Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 85-98). Available also in a digital version from Dissertation Abstracts.

Human ISG15 is a 17 kDa ubiquitin-like protein (Ubl) that is induced by type I interferons (interferons [alpha] and [beta]) and plays a role in antiviral responses. ISG15 is conjugated via its C-terminus to more than 150 cellular proteins, and like ubiquitin, an E1-E2-E3 enzymatic cascade is required for conjugation. Ube1L and UbcH8 were previously identified as the E1 and E2 enzymes for this pathway. My experiments identified Herc5, a HECT domain E3, as the major ligase for ISG15. Like ISG15, Ube1L, and UbcH8, expression of Herc5 is transcriptionally induced by type I interferons. siRNAs against Herc5 abrogated ISG15 conjugation to the vast majority of target proteins in interferon-treated cells. Wild type Herc5, but not the catalytically inactive C994A mutant, supported conjugation of ISG15 in non-interferon-treated cells co-transfected with Ube1L, UbcH8 and ISG15. IQGAP1, a scaffold protein, was identified as another essential component of the ISG15 system. IQGAP1 was discovered to interact with Herc5, and this interaction was mediated by the C-terminal domain of IQGAP1 and the N-terminal RCC1-like repeats of Herc5. IQGAP1 was required for auto-conjugation of ISG15 to Herc5, and I propose a model where IQGAP1 functions, at least in part, by relieving an auto-inhibitory conformation of Herc5. Thus, I have identified two factors that are critical for ISG15 conjugation and my discoveries have increased our understanding of the ISG15 pathway. Identification and characterization of the conjugation apparatus will aid in establishing an in vitro biochemical system for ISG15 conjugation, which in turn, will be important to decipher the biological function of ISG15 modification. / text

Leishmaniasis is a spectrum of diseases caused by several species of protozoan parasites belonging to the genus, Leishmania. The disease, which is transmitted by Sandflies, ranges from self-healing cutaneous lesions to the life-threatening visceral leishmaniasis. Cutaneous leishmaniasis, caused by L. major, is the most common form of the disease. With no vaccine available for use in humans, cutaneous leishmaniasis remains a global public health problem. Since understanding the factors that regulate effective immunity to cutaneous leishmaniasis is critical for the development of an affective vaccine and treatment strategies, the overall aim of my thesis was to decipher the host and pathogen factors that regulate immunity in cutaneous leishmaniasis. Firstly, I show that parasite dose affects the expansion of different T cell subsets following L. major infection; with low dose infection inducing more CD8+ T cells while high dose infection induced more CD4+ T cells. However, although CD8+ T cells were important for optimal primary immunity following low dose infection, they where dispensable during secondary immunity. Secondly, I found that blockade of LIGHT, (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) significantly impaired DC maturation, expression of co-stimulatory molecules, and early cytokine production (IL-12 and IFN-γ) following L. major infection. Interestingly, LIGHT was completely dispensable during secondary immunity in wild type mice but was critical for effective secondary immunity in CD40 deficient mice. Thirdly,I compared disease progression and immune response in CD40 and CD40L deficient mice infected with L. major under identical experimental conditions. I found significant differences in disease progression and immune response between CD40KO and CD40L KO mice infected with virulent L. major and treated with recombinant IL-12. My data revealed a novel pathway (CD40L-Mac-1 interaction) for IL-12 production and resistance to Leishmania major. Collectively, this thesis provides novel insights into the mechanisms involved in the development and maintenance of protective immunity against cutaneous leishmaniasis, which could lead to the development of a more efficient and effective immunotherapeutic and/or vaccination strategies against the disease. / October 2015

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

published_or_final_version / Paediatrics / Master / Master of Philosophy

Severe influenza virus infection, including human infection with highly pathogenic H5N1 viruses is characterised by massive pulmonary inflammation, immunopathology and excessive cytokine production, a process in which macrophages may play a vital role. The aim of this project was to investigate the hypothesis that inhibition of inflammatory responses from infected macrophages, using either alternatively activated bone marrow derived macrophages (BMDMf), or IFNg receptor deficient (IFNgR-/-) mice may ameliorate the devastating immunopathology and inflammation routinely observed in highly pathogenic influenza virus infections. Infection of alternatively activated BMDMf resulted in enhanced positivity for viral proteins, compared with classically activated, inflammatory BMDMf. However, neither subset propagated the infection indicating that while infection is abortive in both classical and alternatively activated BMDMf, the latter may prove more efficient at removing infectious virus from the site of infection due to enhanced infectivity. However, influenza virus was capable of driving expression of proinflammatory mediators such as iNOS and TNFa from classical and alternatively activated BMDMf even in the absence of IFNg signalling. IFNgR-/- BMDMf demonstrated a reduced inflammatory response to infection compared to Sv129 counterparts, suggesting a potentially impaired inflammatory response in vivo. This was investigated by infection of IFNgR-/- mice, which resulted in ameliorated disease, lower viral titres and mild immunopathology, demonstrating that inhibition of IFNg signalling limits the severity of disease. Additionally, mRNA expression for key inflammatory mediators was reduced, demonstrating that inhibition of the overwhelming inflammatory response to influenza virus infection is beneficial to the host, resulting in protection from immunopathology and improved prognosis, without impairing viral clearance.

There is growing evidence that susceptibility to many inflammatory and infectious diseases may be influenced by our genetic make up. Genetic variants in important immune genes may partially explain variation in susceptibility to common diseases. Interferon-γ (IFNγ) is one of the central mediators of the innate and adaptive immunity and has been implicated in a wide range of infectious and inflammatory disease processes. Severe disruptive mutations in coding regions of the IFN-γ receptor 1 gene (IFNGR1) have been found to be associated with fatal but very rare mycobacterial infections. This study looked at common polymorphisms in potentially regulatory non-coding regions of the IFNγ gene and the IFNGR1 gene and investigated their association with susceptibility to severe malaria, a disease for which there have been indications of a genetic component to susceptibility. Malaria is one of the major causes of childhood deaths in Africa. IFNγ and its receptor have been shown to be critically involved in the host response to the malaria parasites. The promoter regions of IFNGR1 and its neighbouring genes, located on chromosome 6q23, and IFNγ and its neighbours, on chromosome 12ql4, were screened for polymorphisms. Haplotypes and linkage disequilibrium maps were constructed, signatures of natural selection were investigated, haplotype tagging SNPs were dentified, and association with disease was analysed. One of these preliminary results was a putative association between the IFNGRl-470ddel allele and susceptibility to severe malaria in the Mandinka ethnic group. This allele was in strong linkage disequilibrium (LD) with markers which are a considerable distance away which might represent a signature of natural selection. To assess the potential functional significance of the IFNGR1-47Q polymorphism, its effects on DNA-protein interactions and gene expression was investigated further in various cell lines. Evidence of tissue-specific nuclear protein binding to this site which seems to be involved in transcriptional regulation was observed.

Hatchery-reared salmonid fish routinely encounter stress due to handling, barging, tagging, and overcrowding. It has been demonstrated that there exists a direct correlation between stress and transient immune suppression which can last for many days in fish. Epizootic viral infections routinely appear in hatcheries and can have a devastating effect on the fish population. The major viral pathogens in salmon and trout are the fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), and the fish birnavirus, infectious pancreatic necrosis virus (IPNV). Vaccines for these viral pathogens are under investigation; however, the fish immune system becomes virtually nonresponsive during episodes of immune suppression. It was necessary to develop a nonantibody mediated, nonimmune method for preventing viral infections. An interferon-like substance has been described for fish which possesses antiviral activity against both IHNV and IPNV. Since interferon administered to cattle has been very effective against vesicular stomatitis virus, a cattle rhabdovirus, an examination of interferon-like activity in fish was initiated. We report here the establishment of in vitro interferon assays. In addition, the salmonid genome contains a multigene family of p-interferon-like genes, much like those in the bovine, equine and porcine genome. The rainbow trout interferon-like genes were found to be inducible in a manner which parallels those seen with bovine and human interferons. In addition to the multigene interferon-like family, it was found that rainbow trout also contain a retroposon multigene family. Retroposons are repetitive elements which appear to have arisen by a reverse transcription event. Two Ll like repetitive elements have been cloned, one of which contains a Drosophila retroposon polymerase sequences never before described for salmonid fish. A number of retroviruses have been described in fish including the walleye dermal sarcoma virus and the Atlantic salmon swimbladder sarcoma virus. Interferon shows prophylactic promise both in vivo and in vitro, against the human retrovirus, HIV. Therefore, research into fish interferon may be even more important if it demonstrates not only anti- IHNV and anti-IPNV, but also anti-fish retrovirus properties. / Graduation date: 1993

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.