smoke-augmented influenza. / Thesis / Doctor of Philosophy (PhD) / Cigarette smoke exposure is well known to have many harmful effects on human health, including through its ability to promote various infectious diseases such as influenza. However, the mechanisms by which it promotes infection are not fully known. This is an important knowledge gap given that over 1.1 billion individuals continue to smoke worldwide, and a large number of people are exposed to the harmful effects of second-hand smoke, both with fatal consequence. The central goal of this thesis was to gain a better understanding of this relationship between cigarette smoke and infectious disease, specifically by assessing how smoke exposure impacts immune responses in the upper and lower airways. In the first study, we found that smoke exposure interferes with the ability to activate immunoglobulin (Ig)A antibody responses in the nasal passages of mice, which may have important implications for human nasal vaccination strategies. The second study investigated different methods with which to best measure antibodies in human respiratory samples. Finally, in the third study we defined a role for a specific molecule, CSF3, in worsening health in a mouse model of concurrent cigarette smoke and influenza infection. Overall, this work provides new insights into the ways in which smoking can increase the risk of respiratory infection, thereby informing the future design and testing of vaccines and treatments for use in our highly smoke-exposed global population.

No description available.

Streptococcus pneumoniae is an asymptomatic colonizer of the upper respiratory tract as well as an opportunistic pathogen. Colonization is prerequisite to causing disease in a host, and it often involves formation of biofilms. There are currently two vaccines available against pneumococcus, both of which focus on preventing invasive disease by targeting the polysaccharide capsule of the most invasive serotypes. We hypothesized that by using membrane proteins expressed during the biofilm state, we can formulate an effective vaccine against colonization which would lead to an overall decrease in disease incidence. To do this, we selected protein candidates expressed during biofilm growth based on their ability to elicit an IgA response in human serum. Selected proteins (SP_0459, SP_1114, and SP_1702) were identified and used for further experiments. The proteins identified in this study will be paired with other immunogenic proteins to determine a successful vaccine formulation targeting colonization of Streptococcus pneumoniae.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

Sexual transmission of the Human Immunodeficiency Virus Type 1 (HIV-1) accounts for the majority of newly acquired infections. Vaccination efforts have induced only modest protection in HIV clinical trials. HIV-1 induces a robust local immune response in genital mucosa of exposed individuals. Understanding the function of naturally occurring antibodies against HIV-1 in genital mucosa, the primary site of transmission, might be instrumental to improving vaccines and antibody-based microbicides. This study focused on HIV-specific antibody responses in the male genital tract (MGT), which is underexplored. We characterized antibody subclasses and specificities in genital tract secretions (seminal plasma, urethral secretions) and blood from a cohort of HIV-1-infected men to determine the origin and distinct nature of antibodies in the MGT. We detected similar HIV-1 IgG titers and specificities in all three body fluids, indicating that MGT IgG likely originates from blood. In contrast, gp41-specific IgA was restricted to genital secretions suggesting a local niche of IgA antibody production. Genital secretions from a subset of individuals neutralized cell-free HIV-1 and blocked cell-to-cell HIV-1 transmission. Statistically, these functions correlated positively with gp41 IgA titers. HIV-specific IgA monoclonal antibodies were also effective in these assays. To explore cell-dependent activities of HIV-specific antibodies in genital mucosa we surveyed Fc receptor expression in mucosal epithelial tissue. The IgG-engaging neonatal Fc receptor (FcRn), the IgA receptor FcαRI and the high-affinity intracellular Fc receptor TRIM21 were detected by immunohistochemistry and western blots. In stratified squamous epithelia (foreskin, vagina) FcRn+ epithelial cells were detected primarily in the basal layer, FcαRI+ epithelial cells in suprabasal layers and TRIM21 throughout. Deposits of immunoglobulins in the stratified squamous epithelium colocalized with FcRn, FcαRI and TRIM21. Our findings indicate that the MGT is capable of expressing a local anti-HIV IgA response to achieve antiviral defense through antibody neutralization and cell-dependent functions involving classical immune effector cells and epithelial cells.

Abstract not available

Anoplocephala perfoliata is a species of parasitic worm that belongs to a group known as cestodes, which specifically target equine animals. As with all types of tapeworms, these parasites infect the gastrointestinal tract of their host, with devastating and potentially fatal consequences. The current lack of a sensitive and specific test for this parasite means that it continues to go undetected, hense this project aims to develop a novel and rapid diagnostic test with high sensitivity and specificity to help increase detection, thus precluding economic loss in the equine industry. The project details the development of three unique detection platforms; an ELISA, a lateral flow assay and an impedimetric immunosensor, aimed to detect IgA in saliva, since IgA is the dominant immunoglobulin of the mucosal immune system. IgA was therefore believed to be the ideal marker for rapid, specific and early indication of infection with A. perfoliata. Diagnosis using saliva samples was an integral part of this project, since it would allow for non-invasive sampling, by non-skilled personnel. A highly sensitive ELISA-based detection system was developed in this project for the detection of 3 different types of IgA. The first ELISA was developed to detect non-specific or ‘total’ IgA levels. Using a sandwich ELISA format, IgA was detectable with a LoD of ~0.04 ng/ml. A second ELISA was developed using the crude excretory/secretory (E/S) antigen, cultured from A. perfoliata worms, which were obtained by a vet during post-mortem examination of infected horses. The crude antigen mix was then used to fabricate an ELISA to detect specific IgA in saliva, produced against the E/S antigens. The crude antigen was then employed in a series of SDS PAGE and western blot experiments, which revealed the 12/13 kDa antigen as the main antigen detected by IgA in saliva. The 12/13 kDa was then electroeluted and used to immunise rabbits, in order to obtain anti-12/13 kDa antibodies, which were later used to purify large quantities of the 12/13 kDa antigen from the crude antigen mix. This allowed for the fabrication of the third and final ELISA, to detect IgA specific to the 12/13 kDa antigen. The 3 ELISAs were optimised throughout this project to ensure the most ideal conditions, such as antibody concentrations, sample dilutions, sample diluents, incubation temperatures and times were employed to obtain maximum assay sensitivity, specificity and productivity in a commercial setting. Testing samples (n = 24) using all 3 ELISAs and then standardising the specific IgA levels against the non-specific IgA, allowed for a novel and reliable detection method for A. perfoliata to be developed. This diagnostic test was developed in partnership with Austin Davis Biologics Ltd., who in April 2014 launched a screening programme which now offers horse owners an accurate means of testing their horses for A. perfoliata infections accurately. The second detection platform developed during this project was a lateral flow assay, whereby an immunochromographic strip was used to measure IgA levels in saliva. The studies performed determined the optimal conditions as using 40 µl of a 1:1,000 dilution of saliva using PBS(T) 1% as the sample diluent. The capture and control antibody were used at a concentration of 0.2 mg/ml, which were coated on the nitrocellulose membrane using an automated dispensing system (BioDot). The conjugate was labelled using gold nanoparticles, since it does not require any substrates or wash steps and its aggregation allows for immediate visual detection. A LoD of ~47 ng/ml was obtained for this assay. The final detection system investigated as part of this project was a label-less impedimetric immunosensor, whereby IgA was detected by means of electrochemical impedance spectroscopy (EIS). Polyaniline was the conductive polymer chosen to coat the surface of the screen printed carbon electrode, since the amine groups could be utilised to immobilise biotin molecules. A biotin-avidin complex was employed to ensure the uniform immobilisation of the capture antibody. Using the capture and control antibody at a concentration of 50 µg/ml and 10 mM ferri-ferrocyanide as the redox solution, IgA concentrations over a range of 100 – 0 ng/ml were investigated by Electrochemical Impedance Spectroscopy (EIS).