Browsing by Author "Kidson, Sue H"

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    African mole rats as models for regressive evolution of the eye
    (2006) Nikitina, Natalya; Kidson, Sue H
    Darkness-adapted mammals with reduced eyes can serve as valuable models for the study of regressive evolution, as well as for research into the genetic and developmental processes underlying the "degeneration" of the eye. The aim of this work was to characterize two African mole rate species (Heterocephalus glaber and Bathyergus suillus) and explore their potential use as novel models for evolutionary developmental eye research. To this end, this histological structure of the adult eye, the development of the eye and the expression of several molecular markers were investigated. The important abnormal features noted were: the abnormal shape and nuclear distribution in the lens, the extremely large ciliary body and delay in the formation of the anterior chamber compared to other ocular structures.
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    The biogenesis of erythropoietin during inflammation
    (1995) Leng, Henry Martin John; Folb, Peter I; Keraan, M E; Kidson, Sue H
    Anaemia frequently accompanies chronic inflammatory diseases like rheumatoid arthritis and cancer. It is postulated to result primarily from the suppression of erythropoiesis by inflammatory cytokines. A contributing factor could be the inhibition of erythropoietin synthesis which may also be mediated by cytokines. Erythropoietin is the hormone which regulates erythropoiesis. The aims of this project were to investigate whether cytokines can indeed suppress erythropoietin production, and to determine whether the erythropoietin response in experimental models of acute and chronic inflammation was appropriate for the associated anaemia. Macrophage-conditioned medium, interleukin-1β, interleukin-6, tumour necrosis factor-α, and neopterin were assayed for inhibition of erythropoietin synthesis by HepG2 cells in culture. All, except neopterin, effected dose-dependent reductions in the secretion of the hormone. Interleukin-1β and tumour necrosis factor-α down-regulated erythropoietin gene transcription, whereas interleukin-6 inhibited a post-transcriptional process. Rats with acute inflammation developed a mild anaemia which evoked an increase in their serum levels of erythropoietin. The serum erythropoietin levels were optimal, since rats with acute inflammation and severe phenylhydrazine-induced anaemia did not have lower levels of the hormone than controls with a similar degree of anaemia, but without acute inflammation. Erythropoietin is, therefore, not an acute phase reactant. Mice with cancer developed a progressive anaemia which was not due to bone marrow invasion by tumour cells. During the first fourteen days after inoculating them with cancer cells, the mice responded by increasing their serum levels of erythropoietin as the anaemia worsened. The erythropoietin response was appropriate when compared to mice with the same degree of phenylhydrazine-induced anaemia. Erythropoietin levels measured in mice with tumours older than fourteen days were significantly lower than those of control mice with the same degree of experimental anaemia. These animals were very cachectic, suggesting that a blunted erythropoietin response may depend on disease activity.
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    The cellular basis of the Southern African forms of rufous & tyrosinase-positive oculocutaneous albinism
    (1993) Rawoot, Famida; Kidson, Sue H
    Oculocutaneous albinism is a congenital heritable disorder characterised by hypopigmentation of the eyes, hair and skin, together with visual acuity. Ten albinism have been photophobia, nystagmus and decreased different forms of oculocutaneous described. Of these, the tyrosinasepositive and rufous forms are particularly prevalent in Southern Africa. The tyrosinase-positive form manifests as two distinctly different phenotypes with some individuals developing pigmented freckles (ephelides) on their sunexposed regions and others never developing these freckles. To date, studies on the pathophysiology of tyrosinasepositive albinism have been restricted to examination of hairbulbs rather than skin biopsies. The present study utilises light and electron microscopical investigations of both hairbulb and skin melanocytes from the tyrosinasepositive and rufous forms of oculocutaneous albinism to elucidate the cellular aetiology of these disorders. In addition, melanocyte numbers were quantitated in the tyrosinase-positive skin to establish whether the observed hypopigmentation results from a decrease in the size of the melanocyte population. The melanocyte numbers in regions with ephelides were similarly quantiatated in order to see whether these freckles were a consequence of increased melanocyte numbers. The results show, for the first time, that the hypopigmentation observed in tyrosinase-positive albinos is not a consequence of melanocyte paucity and that the regions of ephelides do not contain more melanocytes. Ultrastructural studies show that regions of ephelides and non-ephelides are distinctly different. In regions of ephelides, numerous fully melanised stage IV eumelanosomes, in addition to unmelanised stage I melanosomes, were seen in the melanocyte cytoplasm. Both stage IV and unmelanised stage I melanosomes are transferred to keratinocytes. In ephelis-free regions the melanocyte cytoplasm was filled with numerous unmelanised stage I melanosomes with no evidence of stage IV melanosomes. This suggests that the defect underlying tyrosinase-positive albinism relates to the melanisation process rather than the process of melanosome assembly. In regions of ephelides, the melanocytes are able to produce numerous stage IV melanosomes, and, because these ephelides occur only on sunexposed regions, it is postulated that U.V. exposure induces a "back mutation" resulting in the restoration of the process of melanosome melanisation in these regions of skin. Numerous aberrant melanocyte organelles were also observed in tyrosinase-positive skin. These included dilated RER, distorted mitochondria and bloated Golgi. These ultrastructural observations support the recent report of a candidate gene for tyrosinase-positive albinism, which, it is speculated encodes an integral melanosomal membrane tyrosine transport protein. A defect in tyrosine transport into melanosomes would explain the observed incomplete melanisation of melanosomes in tyrosinase- positive albino skin. In rufous albinism, several aberrant melanosomal shapes were also seen, including "racquet", "crescent"and "comma"shaped melanosomes, all of which were fairly densely melanised. These melanosomes were also about 30% smaller than normal Negroid melanosomes. Upon transfer to keratinocytes, the melanosomes formed membrane-bound "rosette-like" clusters. These findings that the defect in rufous albinism seem to relates suggest to the melanosomal assembly process rather than the melanisation process.
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    Investigating the role of a FAM111B mutation in hereditary fibrosing poikiloderma (POIKTMP) using induced pluripotent stem cell (iPSC) model
    (2019) Gumede, Dimakatso B; Kidson, Sue H; Ballo, Robea; Mayosi, Bongani M
    Hereditary fibrosing poikiloderma is an autosomal dominant disorder that is characterised by mottled pigmentation and telangiectasia, accompanied by tendon contractures, myopathy and pulmonary fibrosis (POIKTMP). Mutations in POIKTMP cases have been shown to harbour the Family with sequence similarity 111B (FAM111B) gene. However, its function is unknown. The aim of this study was to investigate the causative role of the FAM111B mutation (c.1861T>G) in the multi-systemic fibrosis affecting the South African kindred with POIKTMP. Dermal fibroblasts from two affected siblings and a familial control were reprogrammed into induced pluripotent stem cells (iPSCs) via the Sendai virus vector (SeVdp) packaged with pluripotency transgenes (OCT4; SOX2; KLF4; C-MYC). The derived iPSCs successfully showed a) endogenous expression of pluripotency markers (OCT4; NANOG; TRA-1-60), b) in vitro differentiation into the three germ layers (endoderm; mesoderm; ectoderm) and c) normal karyotyping. Next, the iPSCs from two patients, a Familial control and a Non-familial control were differentiated into mesenchymal stem/stromal cells (iPSC-MSCs) as a cell model in this study. Characterisation of derived iPSC-MSCs indicated positive expression of MSC markers (CD73; CD90; α-SMA). Differentiation of iPSC-MSCs demonstrated adequate osteogenicity but limited adipogenicity. Patient-derived iPSC-MSCs were thereafter analysed by qPCR and collagen staining to determine whether the FAM111B mutation alters endogenous expression of pro-fibrotic markers as well as collagen synthesis in patient cells compared to controls. Messenger RNA expression of pro-fibrotic markers (COL1A1; COL3A1; α-SMA) was similar between patient and control iPSC-MSCs. Collagen staining and quantification also showed no statistical differences between patient and control cells. These results suggest that FAM111B does not directly alter the expression of these profibrotic genes in this in vitro model system. Growth curves were then carried out to investigate if the FAM111B mutation modulates cell proliferation and it was found that patient cells proliferated at a higher rate compared to controls. To explore the mechanisms underlying the rate change, analyses of FAM111B expression during cell cycle progressions were conducted. Extensive optimization experiments using the double thymidine block approach were necessary to establish the appropriate synchronization protocol, keeping in mind the extended doubling time of iPSCMSCs. The results revealed that FAM111B mRNA expression was temporally regulated, with a peak at the S-phase and low at the G2/M phase. While there were no pattern differences between patient and control cells, FAM111B mRNA expression was significantly higher in the patient cells compared to controls at the G1- and S-phase. These results suggest that the mutation in FAM111B might affect the stability or perdurance of the mRNA. Unfortunately, analysis of the FAM111B protein data was inconclusive. Problems related to synchronization of the cells and the specificity of the antibody would have to be rectified in order to follow this further. The overall findings in this in vitro study reveal that the FAM111B mutation does not alter expression of pro-fibrotic markers but does affect the cell proliferation rate of patient cells compared to controls. Future work will focus on further optimisation of iPSC-MSCs synchronisation to determine correlation of FAM111B mRNA and protein expression during cell cycle progression in the patient cells. Furthermore, 3D in vitro cellular models that recapitulate some parts of the POIKTMP phenotype will need to be created. Future work will also explore the gain-of-function hypothesis to further understand the role of FAM111B in fibrosis and cancer phenotype in POIKTMP.