L'Herpesvirus humà 8 (HVH-8) és l'agent etiològic del Sarcoma de Kaposi (SK). Aquesta neoplàsia endotelial multifocal és la primera causa de neoplàsia en pacients de sida. En països on l'HVH-8 no és endèmic (nord d'Europa, EUA), el SK afecta sobretot a homes homo o bisexuals (MSM) VIH positius, entre els quals el virus es transmet sexualment, mentre que en països endèmics (Àfrica central) representa la primera neoplàsia tant en adults com en nens, i el virus es transmet a través de la saliva. Pocs estudis han avaluat la presència del virus en nens en zones no endèmiques. L'HVH-8 s'ha detectat per PCR en cèl·lules mononuclears de sang perifèrica (PBMC), però només en el 50% dels casos de SK associat a la sida. Respecte la detecció de la infecció per l'HVH-8, aquesta tesis es planteja dos objectius; 1) determinar, mitjançant mètodes serològics, quin és l'abast de la infecció per l'HVH-8 en zones no endèmiques durant les dues primeres dècades de vida, per tal de determinar si la infecció primària té lloc durant aquesta etapa en comparació amb el virus d'Epstein-Barr (VEB), y 2) estudiar, mitjançant la detecció del propi virus, quin és l'abast de la virèmia en individus seropositius per l'HVH-8 i el VIH, i el paper d'aquesta en la patogènia del SK. Els resultats indiquen que en zones no endèmiques (Alemanya i Geòrgia, EUA), la taxa de seroprevalença de l'HVH-8 fins els 17 anys d'edat és significativament menor a la observada en zones endèmiques (Nigèria). En comparació amb el VEB, el virus més proper filogenèticament i de transmissió horitzontal, l'HVH-8 no es transmet tan eficientment a través de la saliva. Tot i així, la infecció primària pot tenir lloc durant la infància també en zones no endèmiques, on els anticossos poden no persistir a llarg termini. La tècnica d'immunofluorescència permet detectar millor la infecció per l'HVH-8 que els ELISA basats en pèptids. Per a la segona part de la tesi va ser necessari desenvolupar un assaig de dilucions limitants i una PCR dúplex en temps real (co-quantificació del genoma víric i del nombre de PBMC analitzades) que va permetre demostrar que la proporció de cèl·lules infectades per l'HVH-8 en sang és relativament baixa, fins i tot en pacients amb SK actiu (fins a menys d'una en 6 milions), tot i que pot canviar durant el curs del SK. En les neoplàssies associades al VEB aquesta proporció sol ser major. L'HVH-8 és més fàcilment detectable en les PBMC que en el plasma. Tot i que la detecció de l'HVH-8 ha estat més habitual entre pacients amb SK, la presència de lesions no sempre va acompanyada de la detecció del virus en sang, on el virus podria estar per sota el límit de detecció. El virus pot infectar latentement els limfòcits B però també dur a terme el cicle lític en un percentatge de les PBMC (amb alts nombres de còpies del genoma de l'HVH-8 per cèl·lula infectada i presència del virus en plasma); les PBMC líticament infectades poden contribuir a la disseminació del virus cap a dianes endotelials en la patogènesi del SK. La latència en els limfòcits B in vivo i en cèl·lules de limfoma transformades per l'HVH-8 (BCBL-1) podria estar regulada de manera diferent, com és el cas dels diferents tipus de latència del VEB. Alguns estudis publicats han subestimat la presència de l'HVH-8 en sang. En aquesta tesi s'han establert les condicions més adequades de mostreig i d'anàlisi de mostres de sang per obtenir uns resultats fiables, i es proposa una nova metodologia que permetrà aprofundir en l'estudi detallat de la virèmia per l'HVH-8 i la seva rellevància en la patogènia del SK y d'altres malalties associades a aquest virus. / Human Herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's Sarcoma (KS). This multifocal neoplasm has an endothelial origin and is the first cause of cancer in aids patients. In countries where HHV-8 is not endemic (Northern Europe, USA), KS is mostly found in homo or bisexual men (MSM) who are HIV-positive, among whom the virus is transmitted sexually. On the other hand, in endemic countries (central Africa) KS is the most common neoplasm both in adults and in children, and the virus is spread through saliva. Few studies have assessed HHV-8 acquisition by children in non-endemic areas. By PCR, the HHV-8 genome has been detected in peripheral blood mononuclear cells (PBMC), but only in 50% of aids-KS patients. Focused on detection of HHV-8 infection, this thesis has two main objectives; 1) to determine through serologic methods, the extent of HHV-8 infection among children and adolescents in non-endemic areas in order to determine when the primary infection is occurring and whether the virus is transmitted horizontally in comparison with Epstein-Barr virus (EBV), and 2) to assess through the detection of the virus genome, the extent of HHV-8 viremia in individuals that are seropositive for both HHV-8 and HIV, and what is the role of HHV-8 viremia in KS pathogenesis. Our results show that in non-endemic areas (Germany and Georgia, USA), HHV-8 seroprevalence until 17 years of age is significantly lower than that observed in endemic areas (Nigeria). HHV-8 is not as efficiently transmitted though saliva as EBV, which is the most closely related virus. However, HHV-8 primary infection can occur during infancy and adolescence in non-endemic areas, where specific antibodies may not persist long term. The immunofluorescence assay performed better than two peptide-based ELISA at the detection of HHV-8 infection. To accomplish the second objective it was necessary to develop a limiting dilution assay in combination with a duplex real time PCR (co-quantification of the HHV-8 genome and the number of PBMC tested). Using this method we were able to demonstrate that the fraction of HHV-8 infected blood cells is relatively low, even in patients with active KS (down to less than one in 6 million PBMC), although it can change over time. The frequency of infected cells is usually higher in EBV-related malignancies. HHV-8 was mostly detected in PBMC and not so common in plasma. Although the detection of HHV-8 was more common among aids patients, lesions are not always associated to virus detection in blood, where it could be below the threshold of detection. The virus can latently infect B lymphocytes but also undergo lytic replication in a percentage of the PBMC (with high copy numbers of the HHV-8 genome per infected cell and free virus in plasma); lytically infected PBMC may contribute to the spread of infectious virus to endothelial targets promoting KS pathogenesis. HHV-8 latency in B cells in vivo and in B cells transformed by HHV-8 (BCBL-1) could be regulated differently, perhaps analogous to the several types of EBV latency. The prevalence of the virus in blood cells has been underestimated in previous studies. In this thesis, the most adequate sampling and analysis protocols are proposed so that the virus can be detected reliably. The methods developed in this thesis will be of value in monitoring HHV-8 viremia and establish its relevance in KS pathogenesis, as well as in other HHV-8 associated diseases.

The common type of myxoid soft tissue sarcomas is myxofibrosarcoma. Clinically, increased tumor grading and staging are frequently observed in myxofibrosarcomas after relentless local recurrences, which may eventually lead to metastatic diseases. However, metastatic myxofibrosarcomas are often refractory to current treatment strategies and constitute the primary cause of sarcoma-related death. Immunohistochemistry staining was applied to analyze myxoid tumors of soft tissue in our previous studies, and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) was identified to be significantly upregulated in myxoid soft tissue sarcomas. The PAK1 is a pivotal serine/threonine kinase, which integrates stimuli from various signaling pathways to regulate cell survival, mitosis and cytoskeletal remodeling, etc. We first examined the endogenous PAK1 mRNA and total PAK1 protein levels in various myxoid sarcoma cell lines, including OH931, NMFH1 and NMFH2. This initial screening detected that upregulated PAK1 expression in OH931 and NMFH1, whereas downregulated PAK1 in NMFH2 cells. By wound healing and matrigel transwell assay, we further found that transfection of the expression plasmid carrying wild-type PAK1 gene or PAK1 T423E mutant promoted cell migration and invasion abilities in NMFH2 cells. On the other hand, knockdown of the PAK1 gene by short hairpin RNA interference inhibited the migration rate and invasion ability in NMFH1 cells. By 5-bromo-2-deoxyuridine assay and colony formation assay, we found that either exogenous expression of PAK1 protein or knockdown of PAK1 gene affected cell proliferation and transformation. Interestingly, immunofluorescence demonstrated that treatment with hepatocyte growth factor induced phosphorylation of PAK1 (Thr212) and promoted its nuclear import in NMFH2 cells. In summary, PAK1 plays oncogenic roles in myxoid sarcoma carcinogenesis.

Background: Surgical disease related to HIV is scantily documented with a paucity of data detailing the manifestations of HIV in surgery especially in resource-poor, high prevalence settings such as in South Africa. This review provides an update on the topical issues surrounding HIV and surgery. Objectives: The objective of the study was to determine the incidence, pathogenesis, clinical presentation, aspects of diagnosis and management of: HIV- associated salivary gland disease in particular parotid gland enlargement; Kaposi’s sarcoma (KS) and lower limb lymphoedema; AIDS- related abdominal malignancies due to KS and lymphoma; Acalculous cholecystitis and HIV- cholangiopathy and HIV- associated vasculopathy. Methods: A collective review of the literature was performed and data sourced from a search of relevant electronic medical databases for literature from the period 2000 to the present date. Studies under each section were selected based on inclusion and exclusion criteria. Content analysis was used to analyse data. Results: The HIV pandemic has resulted in an increased frequency of benign lymphoepithelial cysts making it the commonest cause of parotidomegaly in most surgical practices. KS should be considered in the differential diagnosis of a patient with chronic lymphoedema. Lymphoedema may be present without cutaneous lesions, making clinical diagnosis of KS difficult. The gastrointestinal tract is the commonest site of extra- cutaneous KS. Surgical management of the lymphoma patient is restricted nowadays to determining the diagnosis and in some cases to evaluate disease stage. Highly active antiretroviral therapy (HAART) is an important part of the management of biliary tract conditions in addition to relevant surgical procedures. HIV- vasculopathy represents a distinct clinico- pathological entity characterized by a vasculitis with probable immune- mediated or direct HIV- related injury to the vessel wall. Conclusion: The rising incidence of HIV in South Africa and other developing countries has been associated with new and unusual disease manifestations requiring surgical management for diagnostic, palliative or curative intent. It is crucial that surgeons remain abreast of new developments related to the challenging spectrum of HIV and its protean manifestations. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

Soft tissue sarcomas account for about 1% of all malignancies. The increase in incidence of soft tissue sarcomas during the recent decades may predominantly be attributed to AIDS-related Kaposi’s sarcoma; when this tumor is excluded, conclusive evidence for an age-adjusted increase is lacking. Beside the well investigated role of the human immunodeficiency virus 1 (HIV-1) and the human herpesvirus 8 (HHV-8) in the tumorigenesis of AIDS-related Kaposi’s sarcoma and several inherited disorders, considerable evidence support a relationship between occupational chemicals as vinyl chloride, phenoxyacetic acid herbicides, chlorphenols, dioxin, medicinal measures as Thorotrast exposure and therapeutic irradiation, and the development of soft tissue sarcoma. Hormones and chronic repair processes are further probably sarcoma-promoting factors. Considering the rarity of soft tissue sarcomas despite the vast portion that soft tissues comprise in the human body, additional knowledge on the tumorigenesis of soft tissue sarcomas might considerably contribute to the understanding of the etiologic pathways of malignant tumors in humans. / Weichteilsarkome stellen etwa 1% aller bösartigen Neubildungen. Der in den vergangenen Jahrzehnten beobachtete Inzidenzanstieg geht fast ausschließlich auf die rasante Zunahme an AIDS-assoziierten Kaposi-Sarkomen zurück. Bei Außerachtlassung dieses Tumors gibt es bisher keinen schlüssigen Beweis für eine wirkliche alterskorrigierte Häufigkeitszunahme der Weichteilsarkome. Neben der gut untersuchten Rolle des HIV-1-Virus und des humanen Herpes-Virus 8 bei der Entstehung des AIDS-assoziierten Kaposi-Sarkoms und einigen prädisponierenden genetischen Erkrankungen existieren starke Hinweise für einen Zusammenhang zwischen Industriegiften wie Vinylchlorid, Phenoxyessigsäure-Herbiziden, Chlorphenolen, Dioxinen, medizinischen Maßnahmen wie therapeutischer Bestrahlung oder dem Einsatz von Thorotrast, und der Entwicklung von Weichteilsarkomen. Hormone und chronische Reparaturprozesse sind weitere wahrscheinlich fördernde Einflüsse auf die Entstehung von Weichteilsarkomen. Die Tatsache, daß trotz des großen Anteils, den die Binde- und Stützgewebe an der Körpermasse stellen, nur selten maligne Tumoren von diesen Strukturen ausgehen, läßt hoffen, daß ein besseres Verständnis der an der Kanzerogenese von Weichteilsarkomen beteiligten Mechanismen in der Zukunft wichtige Erkenntnisse über die Entstehung menschlicher Tumoren liefern kann. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The Snail1 transcriptional repressor is a key factor responsible in triggering epithelial to mesenchymal transition. Although Snail1 is widely expressed in early development, it is limited in adult animals to a subset of mesenchymal cells where it has a largely unknown function. In this project we have demonstrated that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to TGF-[beta]1 which showed a strong Snail1 dependence. Snail1-depletion in conditional knock-out adult animals caused a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiated to osteoblasts or adipocytes and, in contrast to controls, were resistant to the TGF-[beta]1-induced differentiation block. TGF-[beta]1 was unable to up-regulate most of its targets in Snail1 KO MSCs, an effect that was related, but not limited, to defective PTEN repression and Akt activation. Correspondingly, an analysis of human sarcomas also showed enhanced expression of Snail1 in undifferentiated tumors, which was strongly associated with high expression of TGF-[beta] and poor outcome. These results not only demonstrate a new role for Snail1 in TGF-[beta] response and MSC maintenance but also suggest the involvement of MSCs in sarcoma generation. / El repressor transcripcional Snail1 ha estat descrit principalment com el responsable de la inducció de la transició epiteli mesènquima. Encara que Snail1 s’expressa durant les etapes més primerenques del desenvolupament embrionari, la seva expressió en adults es veu limitada en un conjunt de cèl•lules mesenquimals sense saber-se la seva funció. En aquest projecte hem demostrat que Snail1 es requereix per mantenir el fenotip més indiferenciat de les cèl•lules mare del mesènquima. Aquesta funció la fa en part, per la capacitat de resposta de la citoquina TGF-[beta] la qual mostra una força dependència amb Snail1. Quan s’elimina Snail1 en ratolins adults provoca una clara disminució en el nombre de cèl•lules mare de la medul•la òssia. Aquestes cèl•lules en cultiu presenten una clara diferenciació prematura a osteoblasts i adipòcits. Pel contrari, tractaments amb TGF-[beta]1 aturen la diferenciació. El TGF-[beta]1 es incapaç de incrementar moltes dianes en cèl•lules mare del mesènquima aïllades del ratolí deficient per snail1, aquest efecte en part es degut a la repressió de PTEN i l’activació de AKT. L’anàlisi de sarcomes humans ens ha mostrat una alta expressió de Snail1, el qual també es troba associada amb una alta expressió de TGF-[beta] i baixa supervivència. Aquests resultats no només demostren una nova funció per Snail1 en resposta a TGF-[beta] i el manteniment de les MSC, sinó que també suggereix que Snail1 podria participar en la generació del sarcoma.

Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.

Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.

Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Thesis (Ph.D.)--Ohio University, November, 2004. / Includes bibliographical references (leaves 115-128)