Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 132-142.

Doctor of Philosophy / Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Severe combined immunodeficiency (SCID) is a rare group of inherited disorders characterized by defects in both humoral and cellular immune functions. Naturally occurring SCID has been first described in humans in the 1960s and subsequently identified in horses, mice, and dogs, but never before in pigs. Affected animals are characterized by having loss of functional B and T lymphocytes, and in some cases natural killer (NK) cells, but normal numbers of monocytes, granulocytes, and megakaryocytes. As a result, affected animals fail to produce antibodies and succumb to common disease pathogens after circulating maternal antibodies decay. SCID models are extremely valuable for the understanding of molecular mechanisms of immunological processes during viral and bacterial diseases, cancer, and autoimmunity. SCID mice are widely used as the current model; however, the relevance of the murine SCID model to human and veterinary immune research is limited and there is an increasing need for a more representative model of SCID is imperative. We describe the gross, microscopic, and immunophenotypic characteristics of a line of Yorkshire pigs having naturally occurring SCID. Affected pigs lack T and B lymphocytes, but display circulating NK cells, fail to produce antibodies to viral infection, and lack cell-mediated response to tumor xenotransplants. We also describe response of SCID pigs to porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is the most devastating virus in swine industry, causing losses of billions of dollars annually. Understanding the immunopathogenesis of the disease is imperative in order to develop strategies to combat this devastating virus. PRRSV infected-SCID pigs failed to develop lesions of PRRSV infection, demonstrating the significant role of the adaptive immunity to PRRSV infection. Finally, we describe the preliminary results of the adoptive transfer of purified CD3⁺ T lymphocytes to SCID pigs from SLA-II matched wild-type littermates, with the objective of establishing a porcine model for the study of T cell immunopathogenesis with viral diseases.

Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis and responsible for the deaths of over 200,000 children each year worldwide. HuNoV research has been hampered by the long absence of a readily reproducible cell culture system and a suitable small animal model, while gnotobiotic (Gn) pigs have been a unique animal model for understanding HuNoV pathogenesis and immunity, as well as evaluating vaccine and therapeutics. Recent reports of HuNoVs infection and replication in B cells supplemented with commensal bacteria Enterobacter cloacae and in Blab/c mice deficient in RAG/IL2RG have gained extensive attention, and my studies utilized the well-established Gn pig model to investigate the effects of these two interventions on HuNoV infection. Surprisingly, the colonization of E. cloacae inhibited HuNoV infectivity in Gn pigs, evidenced by the significantly reduced HuNoV shedding in feces and HuNoV titers in intestinal tissues and blood compared to control pigs. Moreover, HuNoV infection of enterocytes but not B cells was observed with or without E. cloacae colonization, indicating B cells were not a target cell type for HuNoV in Gn pigs. On the other hand, using RAG2/IL2RG deficient pigs generated by CRISPR/Cas9 system, with confirmed severe combined immunodeficiency, I evaluated the effects of host immune responses on HuNoV infection. Compared to wild-type Gn pigs, longer HuNoV shedding was observed in RAG2/IL2RG deficient pigs (16 versus 27 days), and higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs. In addition, I evaluated dietary interventions including probiotics and rice bran using Gn pig model of HuNoV infection and diarrhea. While the colonization of probiotic bacteria Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) in Gn pigs completely inhibited HuNoV fecal shedding, the two cocktail regimens, in which rice bran feeding started either 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized Gn pigs, exhibited dramatic anti-HuNoV effects, including reduced incidence and shorter duration of diarrhea, as well as shorter duration of virus fecal shedding. The anti-HuNoV effects of the cocktail regimens were associated with the enhanced IFN-𝛾⁺ T cell responses, increased production of intestinal IgA and IgG, and longer villus length. Taken together, my dissertation work improves our understanding of HuNoV infection and immunity, and further supports for Gn pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions. / Ph. D. / Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis. Using the gnotobiotic pig model of HuNoV infection and diarrhea, we found that (1) the colonization of a commensal bacterium E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs. (2) Increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs, which had severe combined immunodeficiency. (3) The dietary supplementation of rice bran and colonization of two probiotic bacteria significantly reduced HuNoV infectivity and diarrhea, and the beneficial effects were associated with enhanced intestinal immunity and health. Taken together, the dissertation work improves our understanding of HuNoV infection and immunity, and further supports for gnotobiotic pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions.

OBJECTIVES: Models for the development of new treatment options in vestibular schwannoma (VS) treatment are lacking. The purpose of this study is to establish a quantifiable human VS xenograft model in mice. STUDY DESIGN AND METHODS: Both rat malignant schwannoma cells (KE-F11 and RT4) and human malignant schwannoma (HMS-97) cells were implanted near the sciatic nerve in the thigh of severe combined immunodeficiency (SCID) mice. Additionally, human benign VS specimens were implanted in another set of SCID mice. Three-dimensional tumor volumes were calculated from magnetic resonance images over the next 6 months. RESULTS: Mice implanted with malignant schwannoma cells developed visible tumors within 2 weeks. Imaging using a 4.7-tesla magnetic resonance imaging and immunohistopathologic examination identified solid tumors in all KE-F11 and HMS-97 xenografts, whereas RT4 xenografts consistently developed cystic schwannomas. VS xenografts demonstrated variability in their growth rates similar to human VS. The majority of VS xenografts did not grow but persisted throughout the study, whereas two of 15 xenografts grew significantly. Histopathologic examination and immunohistochemistry confirmed that VS xenografts retained their original microscopic and immunohistochemical characteristics after prolonged implantation. CONCLUSIONS: This study describes the first animal model for cystic schwannomas. Also, we demonstrate the use of high-field magnetic resonance imaging to quantify VS xenograft growth over time. The VS xenografts represent a model complimentary to Nf2 transgenic and knockout mice for translational VS research.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Raymond R. R. Rowland / Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most costly virus to the swine industry, worldwide. This study explored the application of deep sequencing techniques to understand better the virus-host interaction. On the virus side, PRRSV exists as a quasispecies. The first application of deep sequencing was to investigate amino acid substitutions in hypervariable regions during acute infection and after virus rebound. The appearance and disappearance of mutations, especially the generation of a new N-glycosylation site in GP5, indicated they are likely the result of immune selection. The second application of deep sequencing was to investigate the quasispecies makeup in pigs with severe combined immunodeficiency (SCID) that lack B and T cells. The results showed the same pattern of amino acid substitutions in SCID and normal littermates and no different mutations were identified between SCID and normal littermates. This suggests the mutations that appear during the early stages of infection are the product of the virus becoming adapted to replication in pigs. The third application of deep sequencing was to investigate the locations of recombination events between GFP-expressing PRRSV infectious clones. The results identified different cross-over occurred within three conserved regions between EGFP and GFPm genes. And finally, the fourth goal was applied to develop a set of sequencing tools for analyzing the host antibody repertoire. A simple method was developed to amplify swine VDJ repertoires. Shared and abundant VDJ sequences that are likely expressed by PRRSV-activated B cells were determined in pigs that had different neutralization activities. These sequences are potentially correlated with different antibody responses.

A imunodeficiência combinada grave (SCID) é uma doença caracterizada por profunda deficiência de células T, que afeta as imunidades celular e humoral e gera anormalidades graves no desenvolvimento e funções do sistema imune. Recém-nascidos com SCID apresentam a doença nos primeiros meses de vida e tem grande susceptibilidade a infecções. Sem tratamento, essas condições são invariavelmente fatais, porém se reconhecidas precocemente, há a possibilidade da realização do transplante de células-tronco hematopoiéticas, o tratamento curativo, o que torna a SCID uma emergência pediátrica. A investigação do defeito genético é uma prerrogativa para o condicionamento adequado do transplante, a terapia gênica, o aconselhamento genético e o diagnóstico pré-natal. No Brasil, o conhecimento sobre SCID é incipiente e não existem dados moleculares sobre pacientes com a doença. Sendo assim, este estudo teve por objetivo investigar defeitos genético-moleculares de pacientes brasileiros com SCID. Foram incluídos 13 pacientes, todos com início precoce dos sintomas e manifestações clínicas esperadas em SCID (principalmente infecções respiratórias, de pele, diarreia crônica e atraso de crescimento). Os patógenos isolados foram vírus, bactérias e fungos oportunistas comumente encontrados em pacientes SCID. A partir da quantificação de TRECS e KRECs e imunofenotipagem de linfócitos, foi montado o perfil imunológico de cada paciente, que guiou o sequenciamento direto de Sangerdos genes mais frequentemente mutados em cada imunofenótipo de SCID. Mutações em 3 pacientes foram identificadas por Sanger e, posteriormente, 8 pacientes cujas mutações não foram encontradas no Sanger foram encaminhados para o sequenciamento completo de exoma, que resultou na identificação do gene afetado em 62,5% dos casos. Ao todo, foram identificadas mutações patogênicas em 8 dos 13 pacientes. Os resultados revelaram 6 alterações em 5 genes de SCID clássica (IL7R, RAG2, DCLRE1C, JAK3, IL2RG), 1 mutação no gene CD3G e 2 alterações em CECR1. Das 9 mutações encontradas, 5 não possuíam registro na literatura. O estudo genético de SCID em nosso país é problemático, principalmente porque ainda hoje, a esmagadora maioria dos pacientes não é diagnosticada. A implementação da quantificação de TRECs e KRECs como triagem neonatal para linfopenias graves é uma ferramenta fundamental para que os pacientes SCID possam ser identificados, investigados e tratados adequadamente. / Primary immunodeficiencies are a heterogeneous group of genetic diseases that lead to increased susceptibility to infections and affect mostly children. Severe Combined Immunodeficiency (SCID) is the most severe of all these diseases and is characterized by profound T cell deficiency, which affects cellular and humoral immunities and leads to severe abnormalities in the development and function of the immune system. Newborns with SCID present the disease in the first months of life and are highly susceptible to infections. Without treatment, these conditions are invariably fatal, but if recognized early, there is the possibility of hematopoietic stem cell transplantation, the curative treatment, which makes SCID a pediatric emergency. Identifying the genetic defect of SCID patients is a prerequisite for proper transplant conditioning, gene therapy, genetic counseling and prenatal diagnosis. Knowledge about SCID is still incipient in Brazil, and there are virtually no molecular data on patients with the disease. Therefore, this study aimed to investigate genetic-molecular defects of Brazilian patients with SCID. Thirteen patients were recruited, all with early onset of symptoms and clinical manifestations expected of classic SCIDs (mainly respiratory and skin infections, chronic diarrhea and failure to thrive). The pathogens isolated were opportunistic viruses, bacteria and fungi often reported in SCID patients. The immunological profile from each patient was defined by the quantification of TRECS and KRECs and lymphocyte immunophenotyping, which was meant to guide direct sequencing by Sanger of the most frequently mutated genes of each SCID immunophenotype. Mutations in 3 patients were identified by Sanger and, subsequently, 8 patients whose mutations were not identified by Sanger were referred for whole exome sequencing, which resulted in the identification of the affected gene in 62,5% of cases. Pathogenic mutations were identified in 8 of the 13 patients. The results revealed 6 mutations in 5 genes associated to classical SCID genes (IL7R, RAG2, DCLRE1C, JAK3, IL2RG), 1 mutation in the CD3G gene, and 2 mutations in CECR1. Five of the 9 mutations found had no record in the literature. SCID genetic investigation in our country is troublesome, mainly because even nowadays, the vast majority of patients are not diagnosed properly. Newborn screening for SCID and other severe lymphopenias by the quantification of TRECs and KRECs is key for the identification, investigation and proper treatment of SCID patients.

The development of new high throughput technologies able to multiplex disease biomarkers as well as advances in medical treatments has lead to the recent expansion of the newborn screening panel to include DNA-based targets. Four rare disorders; deletion 22q11.2 syndrome and Spinal Muscular Atrophy (SMA), Severe Combined Immunodeficiency (SCID) and Congenital Cytomegalovirus (CMV), are potential candidates for inclusion to the newborn screening panel within the next few years. The major focus of this study was to determine whether 5’-hydrolysis assays developed for the four distinct disorders with specific detection needs and analytical ranges could be combined on the OpenArray system and in multiplexed qPCR reactions. SNP detection of homozygous SMN1 deletions in SMA, CNV detection in the 22q11.2 critical region, and quantification of the SCID biomarker, T-cell receptor excision circles (TRECs) and CMV were all required for disease confirmation. SMA and 22q11.2 gene deletions were accurately detected using the OpenArray system, a first for the technology. The medium density deletion 22q11.2 multiplex successfully identified deletion carriers having either the larger 3 Mb deletion or the smaller 1.5 Mb deletions. Both TREC and CMV targets were detected but with a decrease in sensitivity when compared to their singleplex counterparts. Lastly, copy number detection of the TBX1 was performed when multiplexed with the TREC assay, without a decrease in detection limit of either assay. Here, we provide proof of principal that qPCR multiplexing technologies are amenable to implementation with a newborn screening laboratory.