Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 127-137. Available online via the Internet.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The Y402H polymorphism in complement factor H (CFH) is a common and important risk factor, where CFH is an inhibitor of the alternative complement pathway. The disease-associated protein variant (CFH402H) binds poorly to aged human macular Bruch’s membrane (BM), a site of AMD formation. Heparan sulphate (HS) is the major binding site for CFH in this extracellular matrix. Unlike CFH402Y, CFH402H binds poorly to lowly sulphated HS. The aim of this research was to investigate the presence and distribution of proteoglycan (PG) core proteins and glycosaminoglycans (GAGs) in the normal adult human macula, and to analyse potential changes with age in the quantity and composition of HS and other potential molecular determinants of disease in BM. Post mortem human eye tissue was obtained from consenting donors (age range 18-93 years), and either dissected into tissue layers or used to produce frozen macular tissue sections. Proteomic analysis of different retinal tissue layers was performed by tandem mass spectrometry. Immunofluorescence microscopy was undertaken on the macular tissue sections. Compositional analysis of HS in BM was performed by 2-aminoacridone labelling of HS disaccharides and reverse phase high performance liquid chromatography against reference HS disaccharide standards. PG core proteins were identified in BM and other macular tissue layers, including members of the basement membrane, hyalectan and short leucine-rich repeat PG families. HS, chondroitin sulphate, dermatan sulphate and hyaluronan were present throughout the retina and choroid, but keratan sulphate only in the sclera. The mean quantity of HS in BM was 47% lower (p=0.006) in old donors (n=13, 64-92 years), compared to young donors (n=6; 26-39 years). The mean level of HS sulphation was also lower in old donors, e.g. 34% vs. 39% (p=0.02) N-sulphated HS. The mean level of HS in macular BM by immunohistochemistry was approximately 50% lower (p=0.02) in old donors (n=10, 18-93 years), and the mean level of the HS PG core protein perlecan was reduced by 85% (p=0.01; n=18, 27-90 years). High levels of complement activation (C3b and membrane attack complex) were observed in some young donors. Reduced HS was associated with increased complement activation in some donors (r2 0.30). A combination of proteomics and immunohistochemistry approaches has provided the first comprehensive analysis of the presence and distribution of PG core proteins and their associated GAG chains throughout the macular layers of the normal adult human retina. These demonstrate a differential distribution according to PG core protein, GAG class and GAG sulphation state. The quantity of HS decreases substantially with age in human BM, and its sulphation level also decreases. The presence of less HS in old BM would make fewer binding sites available for CFH, and could contribute to AMD pathogenesis through increased complement activation. This idea is supported by the observation that reduced HS is associated in some individuals with increased C3b in BM. These findings have important implications for unravelling mechanisms of ocular disease and planning novel therapeutic strategies, particularly in the case of AMD.

C1QTNF5 is a 25kDa short chain collagen of unknown function which is mutated in late-onset retinal macular degeneration (L-ORMD). L-ORMD is an autosomal dominant disease characterised by sub-retinal pigment epithelial deposits leading to photoreceptor death and visual loss and shows several similarities to age-related macular degeneration (AMD). A Tyr402His polymorphism in complement factor H (CFH), a regulatory protein in the innate immune system, has been associated with increased risk of AMD. C1QTNF5 and CFH are both expressed and secreted by the retinal pigment epithelium (RPE) which supports photoreceptors and is responsible for phagocytosis of shed rod photoreceptor outer segments (ROS). The properties of the normal C1QTNF5 and disease-associated Ser163Arg mutation were examined in detail, including protein characterisation, cellular processing and function. Recombinant wild type and mutant C1QTNF5 were produced and their multimerisation and solubility functions compared. Both proteins were found to be soluble and to form similar multimeric species which were resistant to reducing conditions, as seen in other short chain collagens. Due to the similarities between LORMD and AMD, a proposed interaction between C1QTNF5 and CFH was investigated. CFH is composed of 20 short consensus repeats (SCR) and interactions were confirmed between C1QTNF5 and both CFH and SCR modules 7-8 and 19-20. CFH showed a greater affinity for mutant C1QTNF5 compared with wild type on the basis of surface plasmon resonance assays. Stably transfected RPE-derived cell lines were created which expressed either wild type or mutant C1QTNF5. Both proteins were found to be secreted and showed similar cellular processing with no evidence of aggregation or retention of the mutant protein within the endoplasmic reticulum. In order to investigate C1QTNF5 function, phagocytosis of ROS by the stably transfected cell lines was carried out. Cells expressing wild type C1QTNF5 showed greater ROS phagocytosis compared with mutant C1QTNF5-expressing or untransfected cells. Addition of anti-C1QTNF5 antibody increased ROS phagocytosis further. In summary, it is proposed that wild type and mutant C1QTNF5 are secreted by the RPE where they interact with CFH. C1QTNF5 is also shown to have a role in ROS phagocytosis, with mutation in C1QTNF5 affecting phagocytosis efficiency, which may contribute to sub-RPE deposit formation. The results suggest that CFH may also be involved in this process, suggesting a common pathogenic pathway between L-ORMD and AMD.

<p>Age-related macular degeneration is one of the leading causes of vision loss in the world. While identification of various environmental risk factors including but not limited to smoking, ethnicity, and diet have been reported to contribute to the complex etiology of AMD, age and genetics remain the largest susceptibility factors in its pathogenesis. Initially, with the identification of the common Y402H variant in CFH, approximately 35% of the genetic determinants of AMD had been identified with the majority remaining unknown. Therefore, we set forth to A) identify additional AMD susceptibility genes that contribute to AMD through the use to next generation sequencing technologies and B) to assess associated alleles for pathogenicity in the attempt to interpret their functional contributions to AMD outcome as observed via patient serum and zebrafish analysis. In doing such, we have identified both common and rare variants that contribute to the heritability of AMD. Additionally, we report one of the first instances of a rare variant significantly increasing disease onset and a gene with increased rare mutational burden in AMD patients. All together adding to our understanding of the genetics of AMD and potentially leading to putative therapeutic targets.</p> / Dissertation

Ng, Tsz Kin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 151-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (M.P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Masters Abstracts International, Volume: 46-06, page: 3221. Adviser: Eric Boerwinkle. Includes bibliographical references.

Orientador: José Paulo Cabral de Vasconcellos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T03:32:34Z (GMT). No. of bitstreams: 1 Medina_FlavioMacCord_D.pdf: 2571030 bytes, checksum: 442e6e86f7bd3a0d6fae088740a1e85e (MD5) Previous issue date: 2013 / Resumo: Introdução: O fator de complemento H (CFH) é um componente do sistema imunológico que possui ação imunomoduladora sobre a resposta inflamatória. A gravidade da degeneração macular relacionada à idade (DMRI) é determinada em parte por um estado inflamatório sustentado por atividade aberrante da via alternativa do complemento. As evidências na literatura da relação entre o polimorfismo Y402H do gene CFH e a resposta ao tratamento da DMRI exsudativa permanecem controversas. Objetivo: Avaliar a associação entre as variantes do polimorfismo Y402H do gene CFH e os efeitos funcional e morfológico a curto prazo, assim como a evolução a longo prazo, dos antiangiogênicos em pacientes com DMRI exsudativa. Métodos: Vinte e cinco pacientes recém diagnosticados com DMRI exsudativa foram avaliados em um estudo de curto prazo com acuidade visual medida pela tabela do ETDRS e espessura retiniana central por tomografia de coerência óptica (OCT) de alta resolução, submetidos a injeção intravítrea de bevacizumabe e prospectivamente reexaminados em 7 e 28 dias. Quarenta e seis pacientes previamente submetidos ao tratamento com antiangiogênicos tiveram seus prontuários e exames retrospectivamente avaliados em um estudo de longo prazo quanto às evoluções funcional e morfológica ao longo de um ano. Esses parâmetros foram comparados com o genótipo do CFH, cuja análise molecular do polimorfismo Y402H foi realizada por meio da reação em cadeia da polimerase (PCR) e sequenciamento direto. Resultados: No estudo de curto prazo, houve melhora da acuidade visual no dia 28 em relação ao valor inicial (D0 vs. D28) em todos os genótipos. Entretanto, no grupo homozigoto para o alelo de risco (CC), ocorreu diferença apenas no dia 28 em relação ao dia 7 (D7 vs. D28), enquanto nos grupos CT e TT, a acuidade visual melhorou mais precocemente, no dia 7 em relação ao valor inicial (D0 vs. D7). A espessura retiniana central apresentou redução nos grupos CT (D0 vs. D7 e D0 vs. D28) e TT (D0 vs. D28), enquanto não houve mudança significativa no grupo CC. No estudo de longo prazo, foi evidenciada melhora da acuidade visual ao longo de um ano de acompanhamento apenas no grupo de pacientes sem o alelo C, sem diferença significativa no grupo de pacientes com o alelo de risco. A espessura retiniana central apresentou redução nos genótipos CT e TT, enquanto que no grupo CC não houve significância. Número de injeções, persistência de atividade neovascular e percepção subjetiva de melhora não diferiram entre os genótipos. Conclusão: O perfil de genótipo do CFH parece influenciar o efeito funcional e morfológico da injeção intravítrea de bevacizumabe com uma ação mais precoce em pacientes sem o genótipo de risco. A presença do alelo de risco parece estar relacionada à ausência de melhora visual ao longo de um ano de tratamento com inibidores do VEGF. Esses resultados sugerem que o perfil do genótipo do CFH possa exercer efeito farmacogenético nesse grupo de pacientes brasileiros, influenciando negativamente a resposta ao tratamento da DMRI exsudativa com antiangiogênicos / Abstract: Introduction: The complement factor H (CFH) is a component of the immune system that has immunomodulatory action on the inflammatory response. The severity of age-related macular degeneration (AMD) is determined in part by an inflammatory state sustained by aberrant activity of the alternative complement pathway. Evidences in the literature of the relationship between the Y402H polymorphism of CFH gene and response to treatment of wet AMD remain controversial. Purpose: To evaluate the association between variants of the Y402H polymorphism of CFH gene polymorphism and the short-term functional and morphological effects, as well as long-term evolution, of antiangiogenic drugs in patients with exudative AMD. Methods: Twenty-five patients with newly diagnosed exudative AMD were evaluated in a short-term study with visual acuity on ETDRS chart and central retinal thickness measured with high resolution optical coherence tomography (OCT), underwent intravitreal injection of bevacizumab and were prospectively reviewed in 7 and 28 days. Forty-six patients previously submitted to treatment with VEGF inhibitors had their medical charts retrospectively evaluated in a long-term study about the functional and morphological evolutions over one year. These parameters were compared with the CFH genotype, whose molecular analysis of Y402H polymorphism was performed by polymerase chain reaction (PCR) and direct sequencing. Results: In the short-term study, there was improvement in visual acuity at day 28 compared to baseline (D0 vs. D28) in all genotypes. However, in the group homozygous for the risk allele (CC), differences occurred only on day 28 compared to day 7 (D7 vs. D28), while the CT and TT groups, visual acuity improved earlier in the day 7 compared the initial value (D0 vs. D7). The central retinal thickness decreased in groups CT (D0 vs. D7, D0 vs. D28) and TT (D0 vs. D28), while there was no significant change in group CC. In the long-term study, it was noticed improvement in visual acuity over one year of follow-up in the group of patients without the C allele and no significant difference in the group of patients with the risk allele. The central retinal thickness decreased in the CT and TT genotypes, whereas in the CC group the difference was not significant. Number of injections, persistent neovascular activity and subjective perception of improvement did not differ between genotypes. Conclusion: The profile of the CFH genotype seems to influence the functional and morphological effect of intravitreal injection of bevacizumab with an earlier action in patients without the risk genotype. The presence of the risk allele seems to be related to the lack of visual improvement over one year of treatment with inhibitors of VEGF. These results suggest that the profile of the CFH genotype may present pharmacogenetic effect in this group of Brazilian patients, negatively influencing the response to treatment of exudative AMD with antiangiogenic drugs / Doutorado / Oftalmologia / Doutor em Ciências Médicas

The brain and spinal cord make up the central nervous system (CNS), and as an immune-privileged site, it requires special immune surveillance and regulation. The complement system is a component of innate immunity produced locally in the CNS, since size restrictions from the blood brain barrier prevent complement proteins from easily passing through from the rest of the body. The complement pathway contributes to inflammatory cell recruitment, cell lysis, and opsonization, and thus requires regulation to avoid inappropriate activation. Despite its important role in innate immunity, very little is known about complement production, regulation, and function in the CNS of healthy or diseased individuals. For this dissertation, the central goal was to investigate and characterize the regulation of complement factor H (CFH), a regulator of the alternative pathway of complement activation. CFH polymorphisms have been associated with a number of diseases including atypical hemolytic syndrome, age-related macular degeneration, and Alzheimer's disease, but the regulation of CFH is not well understood, especially in the CNS. To investigate the role of CFH in the CNS, mRNA and protein production in glial cells was first established. The murine CFH (mCFH) promoter was cloned and the transcription start site was identified in astrocytes, microglia, and liver tissue. The mCFH promoter was truncated and different regions were investigated for enhancer or silencer activity. Database mining identified potential transcription factor binding sites, and mutagenesis studies and binding assays identified transcription factor binding candidates. Specifically, the activating protein-1 (AP-1) transcription factors c-Jun and c-Fos bound to a region of the mCFH promoter between – 416 base pairs (bp) and – 175 bp in an electrophoretic mobility supershift assay. Cytokine stimulation increased mCFH mRNA and protein production, as well as the mRNA production of c-Jun and c-Fos and the protein production of c-Jun. These results suggest a relationship between cell cycle and complement regulation, and the investigation of how these transcription factors and CFH affect disease will be a valuable area of research for CNS immune regulation.

The aims of this study were to analyse known and potential biomarkers of common and genetically complex human disorders and to identify genetic and environmental variation associated with plasma biomarker concentrations. Two groups of protein biomarkers were analysed. First, plasma complement factor H (CFH) was selected as a potential biomarker for age-related macular degeneration (AMD), since common variants in the CFH gene show strong association with this disorder. Secondly, two isoforms of amyloid-β (Aβ40 and Aβ42) were selected as biomarkers for Alzheimer disease (AD) since Aβ deposits are major constituents of the amyloid plaques characteristic of this disorder. Physiological and anthropometric measurements and samples of human and genomic DNA were collected from a population sample of 1,021 individuals from the Croatian island of Vis. Quantitative determination of plasma Aβ40 and Aβ42 concentrations was performed using enzyme-linked immunosorbent assays. Heritabilities and significant covariate effects were estimated for each trait in the Croatian data set. Genome-wide linkage and association analyses were conducted for the biomarker traits. A novel finding was the genome-wide significant association between a CFH and several polymorphisms close to and within the CFH gene. The strongest association was with an intronic SNP within CFH, which explained 28% of the total trait variance (P < 10-50). The association was also replicated in a Dutch sample set. A SNP haplotype was identified which accounted for a higher proportion of the phenotypic variance. Conditional haplotype analysis showed that the effect of this haplotype on plasma CFH concentration was independent of the CFH Y402H variant, and significantly stronger than a deletion of the adjacent CFHR3/CFHR1 which was already known to affect AMD susceptibility. Genetic analysis of 382 AMD cases and 201 controls was consistent with the CFH Y402H variant being the strongest AMD susceptibility locus. Variation in plasma CFH concentration was found to explain up to 1.8% of the variation in susceptibility to AMD with an odds 2.1 (95% C.I. 1.3-3.4, P = 0.003). SNPs that were strongly associated with a CFH concentration also influenced AMD susceptibility (P < 0.05) independently of the CFH Y402H polymorphism. Functional analysis of genomic regions associated with plasma CFH is needed to identify the causal variants. Associations were observed between plasma Aβ40 concentration and several novel candidate loci, spanning regions of approximately 0.2 Mb, on chromosomes 9 and X. Similarly, novel associations with plasma Aβ42 were found in several regions, each spanning 0.2-0.4 Mb, on chromosomes 2, 5, 9, 15 and 20. The proportion of the phenotypic variance in plasma Aβ42 explained by these putative associations ranged between 1.8 and 2.8%. However, none of the associated SNPs was significant after correction for multiple testing, therefore replication is required. Finally, attempts were made to identify and quantitate new protein biomarkers of disease in human plasma using mass spectrometry. Development and optimisation of techniques was initially undertaken to deplete high-abundance plasma proteins and improve signal:noise ratio. This allowed the assessment of downstream proteomic approaches including MALDI-TOF mass spectrometry (MS), capillary electrophoresis (CE) and ion exchange chromatography (IEC), each with the potential for large-scale quantitation of plasma proteins. Although the analysis of single protein analytes, using CE and IEC proved promising, the results highlighted the difficulty associated with MALDI-TOF and protein ionisation techniques in analysing complex mixtures such as plasma.