The objective of this study was to determine whether neutrophils play a significant role in anaphylaxis or in the response to the anaphylactic mediator platelet activating factor (PAF) in the rabbit. Vinblastine and anti-neutrophil antibodies were compared as neutrophil depleting agents, and 0.35 mg/kg vinblastine was selected as optimal for efficiency and specificity of depletion. Anaphylaxis was induced in sensitized rabbits by intravenous antigen challenge. Neutrophil depletion to 399 ± 101 cells/mm³ blood (14 ± 3%) did not significantly inhibit the physiologic and hematologic events associated with anaphylaxis except tachycardia. However, vinblastine pretreatment significantly reduced tachycardia and the right ventricular pressure increase and abolished the increase in pulmonary resistance caused by intravenous PAF. We conclude that although neutrophils do not play a significant role in IgE-anaphylaxis, they are important in the PAF-induced increases in right ventricular pressure and pulmonary resistance. PAF may not be a major mediator of these two physiologic alterations in IgE-anaphylaxis.

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The acquisition of cellular adhesion machinery likely represented a key factor in the evolutionary transition from unicellular to multicellular organisms. Within metazoa, cellular adhesion is an integral aspect of organismal integrity through its regulation of a wide range of processes, including tissue patterning, cellular proliferation, and migration. As such, dysregulation of adhesion has been linked to diverse pathologies including cancers and neurodegenerative diseases. At the molecular level, adhesion is mediated by specific transmembrane cell adhesion molecules (CAMs) and intracellular complexes that create a dynamic link between the extracellular milieu and the intracellular cytoskeleton. At the sequence level, immunoglobulin domains act to mediate homo- and heterophilic interactions among CAMs and thus adhesion between neighboring cells. LIGs, a family of Ig-containing proteins that contain Leucine-rich repeats, represent candidates for novel CAMs with functions in axonal regeneration and synaptic pathfinding - all of which are highly dependent on cellular adhesion. In Drosophila, two LIG family members, Kekkon1 (Kek1) and Kekkon5 (Kek5) have been been implicated in EGF signaling, and Bone Morphogenetic Protein signaling as well as cellular adhesion, respectively. To investigate the putative role of Kek5 as a CAM, characterization of Kek5 activity was carried out at the cellular and molecular level. From this it was discovered that Kek5 is able to induce a dramatic upregulation of the adherens junction component Armadillo, in addition to epithelial extrusion and cell enlargement. Together, the studies presented within support a model in which Kek5 acts in a homophilic fashion to upregulate Arm and that this activity is functionally separable from other observed effects (epithelial extrusion and cell enlargement).

Introdução: O aumento no consumo mundial de imunoglobulina humana tem desafiado os sistemas de saúde no estabelecimento de padrões de utilização adequados a esta terapia. O conhecimento das políticas públicas pelos profissionais de saúde, aderidos a uma conscientização ao uso racional e estudos baseados em evidências científicas é fundamental para assegurar o acesso adequado e uma maior segurança e efetividade de tratamento. Objetivo: Avaliar a utilização de imunoglobulina humana em hospital universitário de alta complexidade do sul do país e suas indicações relacionando as mesmas aos protocolos clínicos estabelecidos. Métodos: Trata-se de um estudo transversal, retrospectivo, baseado na busca de informações através do prontuário eletrônico dos pacientes do Hospital de Clínicas de Porto Alegre no período de janeiro à dezembro de 2015 Resultados: Foram identificadas 191 prescrições de imunoglobulina humana endovenosa, totalizando 116 pacientes. Desses pacientes, 23% apresentaram síndrome de Guillain Barré, púrpura trombocitopênica idiopática, miastenia gravis, transplante renal, imunodeficiência com aumento de IgM e outras anemias hemolíticas autoimunes. Todas essas situações clínicas tem indicação de uso de acordo com os protocolos estabelecidos pelo Ministério da Saúde. Os demais casos identificados (77%) não constam nas indicações previstas nos protocolos do Ministério da Saúde. Conclusão: Foi possível identificar a utilização de imunoglobulina humana endovenosa em hospital de alta complexidade e quantificar os casos clínicos que fazem uso desse medicamento e que apresentam protocolos nacionais orientando os profissionais de saúde quanto a correta administração desse medicamento. Observou-se que a maioria dos casos identificados no estudo não apresentam regulamentos oficiais que autorizem a sua administração. / Introduction: The increase in the world consumption of human immunoglobulin has challenged the health systems in establishing appropriate standards of use for this therapy. Knowledge of public policies by health professionals adhering to rational use awareness and evidence-based studies is critical to ensure adequate access and greater safety and effectiveness of treatment. Objective: To evaluate the use of human immunoglobulin in a university hospital of high complexity in the South of the country and its indications relating them to the established clinical protocols. Methods: This is a cross-sectional, retrospective study based on the search of information through the electronic medical records of patients from the Hospital de Clínicas of Porto Alegre from January to December 2015 Results: 191 prescriptions of intravenous human immunoglobulin were identified, totaling 116 patients. Of these patients, 23% had Guillain Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, renal transplantation, immunodeficiency with increased IgM and other autoimmune hemolytic anemias. All of these clinical situations are indicated for use according to protocols established by the Ministry of Health. The other cases identified (77%) are not included in the indications provided for in the protocols of the Ministry of Health. Conclusion: It was possible to identify the use of intravenous human immunoglobulin in hospital of high complexity and to quantify the clinical cases that use this medicine and that present national protocols guiding healthcare professionals about the correct administration of this medicine. It was observed that the majority of the cases identified in the study do not present official regulations that authorize its administration.

Immunoglobulin (Ig) gene diversification plays an essential role in adaptive immunity. Faced with a continuous yet varied stream of self, non-self, and possibly harmful molecules, many organisms have mechanisms in their arsenal that have evolved to match the diversity of the antigens they encounter. In humans and mice, developing B and T lymphocytes go through a first round of genomic alteration — V(D)J recombination — in the bone marrow and the thymus, respectively. B cells can subsequently undergo two additional Ig gene diversification processes in secondary lymphoid tissues. Through somatic hypermutation (SHM), Ig variable regions of stimulated germinal center (GC)-forming B cells are mutated and further diversified, enabling affinity maturation. During class-switch recombination (CSR), on the other hand, B cells in the GC or prior to entering the GC recombine Ig constant regions, swapping the IgM-encoding locus for another isotype constant regions gene (e.g., IgG1, IgG3, IgE, IgA) to allow for different effector functions. Both B cell-specific genomic alterations are initiated when the single-stranded DNA (ssDNA) mutator enzyme activation-induced cytidine deaminase (AID) catalyzes the removal of the amino group off deoxycytidine residues, resulting in deoxyuridines and dU:dG mismatches. Low-fidelity cellular responses to the presence of dU, including the mismatch repair (MMR) and the base-excision repair (BER) pathways, are then thought to introduce mutations in SHM and CSR, as well as cause double-strand breaks (DSBs) repaired through canonical and alternative non-homologous end-joining in CSR. Though necessary for proper physiological function, these lymphocyte genome diversification processes are rife with danger for B cells and there is strong selective pressure to carefully orchestrate and target them so as not to threaten the genomic integrity of the cells through breaks or other mutations at non-Ig loci. Yet, these events can still occur, as demonstrated by the implication of AID with translocations found in some cancers (e.g., c- MYC:IGH in Burkitt’s lymphoma). Therefore, the mechanisms underlying AID mutagenic activity targeting to physiological deamination substrates have been the focus of several studies. Protein kinase A (PKA)-dependent phosphorylation of AID at its serine 38 residue has been shown to enable its interaction with replication protein A (RPA) before binding to ssDNA. Others have reported that SPT5 helps target AID to sites of RNA polymerase II (Pol II) stalling, such as the Ig switch sequences. Another cofactor, the RNA exosome complex, helps target the ssDNA mutator AID to both strands of DNA in vivo. The RNA exosome had hitherto been described in the context of RNA processing and degradation as 3’ → 5’ exoribonuclease. Sterile transcript-generating transcription at Ig loci was known to be required for proper AID catalytic activity; the newly described link between the RNA exosome and AID activity raised the prospect that RNA processing, and not mere transcription, might be playing a role in shaping the diversification of the immune repertoire in B lymphocytes. During CSR, transient three-strand structures called R loops are generated. R loops are formed as the nascent transcript invades the DNA duplex, hybridizing to the template strand, and displacing the non-template one. The G-rich nature of the non-template strand is posited to help stabilize the R loop, which allows the ssDNA mutator AID to use the exposed, non-template strand as a substrate. AID must then access the template strand. Here, we investigate the role that the RNA exosome and a potential cofactor, the putative RNA/DNA helicase senataxin (SETX), play in the sequence of biological events that result in CSR while protecting the cell from R-loop accumulation-associated genomic instability.

published_or_final_version / Zoology / Master / Master of Philosophy

In Canada, and more specifically in Canadian-born Aboriginals and foreign born populations, high incidence of tuberculosis (TB) causes significant morbidity and mortality. The presence or absence of specific killer immunoglobulin-like receptor (KIR) genes, individually or in conjunction, may be associated with tuberculosis (active, latent, or uninfected disease status) as well as ethnicity of an individual. It is hypothesized that the differences in KIR profiles, gene frequencies, and/or haplotypes in Canadian-born Aboriginal, Canadian-born non-Aboriginal, and foreign born individuals elicits a differential activation or inhibition profile, resulting in differential cytokine expression and eventually contributes to the outcome of TB infection. In this study we examined the enrichment or depletion of KIR genes in different ethnic populations in Manitoba with special focus on active, latent, and uninfected TB status. In addition, we sought to explore the statistical correlation between TB status and inhibitory/stimulatory KIR haplotypes.