An experimental study of human melanoma cells cutured in vitro

This thesis records the results of a series of experiments that were designed to examine the biology of human malignant melanoma cells cultured in vitro. The studies were so planned as to document phenotypic differences that exist between melanomas, to define respects in which melanoma cell differentiation could be modulated and to correlate biochemical variability with in vivo behaviour as measured in the nude mouse. Melanoma cell lines were established from biopsy material obtained from 7 patients at Groote Schuur Hospital. Two of these lines synthesized tyrosinase and melanin at a rate that was directly related to cell density. The five remaining lines did not pigment. ii All of the lines showed aneuploidy; 5 of the 7 showed anchorageindependent growth; and 6 of the 7 grew as lethal tumours in nude mice. As has been found with all other melanomas studied, these cells released a plasminogen activator that was chemically and immunologically identical to tissue activator. One of the lines proved to be an exception to this general rule in that it synthesized urokinase-type enzyme. Unlike most other human cells cultured in vitro, melanoma cells proved to be relatively refractory to hormonal stimuli. Addition of estrogen, progesterone, testosterone, dexamethasone or melanocyte-stimulating hormone to the culture medium had very little effect on cellular release of plasminogen activator, upon cell growth, or upon cellular morphology. Although remarkably resistant to hormonal influences, cellular release of plasminogen activator did appear to be inhibited to a striking degree by cocultivation with normal skin fibroblasts. This observation led to the discovery of a phenomenon in which fibroblasts of many types bound tissue-type plasminogen activator and so removed it from the medium. This was accompanied by an apparent change in molecular weight of the melanoma cell enzyme from 72K daltons to approximately 115K daltons, suggesting the presence of a 40-SOK binding molecule. iii In an attempt to influence in vitro differentiation, the tumour promoter tetradecanoylphorbol acetate, and the differentiation-inducing retinoid, retinoic acid, were added to the two pigmenting cell lines. The effects of these compounds on induction of tyrosinase activity, morphological change or plasminogen activator release differed. In the one cell line, tetradecanoylphorbol acetate caused morphological maturation with a decrease in the rate of plasminogen activator release and no obvious effect upon pigmentation. This line was relatively resistant to the action of retinoids. The other pigmenting line responded hardly at all to the tumour promoter. Retinoic acid, on the other hand, inhibited the induction of tyrosinase activity, yet caused an inhibition of growth and plasminogen activator release. A number of interesting observations could be made in experiments in which melanoma cells were inoculated into nude mice. Firstly, the growth rate of the tumours ~n vivo correlated poorly with the doubling times of the corresponding cells cultured in vitro. Secondly, despite a marked inhibitory effect of fibroblasts on plasminogen activator in vitro, coinjection of fibroblasts and melanoma cells in vivo greatly enhanced tumour growth when small tumour cell inocula were used and shortened the latent period for tumour appearance with larger inocula. Thirdly, melanomas growing in nude mice differed strikingly in their ability to elicit a desmoplastic response. Tumours in which large amounts of host connective tissue were deposited tended to be heavily contaminated with murine fibroblasts when re-established in vitro. This contamination was not seen with tumours that contained very little connective tissue. These results point to the existence of a melanoma-associated fibrogenic factor. Finally, by excision of the primary tumour, it was possible to avoid death of the animal from local complications and so allow time for metastases to develop. In three mice, metastatic melanoma deposits could be detected by this device, so establishing a protocol for the use of nude mice as valid models for the experimental study of metastatic spread of human tumours

Putting RFMix and ADMIXTURE to the test in a complex admixed population

CITATION: Uren, C., Hoal, E. G. & Moller, M. 2020. Putting RFMix and ADMIXTURE to the test in a complex admixed population. BMC Genetics, 21:40, doi:10.1186/s12863-020-00845-3.The original publication is available at https://bmcinfectdis.biomedcentral.comPublication of this article was funded by the Stellenbosch University Open Access FundBackground: Global and local ancestry inference in admixed human populations can be performed using computational tools implementing distinct algorithms. The development and resulting accuracy of these tools has been tested largely on populations with relatively straightforward admixture histories but little is known about how well they perform in more complex admixture scenarios. Results: Using simulations, we show that RFMix outperforms ADMIXTURE in determining global ancestry proportions even in a complex 5-way admixed population, in addition to assigning local ancestry with an accuracy of 89%. The ability of RFMix to determine global and local ancestry to a high degree of accuracy, particularly in admixed populations provides the opportunity for more accurate association analyses. Conclusion: This study highlights the utility of the extension of computational tools to become more compatible to genetically structured populations, as well as the need to expand the sampling of diverse world-wide populations. This is particularly noteworthy as modern-day societies are becoming increasingly genetically complex and some genetic tools and commonly used ancestral populations are less appropriate. Based on these caveats and the results presented here, we suggest that RFMix be used for both global and local ancestry estimation in worldwide complex admixture scenarios particularly when including these estimates in association studies.https://bmcgenet.biomedcentral.com/articles/10.1186/s12863-020-00845-3Publisher's versio

The critical needs and challenges for genetic architecture studies in Africa

Human genetic studies have long been vastly Eurocentric, raising a key question about the generalizability of these study findings to other populations. Because humans originated in Africa, these populations retain more genetic diversity, and yet individuals of African descent have been tremendously underrepresented in genetic studies. The diversity in Africa affords ample opportunities to improve fine-mapping resolution for associated loci, discover novel genetic associations with phenotypes, build more generalizable genetic risk prediction models, and better understand the genetic architecture of complex traits and diseases subject to varying environmental pressures. Thus, it is both ethically and scientifically imperative that geneticists globally surmount challenges that have limited progress in African genetic studies to date. Additionally, African investigators need to be meaningfully included, as greater inclusivity and enhanced research capacity afford enormous opportunities to accelerate genomic discoveries that translate more effectively to all populations. We review the advantages, challenges, and examples of genetic architecture studies of complex traits and diseases in Africa. For example, with greater genetic diversity comes greater ancestral heterogeneity; this higher level of understudied diversity can yield novel genetic findings, but some methods that assume homogeneous population structure and work well in European populations may work less well in the presence of greater heterogeneity in African populations. Consequently, we advocate for methodological development that will accelerate studies important for all populations, especially those currently underrepresented in genetics.Peer reviewe

Genetic resistance to Mycobacterium Tuberculosis infection and disease

CITATION: Möller, M. et al. 2018. Genetic resistance to Mycobacterium tuberculosis infection and disease. Frontier in Immunology, 9:2219, 1-13. doi:10.3389/fimmu.2018.02219.The original publication is available from https://www.frontiersin.org/journals/immunology#Natural history studies of tuberculosis (TB) have revealed a spectrum of clinical outcomes after exposure to Mycobacterium tuberculosis, the cause of TB. Not all individuals exposed to the bacteriumwill become diseased and depending on the infection pressure, many will remain infection-free. Intriguingly, complete resistance to infection is observed in some individuals (termed resisters) after intense, continuing M. tuberculosis exposure. After successful infection, the majority of individuals will develop latent TB infection (LTBI). This infection state is currently (and perhaps imperfectly) defined by the presence of a positive tuberculin skin test (TST) and/or interferon gamma release assay (IGRA), but no detectable clinical disease symptoms. The majority of healthy individuals with LTBI are resistant to clinical TB, indicating that infection is remarkably well-contained in these non-progressors. The remaining 5–15% of LTBI positive individuals will progress to active TB. Epidemiological investigations have indicated that the host genetic component contributes to these infection and disease phenotypes, influencing both susceptibility and resistance. Elucidating these genetic correlates is therefore a priority as it may translate to new interventions to prevent, diagnose or treat TB. The most successful approaches in resistance/susceptibility investigation have focused on specific infection and disease phenotypes and the resister phenotype may hold the key to the discovery of actionable genetic variants in TB infection and disease. This review will not only discuss lessons from epidemiological studies, but will also focus on the contribution of epidemiology and functional genetics to human genetic resistance to M. tuberculosis infection and disease.https://www.frontiersin.org/articles/10.3389/fimmu.2018.02219/fullhttps://doi.org/10.3389/fimmu.2018.02219Published review articlePublishers versio

Determining ancestry proportions in complex admixture scenarios in South Africa using a novel proxy ancestry selection method

Admixed populations can make an important contribution to the discovery of disease susceptibility genes if the parental populations exhibit substantial variation in susceptibility. Admixture mapping has been used successfully, but is not designed to cope with populations that have more than two or three ancestral populations. The inference of admixture proportions and local ancestry and the imputation of missing genotypes in admixed populations are crucial in both understanding variation in disease and identifying novel disease loci. These inferences make use of reference populations, and accuracy depends on the choice of ancestral populations. Using an insufficient or inaccurate ancestral panel can result in erroneously inferred ancestry and affect the detection power of GWAS and meta-analysis when using imputation. Current algorithms are inadequate for multi-way admixed populations. To address these challenges we developed PROXYANC, an approach to select the best proxy ancestral populations. From the simulation of a multi-way admixed population we demonstrate the capability and accuracy of PROXYANC and illustrate the importance of the choice of ancestry in both estimating admixture proportions and imputing missing genotypes

An evaluation of commercial fluorescent bead-based luminex cytokine assays.

The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25-50 microl sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-gamma measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-gamma Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques

BDNF Val66Met and DRD2 Taq1A polymorphisms interact to influence PTSD symptom severity: A preliminary investigation in a South African population

BACKGROUND: We evaluated the role that selected variants in serotonin transporter (5-HTT), dopamine receptor 2 (DRD2) and brain-derived neurotrophic factor (BDNF) genes play in PTSD symptom severity in an at-risk population. We also investigated the interaction between the genetic variants to determine whether these variables and the interactions between the variables influenced the severity of PTSD symptoms. METHODS: PTSD symptoms were quantitatively assessed using the Davidson Trauma Scale (DTS) in 150 participants from an at-risk South African population. All participants were genotyped for the 5-HTTLPR, DRD2 Taq1A and BDNF Val66Met polymorphisms. Gene–gene interactions were investigated using various linear models. All analyses were adjusted for age, gender, major depressive disorder diagnosis, level of resilience, level of social support and alcohol dependence. RESULTS: A significant interaction effect between DRD2 Taq1A and BDNF Val66Met variants on DTS score was observed. On the background of the BDNF Val66Val genotype, DTS score increased significantly with the addition of a DRD2 Taq1A A1 allele. However, on the BDNF Met66 allele background, the addition of an A1 allele was found to reduce total DTS score. CONCLUSIONS: This study provides preliminary evidence for an epistatic interaction between BDNF Val66Met and DRD2 Taq1A polymorphisms on the severity of PTSD symptoms, where both too little and too much dopamine can result in increased PTSD symptom severity.Web of Scienc