Dissertations / Theses on the topic 'Kaposi's sarcoma'

Over the past two decades there has been an explosion in the number of cases of Kaposi’s sarcoma (KS) in parts of sub-Saharan Africa, where Kaposi’s sarcoma associated-herpesvirus (KSHV) and HIV are relatively prevalent. Currently KS is the most commonly reported cancer in Uganda causing significant morbidity and mortality. Limiting KSHV transmission or halting disease progression could prevent KS. Here, I describe an investigation of factors that might impact on transmission of KSHV and report the first prospective study of antibody titre to KSHV to determine risk of KS from Africa. Stored samples from Medical Research Council, Uganda cohorts were tested using an ELISA to KSHV antigens. Results from a birth cohort found that among both mothers and children malaria parasitaemia was identified as a novel association with KSHV seropositivity. Among children HIV exposure and HIV infection was associated with antibodies to KSHV. A random effects meta-analysis conducted to clarify wider evidence of an association between KSHV and HIV found that HIV was associated with an increased prevalence of antibodies to KSHV in mothers and children. A case-control study nested within a longitudinal HIV cohort found among individuals who develop KS, antibody titres to KSHV are higher and increased over time compared to adults who did not. It is plausible that control of malaria may also reduce the spread of KSHV. How malaria may interact with KSHV and if malaria control will reduce transmission are key future questions. Prevention and treatment of HIV with anti-retroviral therapy may lower KSHV transmission between mothers and children. For individuals with HIV-KSHV co-infection, increasing antibody titre to KSHV precedes development of KS. Research is required to elucidate co-factors driving progression to cancer. A clinically valid tool to screen for risk of HIV-associated KS is urgently needed.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus associated with a number of malignancies, including Kaposi’s sarcoma. Similar to all herpesviruses, KSHV establishes either latent or lytic infections in host cells. The latent stage involves minimal viral gene expression, enabling the virus to remain dormant, maintaining genome integrity and enabling viral persistence. Conversely, lytic replication is characterised by the expression of a highly regulated and coordinated cascade of viral gene expression, ultimately resulting in the production of new mature, infectious virions. Importantly, lytic replication is necessary for the development and spread of Kaposi’s sarcoma. State of the art high throughput, high resolution “omics” approaches, such as mass spectrometry-based quantitative proteomics and next generation sequencing-based transcriptomics, are rapidly becoming the techniques of choice for discovering novel biological phenomena due, in part, to their sensitivity and the ability to repeatedly ask new questions of an existing dataset. Herein, three high throughput, high resolution approaches, namely SILAC-based quantitative proteomics, miRNA sequencing and mRNA sequencing are employed in an attempt to identify and characterise novel interactions between KSHV and the host cell. Utilising quantitative proteomics, the essential host cell splicing factor Prp19 is identified as a novel interacting partner of the lytic KSHV ORF57 protein in subnuclear bodies. This interaction, surprisingly, does not contribute to viral mRNA maturation, but instead has implications for the cellular DNA damage response, appearing to limit the effectiveness of this important pathway, possibly to reduce any negative effects on viral replication. Proteomic analyses also highlighted a link between KSHV lytic replication and host miRNA biogenesis pathways. Through the application of miRNA sequencing, two host miRNAs, namely miR-151a-5p and miR-365a-3p, were found to be dysregulated during KSHV infection. Importantly, neither of these miRNAs appear to represent a host antiviral response. Instead, cellular target mRNAs are identified for miR-365a-3p, through the use of mRNA sequencing. These targets, termed DOCK5 and PRUNE2 are rapidly degraded during KSHV lytic replication via the viral-mediated upregulation of miR-365a-3p expression. Subsequent analysis of DOCK5 function during lytic replication suggests this interaction may promote viral egress. The data presented herein sheds light on previously unidentified mechanisms employed by KSHV to hijack the host cell, and may aid in the development of novel therapeutics against this important pathogen.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), has been been identified in all epidemiological forms of KS as well as in tissue obtained from primary effusion lymphoma (PEL) and Multicentric Castleman's disease (MCD). The KSHV genome contains several putative oncogenes, suggesting that viral infection may induce cellular transformation and tumorgenesis. Herpesviruses encode a number of different surface glycoproteins, which are involved in virus-host interactions. Studies have shown that the viral glycoproteins H and L form a complex that plays an essential role in viral attachment and cell to cell fusion. Both glycoproteins have been identified in KSHV and were expressed in mammalian cells. Expression studies revealed that KSHV gH and gL exhibit similar features to those seen in other herpesviruses. However, KSI-IV gL appears to traffic independently and may function in cell to cell fusion processes even when expressed alone. KSI-IV de novo infections are rare and the lack of a reliable cell culture system has delayed pathogenesis studies. As part of this thesis the hepatoma cell line HepG2 has been shown to allow limited KSHV infection, as judged by nested PCR. Studies have shown that infection leads to increased apoptosis, although viral replication could not be detected. Furthermore, Epstein Barr Virus (EBV) appeared to modulate the ability of KSHV to infect HepG2 cells. Finally, a microtitre plate assay has been established for the quantification of the KSHV genome. A comparison of plasma and serum samples obtained at the same time point showed that plasma is more reliable in testing for KSHV, the DNA copy number in serum samples being reduced up to 10 fold. In conclusion, this new assay is a potentially useful tool for both diagnostic proposes and research studies.

Kaposi's sarcoma associated herpesvirus (KSHV), is a gammaherpesvirus from the Herpesviridae family and causes a number of proliferative diseases such as Kaposi sarcoma, primary effusion lymphoma and Castleman disease. The prime effectors of the cell proliferation occurring during KSHV infections are the latent gene products LANA, vCyclin and vFLIP. The analysis of the mRNA latent gene transcripts produced by KSHV showed no monocistronic transcript encoding vFLIP suggesting its expression was regulated by a non-canonical translation mechanism. Indeed, previous studies proposed that an IRES element located within the vCyclin ORF is responsible for vFLIP expression. We have now shown that a minimum fragment of 252 nt of within the vCyclin ORF and directly upstream of vFLIP can function as IRES. The KSHV IRES 252 fragment showed IRES activity in Rabbit Reticulocyte Lysate (RRL) but not in KSderived cell lines. Due to lack of information on the mechanism of internal initiation mediated by the KSHV IRES, we have analyzed the role of eIF4A, eIF4E and eIF4G for the IRES function in RRL. Surprisingly, the KSHV IRES require the whole eIF4F complex for it to function. In addition, several IRES trans-acting factors (ITAFs) have been shown to interact with KSHV IRES by Mass spectrometly analysis. These are Ybox protein 1 (YB-l), eukaryotic translation elongation factor la (EEF1Al and EEF1A2), heterogenous nuclear ribonucleoprotein K (hnRPK), heterogenous nuclear ribonucleoprotein Cl/C2 (hnRP Cl/C2), and Poly(rc) binding protein 1 (PCBP1). Although the interaction between the KSHV lRES and IT AFs has yet to be confirmed by direct interaction studies, analysis on the YB-l showed an interaction with KSHV lRES based on Western blot result. With several ITAFs interacting with the KSHV IRES and the requirement for eIF4F, we then characterized the RNA secondary stmcture of the KSHV IRES in solution to understand how the IRES stmcture could coordinate such interactions. Chemical and enzymatic probing, combined with structure folding prediction using Mfold revealed 3 domains with domain I is the most structured domain. In search of structure similarities among IRESes, X-linked inhibitor of apoptosis (XIAP) was found to be the closest, although the XIAP IRES contains only two domains. The two IRESes also share similarities for a polypyrimidine sequence located in domain II. For the first time, we have showed the requirements of the KSHV IRES for canonical, non-canonical initiation factors and preliminary prediction of secondary structure which shows that the KSHV IRES does not belong to any existing functional groups of IRESes and a novel DNA vims IRES.

Mechanistic insights on molecular and cellular mechanisms whereby KSHV induces Kaposi?s sarcoma (KS) are key for our understanding of KS tumors and for the development of new therapies. We have previously developed an animal model for KSHV induced KS using murine bone marrow cells transfected with a KSHVBac36. We found that although these cells lacked attributes of transformed cells in vitro, they were able to cause KS-like tumors in vivo. In vivo tumorigenesis correlated with upregulation of both KSHV lytic genes and host angiogenesis suggesting that that cues provided from the microenvironment played a major role in regulating viral and host gene expression related with KSHV-induced tumorigenesis. Our goal thus, was to identify these molecular cues regulating pathogenic gene expression in KSHV infected cells in vivo. An important difference between cells kept in vitro versus in vivo is the lack of environmental extracellular matrix (ECM) signals. Therefore the mECK36 cells were cultured in vitro in matrigel, a basement membrane preparation rich in ECM proteins and its individual components, to discern the effect of host signaling by the ECM on KSHV infected cells. Investigation of gene expression through Real Time RT-PCR identified several viral and host genes associated with tumorigenesis such as KSHV vGPCR and angiogenesis associated VEGF and EGF- receptors were upregulated in response to this environment. Further analysis of the molecular activity of the cell indicated the change in transcription was due to the activation of integrin signaling, as assessed by phosphorylation of the Focal Adhesion Kinase (FAK) protein. Our results show that integrin signaling occurring in vivo through interaction with ECM serves to enhance the pathogenic viral and host gene expression of KSHV infected cells and that EGFR upregulation can be correlated with these conditions. These results points to the integrin signaling pathway or the EGF-Receptor as promising targets for therapy and prevention of KS tumors.

O HHV-8 (herpesvírus 8 humano) é o agente etiológico do sarcoma de Kaposi (SK). Diferentemente dos outros herpesvírus, o HHV-8 é distribuído de modo não ubíquo ao redor do mundo. São sete os principais genótipos de HHV-8, de acordo com o padrão de variabilidade da ORF K1: A, B, C, D, E, F e Z. Estudos da variabilidade genética do HHV-8 poderão trazer melhores interpretações sobre o potencial patogênico dos genótipos de HHV-8 e das variações genotípicas funcionais. Dados sobre a variabilidade genética do HHV-8 no Brasil, em que o SK é associado ao HIV, permanecem escassos. Pelo nosso conhecimento, esse é o primeiro estudo que compara a variabilidade genética de HHV-8 em indivíduos infectados por HIV com SK e sem SK no Brasil. Objetivo. O estudo visou analisar a variabilidade genética do HHV-8 entre indivíduos infectados por HIV com SK e sem histórico de SK. Métodos. Sequências de DNA de HHV-8 foram investigadas em amostras criopreservadas de células mononucleares do sangue periférico a partir de 37 indivíduos infectados por HIV com SK (grupo 1); e de amostras de saliva de indivíduos sem SK (grupo 2), as quais foram selecionadas por meio da detecção positiva de DNA/ORF73/HHV-8 a partir de um total de 751 indivíduos. Dados demográficos e clínicos do estadio e evolução do SK, assim como parâmetros laboratoriais foram caracterizados. As análises moleculares e reconstruções filogenéticas foram baseadas nas ORFs K1 e K12 do HHV-8. Resultados. Foram obtidas sequências de DNA dos loci K1 e/ou K12 de 75 indivíduos, 34 indivíduos do grupo 1 e 41 do grupo 2. O sistema de primers empregado foi capaz de detectar os genótipos A, B, C, F e amplo perfil de subgenótipos de K1/HHV-8. Os dados não mostraram associação de genótipos de HHV-8 com a presença de SK ou estadio de SK. Todavia, o subgenótipo B1 predominou naqueles em que não houve registro de piora de SK (p=0,04). Os subgenótipos B1 e C3 foram igualmente predominantes em ambos os grupos. As frequências do genótipos A foram de 24% e 12,2% e dos genótipos B e C foram de 34,1 e 35,3%, nos grupos 1 e 2, respectivamente. O genótipo F foi descrito pela primeira no Brasil, um caso de cada grupo. Um amplo perfil de subgenótipos de C no grupo 2 sem SK foi encontrado (C1, C2, C3, C5 e C7). Subgenótipos K1 C5 e C7, exclusivos do grupo 2 (7%), foram confirmados como recombinantes. Não houve variabilidade genotípica de HHV-8 em amostras biológicas diferentes do mesmo indivíduo em oito casos estudados. Sítios polimórficos (6/59) em regiões codificadoras de miRNA do locus K12 foram observados, sendo 70% presentes exclusivamente em sequências de HHV-8 do grupo com SK e protótipos de SK. Conclusão. Embora não houvesse associação entre genótipos de HHV-8 e presença ou estadio de SK, o subgenótipo B1 foi significativamente relacionado ao melhor prognóstico de SK. Alguns recombinantes foram observados no locus K1 de HHV-8 em indivíduos do grupo 2 sem SK. A presença de SNPs em regiões codificadoras de miR12-12 e miR12-10 predominou em sequências de HHV-8 de indivíduos com SK, grupo 1, e protótipos de SK epidêmico, endêmico e clássico. A escoha de primers foi importante para garantir a amplificação de todos os genótipos e amplo perfil de subgenotipos de HHV-8.
HHV-8 (Human Herpes Virus 8) is the etiological agent of Kaposi\'s sarcoma (KS). Unlike other herpesviruses, the distribution of HHV-8 is not so ubiquitous around the world. There are seven major HHV-8 genotypes, according to the variability pattern of the ORF K1: A, B, C, D, E, F and Z. Studies on the genetic variability of HHV-8 may help to better understand the pathogenic potential of HHV-8 genotypes and their functional genotypic variations. Data on the genetic variability of HHV-8 in Brazil, where KS is associated with HIV infected people, remain scarce. To our knowledge, this is the first study comparing the genetic variability of HHV-8 among HIV-infected individuals with KS and without KS in Brazil. Objective. The study aimed to analyze the genetic variability of HHV-8 among HIV-infected individuals with and without KS. Casuistry and Methods. HHV-8 DNA sequences were obtained from samples of cryopreserved peripheral blood mononuclear cells from 37 individuals infected with HIV-KS (group 1); and saliva from individuals without KS (group 2) who were selected by means of detection of positive DNA/ORF73/HHV-8 from a total of 751 individuals. Demographic and clinical data (stage and progression of KS), as well as laboratory parameters were characterized. Molecular analysis and phylogenetic reconstructions were based on sequences of the ORFs K1 and/or K12 of HHV-8. Results. K1 and/or K12 DNA sequences of HHV-8 were obtained from 75 subjects, 34 from group 1 and 41 from group 2. The primer system used was able to detect the genotypes A, B, C, F and a wide profile of HHV- 8 K1 subgenotypes. Data showed no association of genotypes with the occurrence of KS or with visceral KS either. However, subgenotype B1 predominated in individuals who did not report any progression of KS (p=0.04). Subgenotypes B1 and C3 were equally prevalent in both groups. Genotype A frequencies were 24 % and 12.2% and genotypes B and C were 34.1 and 35.3 % in groups 1 and 2, respectively. We also described here for the first time the genotype F of HHV-8 in Brazil. A wide profile of subgenotypes C (C1, C2, C3, C5 and C7) in the group without KS was found. HHV-8 K1 DNA sequences of group 2 (7%) belonging to subgenotypes C5 and C7 were confirmed as recombinants. Our findings did not show virus variability in the same patient in samples collected at different times or from different biological material in the eight cases studied here. There was no statistical difference regarding the presence/absence of a given SNP from locus K12 between groups with and without KS. However, there were a total of 6/59 polymorphic sites in coding regions of miRNA, 70% of which present only in the HHV-8 DNA sequence of group with KS and KS prototypes. Conclusion. Although there was no association between HHV-8 genotypes and the presence of KS and KS clinical stage, subgenotype B1 was significantly related to the absence of progression of KS. Some recombinants in K1/HHV-8 locus were observed in the group without KS. The presence of SNPs in coding regions of miR12-12 and miR12- 10 predominated in sequences of HHV- 8 of SK cases (group 1) and of epidemic, endemic and classic KS prototypes. The choice of primers was essential to ensure amplification of all HHV- 8 genotypes and wide profile de subgenotypes.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the 8th human herpesvirus discovered in 1994. After primary infection, KSHV establishes latency and, in the context of immunosuppression, has been associated with specific malignancies: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Seroepidemiological surveys suggest that KSHV is not a ubiquitous virus and several transmission routes and risk factors must exist to explain its global distribution. The increased risk of KSHV-associated cancers in human immunodeficiency virus (HIV)-infected individuals, and the decrease of KS incidence after the introduction of highly active anti-retroviral therapy (HAART) in 1997 suggest that cellular immunity plays an important role in controlling KSHV. In this thesis, seroepidemiological studies were conducted in African, Middle Eastern, Mediterranean European and South American countries to determine the infection rate, and to determine risk factors and possible routes of KSHV transmission. A new quantitative real-time PCR method was developed to detect KSHV in clinical samples. This assay has diagnostic and prognostic implications for the management of KSHV-associated diseases. Anti-viral and immunological responses were measured and analysed, and cellular immune responses to KSHV were demonstrated in a cohort of HIV infected individuals undergoing HAART treatment. The interaction between KSHV and HIV was evaluated and the impact of HAART on KSHV immune reconstitution was investigated.

The latency-associated nuclear antigen (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a multi-function protein involved in maintenance of the viral episome and has been shown to interact with several proteins including p53 and pRB. It is likely that the multifunctional role of LANA1 exceeds these observations therefore the work in this thesis aimed to study LANA1 at both the transcription and protein-protein interaction level. I developed a lentiviral system for the infection of primary endothelial cells with LANA1, to analyse changes in gene expression profiles by gene expression microarrays. While the system was successfully developed, no significant changes in gene expression could be attributed to LANA1. Subsequently, using the yeast two-hybrid system and large-scale immunoaffinity purification coupled to mass spectrometry, I identified 26 novel binding partners of the KSHV LANA1 complex. The majority of these proteins have functions in splicing or mRNA processing and further analysis lead to the discovery of predominant protein domains. Several of the identified proteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, A2/B1, D and I, constitute members of the human H-complex. hnRNP A1, A2/B1 and D are also implicated in telomere biogenesis. I show that LANA1 binds UP1, the proteolytic derivative of hnRNP A1, which modulates telomere elongation and maintenance. Furthermore, I show an in vivo interaction between LANA1 and human telomerase reverse transcriptase (hTERT) and the ability of LANA1 to recover telomerase activity from cell lysates. These findings suggest a function for LANA1 in the maintenance of telomeres and may have important implications for the role of LANA1 in KSHV-related tumours. I propose models for the role of these complexes in splicing and telomere biogenesis.

In humans, Kaposi's Sarcoma-associated Herpesvirus (KSHV) is capable of establishing latent infection in B-cells. During latency no infectious virions are produced, the viral episome is maintained and few viral genes are expressed. Latently infected cells, however, retain the capacity to enter the lytic cycle. KSHV ORF50 encodes the transcription factor RTA whose expression is sufficient to initiate the full lytic cycle. RNA interference (RNAi) involves the sequence specific silencing of gene expression. Although extensively validated and widely available at present, lentiviral vector-mediated RNAi was unavailable in 2002. This thesis describes the generation and characterisation of shRNA expression cassettes that can be delivered using self- inactivating lentiviral vectors. Using shRNAs designed to interfere with KSHV ORF50 expression, the number of cells expressing RTA following chemical induction of the lytic cycle was greatly reduced. Analysis of viral lytic antigens and virus production suggests that reducing RTA expression prevents latent KSHV from initiating the lytic cycle. Although KSHV infects B-cells in vivo, most B-cell lines are recalcitrant to KSHV infection in vitro. This thesis describes the in vitro susceptibility of a panel of B-cell lines, representing different stages of B-cell differentiation, and other common cell-lines to recombinant KSHV infection. Interestingly, all the adherent cell-lines examined were susceptible to recombinant KSHV infection whereas we were unable to identify any B-cell lines which were efficient targets for recombinant KSHV infection KSHV positive primary effusion lymphoma (PEL) cells are phenotypically similar to plasmablasts, which represent a late stage in B-cell differentiation, immediately preceding terminal differentiation into plasma cells. This thesis examines the stage at which B-cell development is arrested in PEL, focussing on the transcription factor X- box binding protein-1 (XBP-1) and the unfolded protein response (UPR). Using exogenously expressed XBP-1, the role of XBP-1 as the possible molecular switch linking terminal differentiation and KSHV lytic reactivation is considered.

Thesis (Ph. D.)--University of Sydney, 2004.
Title from title screen (viewed 5 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Medicine. Includes list of published articles and presentations. Includes bibliographical references. Also available in print form.

Kaposi�s sarcoma (KS) is a peculiar vascular neoplasm that occurs mainly in elderly Mediterranean men and patients with acquired immunodeficiency syndrome (AIDS). The current literature indicates that KS is initiated by the human herpes virus 8 (HHV8) as a reactive polyclonal process but with deregulation of oncogene and tumour suppressor genes, it can progress to a true malignancy with monoclonality. Clinically, classical KS often presents as an indolent disease affecting mainly the lower extremities whereas AIDS-related KS has no site predilection and can progress rapidly with systemic involvement. Histologically, KS can be classified into patch, plaque and nodular stages. Interestingly, classical and AIDS-related KS are indistinguishable histologically and this suggests that AIDS-related KS and classical KS might be initiated by a common aetiology but given their different clinical courses, they may progress through different mechanisms. In view of the importance of the cell cycle proteins in the development and progression of many human malignancies, this thesis aims to examine the role of these proteins in the progression of the two main clinical subtypes of KS. The cell cycle protein expressions in a cohort of 47 patients with KS with welldocumented clinical and histological features were studied. Using a monclonal antibody against the latent nuclear antigen-1 molecule of HHV8, HHV8 was detected in 78% of the cases. The more advanced nodular lesions were found to have a higher level of proliferative activity as measured by the proliferation x marker, Ki-67. This suggests it is valid to use the histological specimens as a tumour progression model of KS. The role of the Rb/cyclin D1/p16 pathway was examined. The more advanced nodular stage KS lesions were more likely to be positive for cyclin D1, suggesting that cyclin D1 is important in the progression from patch stage to nodular stage. p16 acts as a tumour suppressor and it has an inhibitory effect on cyclin D1. The p16 expression rate was low in early stage KS but high in the more advanced lesions. It seems that reduced p16 expression occurs early in KS and may be important in its development. The rate of Rb expression, on the other hand, did not differ significantly among the histological subtypes. The results revealed the significant role of the Rb/cyclin D1/p16 pathway in the progression of KS. Of the mitotic cyclins examined, cyclin A expression was correlated with the advanced tumor stage. The rate of p34cdc2 expression was high in the lesions and there was no correlation with histological stage. This suggests that p34cdc2 is important in the early development of the tumour but not necessarily in its progression. Along the p53-apoptotic pathway, mutant p53 expression was significantly more common in the nodular stage. The cyclin G1 (a protooncogene, one of the target genes of p53) expression also paralleled that of mutant p53 with the majority of the KS lesions showing cyclin G1 expression and significant xi correlation between advanced histological stage and increasing rate of cyclin G1 expression. These findings suggest that progression along the p53 pathway may be important in the advanced stage development of KS. On the other hand, expression of the CDK inhibitor, p27, a protein that normally negatively regulates cyclin G1, was reduced in nodular KS. These findings suggest that some KS lesions may progress through a deregulated or abnormal p53 pathway. There were correlations between cyclin D1, cyclin A, cyclin G1, mutant p53 and negative HIV status. The findings suggest that components of both the Rb/cyclin D1/p16 and p53-apoptotic pathways are important in the progression of classical KS. Rb protein was the only cell cycle protein whose rate of expression correlated significantly with HHV8 status in KS. The majority of HHV8 positive lesions were also positive for Rb protein, unlike HHV8 negative lesions. This suggests that some of the HHV8 negative lesions can progress through a defective Rb pathway whereas the role of Rb in the progression may not be as important in the HHV8 positive lesions. This was an unexpected finding given that one of the postulated mechanisms of tumour initiation by the HHV8 virus is via the viral cyclin it produces. The viral cyclin produced by HHV8 acts through the Rb pathway much the same as cyclin D1 and one would have expected that HHV8 positive cases are less likely to be positive for the Rb protein. In summary, the majority of the KS lesions examined in this thesis show HHV8 infection. The Rb/cyclin D1/p16 pathway appears to be important in the progression of the different stages of KS and expression of the proteins involved in the p53 pathway were found to be important in the advanced stages of the development of KS. There were differential expressions of cell cycle proteins between AIDS-related and classical KS, and between HHV8 positive and HHV8 negative lesions. The findings also provided some clues to the possible mechanisms of development in KS lesions that were not initiated by HHV8.

Kaposi�s sarcoma (KS) is a peculiar vascular neoplasm that occurs mainly in elderly Mediterranean men and patients with acquired immunodeficiency syndrome (AIDS). The current literature indicates that KS is initiated by the human herpes virus 8 (HHV8) as a reactive polyclonal process but with deregulation of oncogene and tumour suppressor genes, it can progress to a true malignancy with monoclonality. Clinically, classical KS often presents as an indolent disease affecting mainly the lower extremities whereas AIDS-related KS has no site predilection and can progress rapidly with systemic involvement. Histologically, KS can be classified into patch, plaque and nodular stages. Interestingly, classical and AIDS-related KS are indistinguishable histologically and this suggests that AIDS-related KS and classical KS might be initiated by a common aetiology but given their different clinical courses, they may progress through different mechanisms. In view of the importance of the cell cycle proteins in the development and progression of many human malignancies, this thesis aims to examine the role of these proteins in the progression of the two main clinical subtypes of KS. The cell cycle protein expressions in a cohort of 47 patients with KS with welldocumented clinical and histological features were studied. Using a monclonal antibody against the latent nuclear antigen-1 molecule of HHV8, HHV8 was detected in 78% of the cases. The more advanced nodular lesions were found to have a higher level of proliferative activity as measured by the proliferation x marker, Ki-67. This suggests it is valid to use the histological specimens as a tumour progression model of KS. The role of the Rb/cyclin D1/p16 pathway was examined. The more advanced nodular stage KS lesions were more likely to be positive for cyclin D1, suggesting that cyclin D1 is important in the progression from patch stage to nodular stage. p16 acts as a tumour suppressor and it has an inhibitory effect on cyclin D1. The p16 expression rate was low in early stage KS but high in the more advanced lesions. It seems that reduced p16 expression occurs early in KS and may be important in its development. The rate of Rb expression, on the other hand, did not differ significantly among the histological subtypes. The results revealed the significant role of the Rb/cyclin D1/p16 pathway in the progression of KS. Of the mitotic cyclins examined, cyclin A expression was correlated with the advanced tumor stage. The rate of p34cdc2 expression was high in the lesions and there was no correlation with histological stage. This suggests that p34cdc2 is important in the early development of the tumour but not necessarily in its progression. Along the p53-apoptotic pathway, mutant p53 expression was significantly more common in the nodular stage. The cyclin G1 (a protooncogene, one of the target genes of p53) expression also paralleled that of mutant p53 with the majority of the KS lesions showing cyclin G1 expression and significant xi correlation between advanced histological stage and increasing rate of cyclin G1 expression. These findings suggest that progression along the p53 pathway may be important in the advanced stage development of KS. On the other hand, expression of the CDK inhibitor, p27, a protein that normally negatively regulates cyclin G1, was reduced in nodular KS. These findings suggest that some KS lesions may progress through a deregulated or abnormal p53 pathway. There were correlations between cyclin D1, cyclin A, cyclin G1, mutant p53 and negative HIV status. The findings suggest that components of both the Rb/cyclin D1/p16 and p53-apoptotic pathways are important in the progression of classical KS. Rb protein was the only cell cycle protein whose rate of expression correlated significantly with HHV8 status in KS. The majority of HHV8 positive lesions were also positive for Rb protein, unlike HHV8 negative lesions. This suggests that some of the HHV8 negative lesions can progress through a defective Rb pathway whereas the role of Rb in the progression may not be as important in the HHV8 positive lesions. This was an unexpected finding given that one of the postulated mechanisms of tumour initiation by the HHV8 virus is via the viral cyclin it produces. The viral cyclin produced by HHV8 acts through the Rb pathway much the same as cyclin D1 and one would have expected that HHV8 positive cases are less likely to be positive for the Rb protein. In summary, the majority of the KS lesions examined in this thesis show HHV8 infection. The Rb/cyclin D1/p16 pathway appears to be important in the progression of the different stages of KS and expression of the proteins involved in the p53 pathway were found to be important in the advanced stages of the development of KS. There were differential expressions of cell cycle proteins between AIDS-related and classical KS, and between HHV8 positive and HHV8 negative lesions. The findings also provided some clues to the possible mechanisms of development in KS lesions that were not initiated by HHV8.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi’s sarcoma (KS), the most common malignancy in individuals with untreated HIV/AIDS. Several lines of evidence indicate that KS oncogenesis is associated with loss of T cell-mediated control of KSHV-infected cells. However, the adaptive CD8 and CD4 T-cell responses against KSHV have not been fully characterised. Neither the antigenic repertoire nor the immunodominant targets of CD8 and CD4 KSHV-specific T cells are fully understood, and the phenotypes and functions of these cells remain largely unexplored. To investigate the targets of the CD8 and CD4 T-cell responses against KSHV, a novel approach for a large-scale screen of KSHV antigens was proposed that used lentiviral-transduced monocyte-derived dendritic cells (moDCs) expressing a panel of KSHV open reading frames (ORFs). Transduced moDCs naturally process the KSHV gene products and present the resulting antigenic peptides in the context of MHC class I and II. Transduced moDCs were cultured with autologous T cells and the CD8 and CD4 proliferative responses to each KSHV ORF (or pool of ORFs) were assessed. CD8 and CD4 KSHV-specific responses were investigated in 14 KSHV-seropositive individuals. Unexpectedly, both the CD8 and CD4 T-cell responses against KSHV were found to be skewed towards ORFs expressed in the early and late phases of the viral lytic cycle. The most frequently recognised CD8 target was a pool of late lytic KSHV ORFs, [ORF28/ORF36/ORF37]. Identification of novel KSHV CD8 epitopes from within the late lytic ORF pool was attempted. Peptide-MHC binding and denaturation assays identified peptides that had the highest affinity for HLA-A*0201. Recognition of these potential epitopes was tested in clinical samples by IFNγ ELISpot, and compared with recognition of nine previously published HLA-A*0201-restricted KSHV epitopes. Finally, the use of pentamers as tools to investigate the memory phenotypes and functions of virus-specific T cells was explored.

Human herpes virus 8 (HHV8) is the aetiological agent of all forms of Kaposi's sarcoma (KS). Seven major subtypes (A, B, C, D, E, F, Z) based on genetic variability of open reading frame (ORF)-K1, have been identified. Numerous studies point to differing tumorigenic and pathogenic properties of the HHV8 subtypes. The study objective was to determine the prevalence of the HHV8 subtypes in a cohort of clinical and histologically confirmed KS in Cape Town, South Africa, and analyse associations between the different subtypes, clinico- epidemiological forms and clinical presentation of KS. The clinical data was prospectively collected and recorded on a body diagram and with photographs. Demographic data was retrospectively collected from clinical records. Tissue biopsies were taken for ORF-K1 subtyping. Out of a cohort of 103, eighty six patients were subtyped; 81 AIDS (aquired immune deficiency syndrome)-KS and 5 African endemic. Subtype A5 (42/86) and B2 (16/86) predominated. B1, B3, A1 and A4 subtypes were identified in 10/86, 9/86, 4/86 and 1/86 patients respectively. A5, B1, B2 and B3 were found in African blacks and individuals of mixed ancestry, while subtypes A1 and A4 are found only in whites and individuals of mixed ancestry. Subtype A5 was associated with >10 KS lesions at presentation in the AIDS-cohort (32/38, p=0,050), but not in the African endemic patients (2/4, p=0,600). Subtypes A1 and A4 were less likely to be associated with poor risk tumour extension (p=0,031) and A1 was associated with lower likelihood of lower limb involvement (p=0,004).

Thesis (Ph. D.) -- University of Maryland, College Park, 2007.
Thesis research directed by: Fischell Dept. of Bioengineering . Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Sep. 3, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology." Discipline: Genetics and Molecular Biology; Department/School: Medicine.

Studies on the viral gene, vOX2, have indicated that this 55kDa glycoprotein has a similar structure to the human CD200, an immunoglobulin superfamily gene, containing two extracellular domains, a single transmembrane region and a short cytoplasmic tail. In order to investigate the potential role of vOX2 in an in vivo system, a novel murid herpesvirus, murine herpesvirus 76 (MHV-76), was utilised. MHV-76 is a natural deletion mutant of murine herpesvirus 68 (MHV-68), lacking four unique genes and eight viral tRNA-like genes from the left end of the MHV-68 genome. This deletion has provided an opportunity to generate a recombinant MW-76 virus expressing vOX2 (MHV76-vOX2) and a selection marker (GFP-Hyg^R), thus allowing a functional study of vOX2 through the infection of mice. A control recombinant virus (MHV76-IRES) was also generated, which only expressed the selection marker (GFP-Hyg^R). The genomic structures of all recombinant viruses were verified by Southern blot analysis and the expression of the inserted genes was confirmed by Northern blot analysis. The growth kinetics of MHV76-vOX2 and MHV76-IRES were compared with wild type MHV-76 in both an in vitro single-step and multi-step growth assay. The growth kinetics of the recombinant viruses was not significantly different from that of MHV-76. In vivo studies have indicated that vOX2 has an effect on viral replication; a 10 to 100 fold increase in viral lung titres was observed in mice infected with MHV76-vOX2 compared to the control recombinant virus. Examination of lung sections showed that mice infected with the vOX2 recombinant virus elicited a strong influx of inflammatory cells, particularly peripheral mononucleocytes (PMN) and macrophages, resulting in perivascular and peribronchiolar inflammation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the known human cancer viruses, causing Kaposi's sarcoma and primary effusion lymphoma in immunosuppressed patients. Although of medical concern, the mechanisms through which the virus causes cancer remain poorly understood. Researchers speculate that the lytic phase contributes to the development of human cancers by this virus.

KSHV, like other herpesviruses, is predominantly latent in the human host, but undergoes lytic activation to produce infectious viral particles. In the process, the virus hijacks the host machinery to express large quantities of viral genes via a process known as the host shutoff effect. The virus then replicates its DNA and assembles viral capsids within nuclear viral replication compartments. Viral proteins act in various locations within the cell depending on their function. However little is known about the location of viral transcripts and how their localization relates to their function. Thus I sought to understand the localization of viral transcripts to gain insight into the spatiotemporal regulation of the lytic phase.

Using fluorescence in situ hybridization (FISH) and immunofluorescence (IF), I observed that particular viral transcripts accumulate within the nucleus in or near replication compartments. This occurs late in the lytic phase coinciding with viral DNA replication. My findings indicate that the mechanism is independent of the host shutoff effect and splicing, but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, polyadenylated nuclear (PAN) RNA. PAN RNA is essential for the viral life cycle and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

One key regulator of the KSHV lifecycle is a long noncoding RNA (IncRNA) called the polyadenylated nuclear (PAN) RNA. PAN RNA is an early gene product comprising nearly 80% of total polyadenylated cellular transcripts in lytic infected cells. Studies on its function demonstrate that PAN RNA is a regulator of virion production through modulation of viral genes.

A glimpse into the mechanism comes from recent chromatin isolation by RNA purification (CHIRP) studies on lytic KSHV-infected B lymphocytes. The studies revealed widespread binding of PAN RNA to viral and host chromatin, but could not identify an underlying mechanism. The most feasible approach to study function is a genetic knockout. However, a complete PAN RNA gene deletion is unachievable in the KSHV genome due to an overlapping open reading frame, K7. In a related gammaherpesvirus, rhesus rhadinovirus (RRV), a computational search uncovered a PAN RNA homologue, whose sequence does not overlap with any known genes. I found that RRV PAN RNA is present at about 150,000 copies per cell and organized the purchase of RRV ΔPAN RNA constructs to facilitate study of PAN RNA's mechanism.

Capitalizing on the RRV homolog, Dr. Johanna B. Withers and I compared changes in chromatin association by PAN RNA between homologs and over the lytic phase with CHART (capture hybridization analysis of RNA targets). After careful analysis, the data suggest that chromatin-association by PAN RNA is nonspecific and that the mechanism of regulation by PAN RNA is not primarily related to chromatin remodeling. With this is mind, I looked to another potential mechanism, one related to binding by nuclear relocalized cytoplasmic polyAbinding protein (PABPC).

Both PAN RNA homologs associate with several host proteins, one of which is cytoplasmic polyA-binding protein (PABPC). Upon lytic induction, SOX, the host shutoff mediator, facilitates degradation of messages in the cytoplasm, causing the PABPC to relocalize to the nucleus. Nuclear relocalized PABPC binds KSHV PAN RNA at ~8-10 proteins per RNA molecule. I hypothesize that a function of PAN RNA is to act as nuclear relocalized PABPC sponge to facilitate preferential expression of viral genes and assembly of virions.

I designed mutant to capitalize on a unique feature of PAN RNA, the triple helical stabilization element (ENE). At the 3' end a triple helix (ENE) forms with polyA tail, protecting PAN RNA from deadenylation, stabilizing it. I reduced the length of the polyA-tail to eight adenylates, which permits formation of the triple helix, but falls below the 20-adenylate footprint of PABPC. The RRV PAN constructs would supplement RRV ΔPAN RNA virus to examine if the tailless PAN RNA mutants rescue the loss of virion production seen previously during the downregulation of KSHV PAN RNA. The results from these experiments would yield a deeper understanding of host-virus interactions and will provide insights into the importance of PABP-binding for the function of a nuclear noncoding RNA.

Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
The c-myc proto-oncogene is involved in various cellular processes including cell growth, proliferation, and apoptosis. Overexpression and deregulated expression of the gene have been previously linked to several lineage-unrelated, aggressive, poorly differentiated tumors. Oncogenic expression of c-myc has also been implicated in several vascular neoplasms as having a crucial role in angiogenesis. This gives c-myc a dual oncogenic function in that tumor growth requires both cell proliferation and angiogenesis to ensure survival and confer an effective malignancy. In vitro studies have shown that the c-Myc protein is an important regulatory molecule of spindle cell proliferation and migration in Kaposi's sarcoma (KS), an angioproliferative tumor that is commonly associated with HIV. In light of the above and recent findings demonstrating amplification of c-myc in select angiosarcomas secondary to irradiation or chronic lymphedema, our primary aim was to ascertain the same in KS. We also attempted to determine what correlation existed, if any, between the immunohistochemical (IHC) expression of the c-Myc protein and c-myc gene copy amplification using fluorescent in situ hybridization (FISH). Samples analyzed during this study included archival tissue samples of KS (N=24 ). For FISH analyses, a dual-labeled technique was employed and probes against the c-myc gene and chromosome 8 (CEP-8) were used. For IHC, the monoclonal anti-c-myc antibody, 9E10, was used and tissue from hemangiomas (N=11) and non-radiation induced angiosarcomas (N=6) served as the controls. PCR for detection of KS-associated herpesvirus (KSHV) DNA was performed on all KS cases. While FISH analyses revealed no amplification of c-myc in any of the cases of KS, IHC analyses revealed positive staining for c-Myc in 13/24 cases (54%) with stain localization throughout the cell. As such, no correlation could be found between gene amplification and protein expression. KSHV-PCR analyses revealed that 19/24 cases (79%) were positive for KSHV-DNA. Ten of 24 cases (42%) were positive for c-Myc IHC and KSHV-PCR, while one case (4%) was negative for both indicating a lack of correlation (using McNemar's test for statistical analysis) between c-Myc IHC protein levels and presence of KSHVDNA. Our findings indicate that c-myc gene amplification is not normally found in KS and cannot be correlated with the expression of the c-Myc protein. Thus, unlike other tumors we have discussed where gene amplification was a common occurrence; it seems to have little clinical significance in KS. The absence of c-myc amplification raises the question of why 54% of the samples in this study still exhibited protein expression as determined by IHC. To grasp a further understanding of what is truly going on in these cases, it would be necessary to use techniques such as RT-PCR or in situ hybridization to study c-myc at the RNA level.

Human herpesvirus-8, or KSHV, was discovered in 1994 and is the causative agent of all forms of Kaposi's sarcoma (KS). It is also associated with two lymphoproliferative disorders: primary effusion lymphoma (PEL) and multicentric Castleman's disease. The KSHV viral chemokine receptor, vGPCR, is a homologue of the IL8 receptors CXCR1 and CXCR2. vGPCR is considered a viral oncogene: it transforms fibroblasts in vitro and enhances growth and longevity of primary endothelial cells. vGPCR signals promiscuously via several heterotrimeric G-protein subtypes and activates the MAP and SAP kinases, the Src family kinases, and PI3 kinase. However, little is known about vGPCR in the context of KSHV-infected haematopoietic cells. In this thesis, a tetracycline-inducible vGPCR-expressing PEL line is used to show that vGPCR causes a G0/G1 arrest in PEL cells via inhibition of Cdk2. This lack of Cdk2 function inhibits the chemically mediated KSHV latent-to- lytic switch. We hypothesize that expression of vGPCR outside of the normal lytic phase could inhibit virion production and resultant cell death this would allow the many vGPCR-induced cytokines to have a more prolonged effect on the tumour microenvironment. These effects would result in enhanced angiogenesis, a hallmark of KS, as well as the recruitment of new infectable KSHV cells. The vGPCR is among the most promising targets for rationally designed anti-KSHV therapy. However, a better understanding of vGPCR signalling events is required. This thesis also examines how vGPCR affects the function of the protein tyrosine phosphatases (PTPs), a family of enzymes that regulates many extracellular signalling events. PTPs are an exciting new potential target in anti-infective and anti-tumour therapeutics. Lastly, we examine the inhibition of TGFp signalling by KSHV in PEL cells. PEL cells secrete TGFp but are resistant to its effects due to downregulation of TGFp Rll. This suggests a KSHV-mediated anti-immune strategy that requires further exploration.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS). Like all herpesviruses, KSHV has a bi-phasic life cycle, with a dormant latent phase and a productive lytic phase. The switch from viral latency to lytic replication is mediated by the replication and transcription activator protein (RTA). RTA activates KSHV lytic gene expression via direct and indirect binding to lytic promoters. Moreover, it functions as an E3 ubiquitin ligase, actively degrading repressor proteins, such as Hey1, maintaining the virus in the latent state. The first aim of this study was to determine if RTA functions as a SUMOylation targeted ubiquitin ligase (STUbL), which recognises poly-SUMOylated targets via SUMO interacting motifs (SIMs). Results presented herein demonstrate that the Hey1 repressor protein is SUMO2 modified. Furthermore, mutation of SIM domains within RTA resulted in attenuation of RTA-mediated degradation of Hey1 and lytic reactivation. However, SIM mutation also severely reduced RTA-mediated transactivation. These results suggest that RTA may have STUbL activity but additional work is required to reconcile the effect of SIM mutation upon transcriptional activity. The second aim of this investigation was to identify novel points of interaction between RTA and the host cell. In chapter 4, SILAC-based quantitative proteomics identified hundreds of proteins which demonstrated a significant change in abundance upon RTA expression. Abundance of the cellular protein ARID3B was found to increase over 7-fold in the nuclear fraction. Furthermore, ARID3B was shown to re-localise to viral replication centres upon lytic reactivation. In chapter 5 SILAC-based immunoprecipitations were performed to identify novel RTA interaction partners. The cellular co-activator RBM14 was found to be enriched in two independent SILAC data sets and subsequent investigation demonstrated that RBM14 localisation was altered upon RTA expression. These novel observations highlight the potential significance of cellular factors in KSHV infection. Further investigation is required to fully characterise the role of these proteins in viral reactivation.

Thesis (PhD)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: Renal transplantation is undoubtedly the best treatment for patients with irreversible renal failure. As a prelude to establishing the nature of malignancies in renal transplant patients we sought to determine factors influencing the outcome of renal transplantation. The survival of renal allografts and of recipients is influenced by a number of demographic, clinical and therapeutic factors. Some of these factors have been better studied than others, and we sought to establish the influence of particular factors on our own patients and allografts. The total number and nature of malignancies developing in these patients subsequent to transplantation was also established. All patients transplanted in our unit between April 1, 1976 and March 31, 1999 were included in the study. In the study period, 542 patients received 623 renal allografts. Demographic details were analysed. Patient and graft outcomes were assessed using Kaplan-Meier survival analysis. The survival curves were compared using univariate analysis; results that were significant were subjected to multivariate analysis. The influence of a number of factors on graft and patient survival were assessed and compared. The impact of a variety of variables on the number and behaviour of malignancies was also established. Patient and graft survival were superior in recipients who were aged less than 40 years; cyclosporine improved graft survival but not patient survival. Early graft loss was associated with a high patient mortality rate. Contrary to the experience elsewhere, black and white patients had similar outcomes after renal transplantation. Of the 542 recipients 41(8.1%) developed malignancies with Kaposi's sarcoma occurring, in 21 patients and skin cancers in 13 patients. The relative risk for the Kaposi's sarcoma development was 235. Kaposi's sarcoma was the most common tumour in non-white patients (accounting for 79% of malignancies in this group) and occurred less than 2 years after transplantation. Kaposi's sarcoma was equally common in male and female recipients. Under cyclosporine the latent period to malignancies was reduced but the frequency remained unaffected. Kaposi's sarcoma skin lesions were present in all the affected patients, with the lower limbs the most common site of involvement. Kaposi's sarcoma responded to reduction of immunosuppression without the need for complete discontinuation, and with preservation of renal function. Extracutaneous involvement occurred in over one quarter of the patients and invariably proved fatal in all patients with visceral organ involvement. The histopathology of posttransplant Kaposi's sarcoma was the same as that described in the other epidemiological forms of the disease. White male recipients were at the greatest risk of developing skin cancers after renal transplantation. Squamous cell carcinomas were relatively more common and were found in sun-exposed areas. The lesions were treated only by local excision and none metastasized. Malignant lymphoma, breast cancer and lung cancer occurred in individual patients but the relative risk of all these lesions were close to unity. Patients with preexisting cancers did not develop recurrences following transplantation. SECTION 2 Both immunosuppression and immunostimulation are thought to play a role in the development of Kaposi's sarcoma after renal transplantation. We investigated the quantitative and qualitative aspects of the immune system of patients who had developed Kaposi's sarcoma. The lymphocyte phenotypes were established using flow cytometry while transformation studies were performed using mitogens. Pokeweed was used as the B-cell mitogen, and concanavalin A and phytahaemagglutinin were the T-cell mitogens. Cell mediated immunity was also tested using delayed type hypersensitivity skin tests and the serum immunoglobulin levels were estimated. Firstly, with regard to humoral immunity, 2/3 of the patients had normal serum immunoglobulin levels, although the B-cell count was reduced in all the patients on immunosuppression. B-cell transformation tests with pokeweed mitogen revealed that B-cell function was not impaired in patients with Kaposi's sarcoma. The patients with decreased immunoglobulin levels also appeared to be malnourished as evidenced by low albumin levels. Secondly, CD3 and CD4, but not CD8, cell counts were reduced in patients with Kaposi's sarcoma. The transformation analyses revealed significant differences compared to controls, with reduced responses in patients with Kaposi's sarcoma. Thirdly, natural killer (NK) cell numbers were also reduced in patients with Kaposi's sarcoma. There were no significant differences in delayed type hypersensitivity skin reactions that could not be accounted for by racial differences. Cellular immunity is impaired in patients with Kaposi's sarcoma with a reduction in the number of NK cells. Both of these components of the immune system are important in protection against malignant transformation. SECTION 3 Kaposi's sarcoma is an important complication of renal transplantation. If the human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma, the virus should be present in all Kaposi's sarcoma lesions and be drastically reduced or cleared from involved tissue on remission of the Kaposi's sarcoma. Fourteen renal transplant patients with cutaneous Kaposi's sarcoma, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of Kaposi's sarcoma, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. HHV-8 DNA was detected in all the cutaneous Kaposi's sarcoma and all the visceral Kaposi's sarcoma samples, as well as a number of Kaposi's sarcoma-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-Kaposi's sarcoma cases than that found in African endemic Kaposi's sarcoma cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low-grade viral replication that coincides with clinical remission of the Kaposi's sarcoma lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant Kaposi's sarcoma. SECTION 4 The recently discovered HHV-8 is an important factor in the aetiopathogenesis of Kaposi’s sarcoma. The reason for the exceptionally high prevalence of Kaposi's sarcoma in our area, as well as that of other developing countries, remains unexplained. We investigated the seroprevalence of the virus in the different healthy subjects as well as organ donor-recipient pairs. All recipients were tested at the time of transplantation, as were the paired donors. Control subjects tested were healthy blood donors, Renal Unit staff, and household contacts of patients with Kaposi's sarcoma. An enzyme-linked immunoassay (ELISA) to the whole virus was used for screening and all positives were confirmed using ELISA to the latent ORF 73 antigen. The prevalence of HHV-8 was similar in all groups and averaged less than 6%. After transplantation the seroprevalence increased to almost 20% but neither the transplanted kidney nor blood transfused perioperatively could account for the increase. Kaposi's sarcoma developed in 3 of the 116 patients transplanted. All patients with Kaposi's sarcoma were proven to be HHV-8 seropositive before the development of the disease. Two of the patients who developed Kaposi's sarcoma were seropositive before transplantation. No patient who received a graft from a seropositive donor developed Kaposi's sarcoma. We refute the notion that a high prevalence of HHV-8 in the general population is responsible for the high prevalence of Kaposi's sarcoma in our population or that the donor organ is a major source of infection in renal transplant recipients. Reactivation, rather than primary infection appears to be the source of the virus after renal transplantation.
AFRIKAANSE OPSOMMING: Nieroorplanting is ongetwyfeld die beste behandeling vir pasiente met onomkeerbare nierversaking. As ‘n aanloop om die aard van maligniteite in nierooorplantingspas'fente vas te stel het ons gepoog om die faktore wat die uitkoms van nieroorplantings bemvloed te bepaal. Die oorlewing van oorgeplante niere en van nierontvangers word deur ‘n aantal demografiese, kliniese en terapeutiese faktore bemvloed. Sommige van hierdie faktore is beter ondersoek as ander and ons het gepoog om die invloed van sekere faktore op ons eie pasiente en oorgeplante niere te bepaal. Die getal en aard van maligniteite wat ontwikkel het in hierdie pasiente na nieroorplanting is ook gedokumenteer. Alle pasiente in ons eenheid in wie ‘n nier tussen 1 April 1976 en 31 Maart 1999 oorgeplant was, is in die studie ingesluit. Tydens die studieperiode het 542 pasiente 623 niere ontvang. Demografiese detail is ontleed. Pasient- en nieroorplantings uitkomste is beraam deur gebruik te maak van Kaplan-Meier oorlewing analiese. Die oorlewingskurwes is vergelyk deur gebruik te maak van enkelveranderlike ontledings; noemenswardige resultate is onderwerp aan meerveranderlike ontledings. Die invloed van ‘n aantal faktore op oorgeplante nier- en pasientoorlewing is ondersoek en vergelyk. Die impak van ‘n verskeidenheid veranderlikes op die getal en gedrag van maligniteite is ook ondersoek. Pasient oorlewing asook oorlewing van oorgeplante niere was beter in ontvangers onder die ouderdom van veertig jaar. Vroee verlies van ‘n oorgeplante nier het verband gehou met ‘n hoe pasientmortaliteit. Siklosporien het die oorlewing van oorgeplante niere verbeter, maar nie die van pasiente nie. In teenstelling met die ervaring elders, het swart en wit pasiente soortgelyke uitkomste uitkoms gehad na ‘n nieroorplanting. Van die 542 ontvangers, het 41 (8.1%) maligniteite ontwikkel; Kaposi se sarkoom het in 21 pasiente voorgekom en velkanker in 13 pasiente. Die relatiewe risiko (“relative risk") vir die ontwikkeling van Kaposi se sarkoom was 235. Kaposi se sarkoom was die algemeenste tumor in swart en gekleurde pasiente (verantwoordelik vir 79% van maligniteite in die groep) en het binne twee jaar voorgekom. Kaposi se sarkoom was ewe algemeen in manlike en vroulike ontvangers. Met behandeling deur middel van siklosporien het die latente periode totdat maligniteite ontwikkel het verkort, maar die insidensie daarvan het onveranderd gebly. Velletsels geassosieer met Kaposi se sarkoom was teenwoordig in alle pasiente met die vel van die onderste ledemate die mees algemeen betrokke ligging. Die sarkoom het gereageer op vermindering van immuunonderdrukking, sonder die nodigheid vir volkome onttrekking, en met die bewaring van nierfunksie. Ekstrakutane betrokkenheid het in meer as ‘n kwart van die pasiente voorgekom en was altyd noodlottig in pasiente met viserale aantasting. Die histopatologie van postoorplanting Kaposi se sarkoom was dieselfde as die wat beskryf is vir die ander epidemiologiese vorms van die siekte. Wit mans het die hoogste risiko vir die ontwikkeling van velkankers na nieroorplanting gehad. Plaveiselsel karsinoom was betreklik meer algemeen en het in son-blootgestelde areas voorgekom. Die letsels was uitsluitlik met lokale eksisie behandel en geen pasiente het metastases ontwikkel nie. Maligne limfoom, borskanker, en longkanker het in enkele pasiente voorgekom maar die relatiewe risiko van al die letsels was om en by een gewees. Nie een van die pasiente met vorige maligniteite het herhaling van die tumore na oorplanting ontwikkel nie. AFDELING 2 Die vermoede is dat beide immuunonderdrukking en immuunstimulasie ‘n rol speel in die ontwikkeling van Kaposi se sarkoom na ‘n nieroorplanting. Ons het die kwantitiewe en kwalitatiewe aspekte van die immuunsisteem van pasiente wat Kaposi se sarkoom ontwikkel het na ‘n nieroorplanting, ondersoek . Limfosiet fenotipes is met behulp van vloeisitometrie bepaal, terwyl transformasiestudies uitgevoer is deur gebruik te maak van mitogene. “Pokeweed” is gebruik as die B-sel mitogeen, en konkanavalien A en fitaheemagglutinien was die T-sel mitogene. Sel-gemedieerde immuniteit was ook getoets deur die gebruik van vertraagde tipe hipersensitiwiteit veltoetse. Die serum immunoglobulien vlakke was ook bepaal. Eerstens, met betrekking tot humorale immuniteit, het 2/3 van die pasiente normale serum immunoglobulienvlakke gehad, alhoewel die B-seltelling verminder was in al die pasiente op immuunonderdrukking. B-seltransformasie-toetse met “pokeweed” mitogeen het getoon dat B-sel funksie nie ingekort was in pasiente met Kaposi se sarkoom nie. Die pasiente met verminderde serum immunoglobulinvlakke het ook wangevoed voorgekom soos die verlaagde serum albumienvlakke uitgewys het. Tweedens was CD3 en CD4 seltellings, maar nie CD8 nie, verlaag in pasiente met Kaposi se sarkoom. Betekenisvolle verskille is ook aangetoon met T-sel transformasietoetse in vergelyking met kontroles, met verminderde response in Kaposi se sarkoom pasiente. Derdens was natuurlike dodersel (NK) getalle ook minder in pasiente met Kaposi se sarkoom. Daar was geen noemenswaardige verskille in vetraagde tipe hipersensitiwiteit velreaksies wat nie deur rasseverskille kon verklaar word nie. Sellulere immuniteit is ingekort in pasiente met Kaposi se sarkoom met ‘n verlaging in die aantal NK selle. Beide die komponente van die immuunstelsel is belangrik vir beskerming teen maligne transformasie. AFDELING 3 Kaposi se sarkoom is ‘n belangrike komplikasie van nieroorplanting. As die menslike herpesvirus-8 (HHV-8) Kaposi se sarkoom veroorsaak, behoort die virus teenwoordig te wees in alle letsels en as Kaposi se sarkoom remissie ondergaan behoort dit drasties te verminder of te verdwyn in weefsel waarin dit voorkom. Veertien nieroorplantingspasiente met Kaposi se sarkoom van die vel, insluitend outopsiemateriaal van twee gevalle, is ondersoek vir die teenwoordigheid van HHV- 8. ‘n Tweede velbiopsie van dieselfde anatomiese area as die eerste is uitgevoer op 11 oorlewende pasiente na remissie van die sarkoom. Remissie was deur die vermindering of onttrekking van immuunonderdrukking bewerkstellig. ‘n Perifere bloedmonster is by dieselfde geleentheid as die tweede biopsie geneem. ‘n Geneste polimerase kettingreaksietoets (“nested polymerase chain reaction”) is gebruik om die teenwoordigheid van HHV-8 DNA in die biopsieweefsel en perifere bloed mononukluere selle te bepaal, gevolg deur direkte volgordebepaling (“sequencing”) van die polimerase kettingreaksieproduk om enige nukleotiedveranderings te dokumenteer. HHV-8 DNA is waargeneem in al die kutane Kaposi se sarkoom en al die viserale Kaposi se sarkoom monsters, sowel as in weefsel waar die Kaposi se sarkoom nie voorgekom het nie en waar die teenwoordigheid van die virus nie tevore beskryf is nie, soos die skilklier, speekselklier, en hartspier. Mutasies in die virale DNA kon in alle pasiente aangetoon word. Die mutasies wat gevind is, was nader verwant aan die wat in VIGS-Kaposi se sarkoom beskryf is as die wat in endemiese Kaposi se sarkoom in Afrika gevind word. HHV-8 volgordes kon waargeneem word in bevrore opvolg-velbiopsies van vyf pasiente, maar was afwesig in die ekwivalente formaliengefikseerde monsters. Virus DNA is ook waargeneem in 2 van 11 perifere bloed mononukluere selmonsters wat versamel is tydens die opvolg velbiopsies. Vermindering of onttrekking van immuunonderdrukking laat die immuunsisteem toe om genoegsaam te herstel om virale replikasie te verminder met daaropvolgende teenwoordigheid en laegraadse virale replikasie wat ooreenstem met kliniese remissie van letsels van Kaposi se sarkoom. Dit verskaf verdere bewyse van die belangrike oorsaaklike rol wat deur HHV-8 gespeel word in die patogenese van postoorplanting Kaposi se sarkoom. AFDELING 4 Die onlangs-ontdekte HHV-8 is ‘n belangrike faktor in die etiopatogenese van Kaposi se sarkoom. Die rede vir die buitengewone hoe prevalensie in ons gebied, sowel as die van ander ontwikkelende lande, is nie voor die hand liggend nie. Ons het die seroprevalensie van die virus in verskillende gesonde persone, sowel as orgaan donor-ontvanger pare ondersoek. Alle nierontvangers en hul gepaarde donors is getoets ten tye van die nieroorplanting. Getoetste kontrole persone was gesonde bloedskenkers, niereenheid personeel en huishoudlike kontakte van pasiente met vorige Kaposi se sarkoom. ‘n Ensiemgekoppelde immuuntoets (enzyme-linked immnoassay; ELISA) vir die volledige virus was gebruik vir sifting en bevestiging is verkry vir alle positiewe toetse deur gebruik te maak van van ‘n ELISA vir die latente ORF 73 antigeen. Die prevalensie van HHV-8 was vergelykbaar in alle groepe en was gemiddeld minder as 6%. Na oorplanting het die prevalensie gestyg tot byna 20%, maar nog die oorgeplante nier nog perioperatiewe bloedoortappings kom die styging verklaar. Kaposi se sarkoom het in 3 van 116 van die pasiente wat ‘n oorplanting ondergaan het, ontwikkel. Al die pasiente met Kaposi se sarkoom was HHV-8 seropositief voor die ontwikkeling van die Kaposi se sarkoom. Twee van die pasiente wat Kaposi se sarkoom ontwikkel het was seropositief voor die oorplanting. Geen pasient wat ‘n nier van ‘n seropositiewe donor ontvang het, het Kaposi se sarkoom ontwikkel nie. Ons weerle die stelling dat ‘n hoe prevalensie van HHV-8 in die algemene bevolking verantwoordelik is vir die hoe prevalensie van Kaposi se sarkoom onder ons nieroorplantingspasiente of dat die donornier ’n belangrike bron van infeksie is. Dit wil voorkom of heraktivering, eerder as primere infeksie, ‘n bron van die virus is na nieroorplanting.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus that is the etiologic agent of Kaposi sarcoma (KS). KS is an angioproliferative neoplasm composed of cells of endothelial origin. For the work described in this thesis, KSHV infection of endothelial cells was used as a tractable model to study the role of microRNAs (miRNAs) in endothelial cell biology. Previous work in the laboratory had identified miRNAs which are either upregulated or downregulated upon KSHV infection of lymphatic endothelial cells (LEC). Target prediction analysis of these miRNAs and cross-comparison of predicted targets with expression levels of pro- and anti-angiogenic genes following KSHV infection, revealed the predicted targeting of Delta-like 4 (DLL4) by the miR-30 family. DLL4 is significantly upregulated in KSHV-infected lymphatic endothelial cells (KLEC) whereas the miR-30 family is significantly downregulated. DLL4 is a membrane-bound ligand belonging to the Notch signalling family that plays a fundamental role in vascular development and angiogenesis. Targeting of DLL4 by miR-30b and miR-30c was confirmed by examining mRNA and protein expression following transfection of endothelial cells with miR-30 mimics and inhibitors or infection with miR-30-expressing lentiviruses. The exact target site within the DLL4 3’UTR was identified using a luciferase reporter assay and site-directed mutagenesis. Overexpression of miR-30b in endothelial cells led to increased vessel number and length in an in vitro model of sprouting angiogenesis. Microinjection of miR-30 into zebrafish embryos resulted in suppression of dll4 and subsequent excessive sprouting of intersegmental vessels and reduction in dorsal aorta diameter. Use of a target protector against the miR-30 site within the dll4 3’UTR upregulated dll4 and synergised with Vegfa signalling knockdown to inhibit angiogenesis. Furthermore, restoration of miR-30b or miR-30c expression during KSHV infection attenuated viral induction of DLL4. Overall, the work presented in this thesis demonstrates that the miR-30 family targets DLL4 to regulate angiogenesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contribute to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFκB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. The aims of this study were to confirm the previously identified 252-nt fragment directly upstream of vFLIP as the location of the vFLIP IRES in cellulo and to determine the structure and mechanism of action of the vFLIP IRES. Here we show that a 252-nt, within a coding region, directs translation in HEK293 cells. We have also established its RNA structure using chemical and enzymatic probing of RNA structure in solution and mutational analysis studies revealed that the domain If of the vFLIP IRES is crucial for its activity. Also, we demonstrate that IRES activity requires the presence of eIF4A and the eIF4E-eIF4G interaction. These interactions may define a new paradigm for IRES-mediated translation. Finally, we attempted to identify cellular proteins that may interact with the vFLIP IRES using several types of protein affinity chromatography, but we could detect a protein interacting with vFLIP IRES but yet to be confirmed.