MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / Introduction: Systemic sclerosis (SSc) is a complex autoimmune disease characterised by autoantibody release, leading to microvascular injury, fibroblast activation and increased production of collagen. The genetics of SSc is complex with many genes implicated in the development and maintenance of the extracellular matrix (ECM). The main aim of this study was to test for differential expression of matrix metalloproteinase 1 (MMP1), tissue inhibitor of metalloproteinase 1 (TIMP1) and hepatocyte growth factor (HGF) in SSc patients compared to healthy control individuals and to assess whether the differential expression of these genes could have an impact on clinical features of the disease. Methods: Two skin biopsies were analysed for each of 16 black SSc patients, one from clinically involved skin (lateral forearm) and one from clinically uninvolved skin (back). One skin sample was obtained from 15 ethnically matched control individuals. The differential expression of MMP1, TIMP1 and HGF in the clinically involved and uninvolved patient samples would be compared to control individuals using relative quantification polymerase chain reaction (qPCR). The gene expression profiles were then compared to specific clinical features to deduce whether any of the gene expression profiles is correlated with the manifestation of specific clinical features. Results: MMP1 gene expression was significantly decreased in SSc patients for both involved (p=0.0004) and uninvolved skin (p=0.0004) compared to controls. Conversely, TIMP1 gene expression was significantly increased in SSc patients at both sites compared to controls (p=<0.00001 for both comparisons). A trend of significance was observed for the difference in TIMP1 expression between the involved an uninvolved skin within the patients (p=0.05) with a greater increase in involved skin. HGF had increased gene expression in the patients compared to controls for involved and uninvolved skin (p=0.002 and 0.004, respectively). The difference in gene expression between the involved and uninvolved biopsies was not significant for either MMP1 or HGF (p=0.87 and 0.83, respectively). The only correlates that may have a biological significance are HGF in involved skin correlated with disease activity (r=0.60; p=0.013) and HGF in uninvolved skin vi correlated with skin score (MRSS) with r=0.50 and p=0.048. With regards to the categorical data, two marginally significant observations were found, once again with HGF, which was found to be associated with gender in involved skin (p=0.037) and renal disease in uninvolved skin (0.031). Conclusion: The relative under expression of MMP1 and over expression of TIMP1 reflect the pro-fibrotic state of scleroderma skin. The over expression of HGF suggests that HGF may play a compensatory anti-fibrotic role, although this is not sufficient to overcome the pro-fibrotic state of the skin. This study provides supporting evidence to debunk the myth of uninvolved skin in SSc patients. The altered expression of MMP1, TIMP1 and HGF in the clinically uninvolved skin of SSc patients suggests that all subcutaneous tissue is affected, although to a greater extent in the clinically involved skin of the patients.

Scleroderma, a genus of ectomycorrhizal (ECM) fungi, is often associated with trees in disturbed habitats and is therefore considered to be suitable for use in plantation forestry. This study investigated aspects of Scleroderma and its mycorrhizas with the view to its future use in plantation forestry in south China. Spores were chosen as inoculum as they are preferred by nursery managers in south China, due to the lack of on-site fermentation and storage facilities. To determine the need for inoculation, Eucalyptus plantations in south China were sampled for sporocarps and mycorrhizas over two years. This study revealed a low diversity of ECM fungi consisting of 15 taxa fruiting beneath Eucalyptus plantations. The most common genera were Scleroderma and Pisolithus, but they were infrequent and the extent of root colonization was poor. Bioassay trials with E. urophylla as a bait host, using soils collected from 8 eucalypt plantations, confirmed low levels of inoculum in field soil. It was concluded that introduction of suitable ECM symbionts into eucalypt nurseries in south China is desirable in the future. As the Scleroderma genus has not been well studied in Australasia or SE Asia, over 140 collections gathered mainly from eucalypt plantations in south China and south-western Australia were described using sporocarp and spore morphology. Twelve Scleroderma taxa were recognized from collections made from under eucalypt plantations in south-western Australia and 6 of these were collected from under eucalypt plantations in south China. In conjunction with classical taxonomy, 30 collections, including those used in inoculation trials, were further characterized by phylogenetic analyses of ITS or LSU rDNA sequences. These studies supported classical delineation of some Scleroderma species but not all. Although a limited number of collections were amplified, phylogenetic results showed that most collections in this study were distinct from the European and Malaysian taxa extracted from GenBank (89% bootstrap support for both LSU and ITS regions). In order to optimise spore germination and root colonization, two glasshouse trials were established to examine suitable spore density and spore storage conditions on E. globulus and E. urophylla. A spore density of 105 spores seedling-1 was identified as a suitable dose for promoting root colonization. Spores stored for 5 years at low temperate (4 0C) were almost as effective as freshly collected spores in forming mycorrhizas. As the compatibility of Scleroderma fungi with plantation trees is unknown, a glasshouse experiment examined the ability of 15 collections of Scleroderma to form mycorrhizas with seedlings of six plantation trees (Acacia mangium, A. mearnsii, E. globulus, E. urophylla, Pinus elliottii and P. radiata) in a nursery potting mix. Most collections were able to aggressively colonize eucalypts and pines, while roots of acacias were poorly colonized. As the Australian collections were more effective in colonizing short roots on eucalypts than the Chinese collections, it was concluded Scleroderma should be sourced from outside China for inoculating eucalypts in Chinese nurseries. To optimize nursery practices to meet the demand for high quality seedlings and clonal lines of E. urophylla and hybrids, for outplanting in south China, effects of rooting medium and inoculation with 6 Scleroderma collections on the growth of E. urophylla were examined in a nursery in south China. Four types of soil taken from eucalypt plantations in south China were compared to a potting mix composed of vermiculite, peat and sand. The inoculant Scleroderma fungi were able to out-compete indigenous mycorrhizal fungi in the rooting media. However, the potting mix was superior to soils both for plant growth and ECM development under nursery conditions. This research should facilitate the use of Scleroderma spores in eucalypt nurseries in south China. Spore orchards could be set up in China using Australian Scleroderma spp. from under eucalypts. Spores could be stored dry at 4 0C until they are required for inoculation in potting mixes in containerized nurseries. However, before commercial application, further work on persistence of Scleroderma in the nursery and field, and responses of trees in the field to inoculation, needs to be undertaken.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 1997. / Vita. Includes bibliographical references (leaves 121-129).

The purpose of this research study was to evaluate the effectiveness of the Arthritis Self-Management Program (ASMP), developed by Dr. K. Lorig, on a population of persons with scleroderma. This particular condition is a type of arthritis (also known as progressive systemic sclerosis) involving a disorder of the small blood vessels and connective tissues. It is characterized by the induration and thickening of the skin and by inflammatory, fibrotic, ischemic, and degenerative changes in the tissues throughout the body. Eighteen people, most of which were female, in the Vancouver Lower Mainland with the diagnosis of scleroderma volunteered for this study. Quantitative and qualitative methodological orientations were used to collect and analyze the data. A quasi-experimental, pretest-posttest nonequivalent comparison group design was used. Self-administered, standardized questionnaires were distributed to a sample of subjects to collect the quantitative data, and a standardized open-ended interview questionnaire was used to collect the qualitative data. The quantitative questionnaire comprised research instruments including The Visual Analogue Pain Scale, Health Assessment Questionnaire, Centre for Epidemiological Studies of Depression Scale, Cantril Quality of Life Scale, Arthritis Self-Efficacy Scale, and Health Locus of Control Scale. The quantitative findings indicated that no statistically significant improvements in health status were found. However, clinically significant improvement trends in health status were found. The qualitative findings generally indicated that the experimental subjects enjoyed the ASMP, found it to increase their perceived level of coping with the management of scleroderma, and found the ASMP to be a positive learning experience. With the exception of the ASMP being limited in its specific application to people with scleroderma, it proved to be a feasible patient education course for these people. / Arts, Faculty of / Social Work, School of / Graduate

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Internal Medicine. Johannesburg, 2014 / BACKGROUND: Interstitial lung disease (ILD) is one of the leading causes of death in systemic sclerosis (SSc). PATIENTS AND METHODS: A retrospective review of case records, over 20 years, of SSc patients attending a tertiary Connective Tissue Diseases Clinic. Comparisons between ILD and non-ILD groups at presentation were performed in order to identify baseline associations and predictors of ILD. RESULTS: Of the 151 participants that met inclusion criteria, 60 (40%) had ILD. On multivariate analysis the only three variables to remain significant were median duration of disease (OR 1.2 (1.1-1.3); p<0.001), speckled anti-nuclear antibody (ANA) pattern (OR 2.95 (1.22-7.15); p=0.017) and bibasal crackles (OR 5.4 (2.1- 13.5); p<0.0001). Univariate analysis of baseline variables associated with interstitial lung disease in systemic sclerosis. Baseline Variable ILD (n=60) Non-ILD (n=91) OR (CI 95%) p Bibasal crackles (%) 28 (46.7) 10 (11.0) 7.1 (3.1-16.3) <0.0001 Diffuse disease subtype (%) 49 (81.7) 45 (48.9) 4.6 (2.1-9.9) <0.001 Limited disease subtype (%) 8 (13.3) 38 (41.3) 0.2 (0.1-0.5) <0.001 Anti-centromere antibodies (%) 0 (0.0) 10 (13.0) - 0.006 Cough (%) 21 (35.0) 15 (16.5) 2.7 (1.3-5.9) 0.007 Median duration in years (IQR) 6.1 (8.3) 4.0 (5.0) 2.2 (1.8-2.4) 0.009 Speckled ANA pattern (%) 29 (50.9) 25 (32.5) 2.5(1.2-4.9) 0.010 Dyspnoea (%) 27 (45.0) 24 (26.4) 2.3 (1.1-4.6) 0.014 Gold mining history (%) 5 (8.3) 1 (1.1) 8.2 (0.9-71.9) 0.037 ANA=antinuclear antibody; ILD=interstitial lung disease; IQR= interquartile range; OR=odds ratio Additionally, dyspnoea was associated with ILD severity (p=0.008). Bibasal crackles (p=0.014), increased plasma urea (p=0.041), and reduced serum albumin (p=0.007) were associated with mortality in the ILD group. CONCLUSION: Interstitial lung disease in South African SSc patients is common. The diffuse cutaneous disease subtype appears to drive the disease process. There should be a high index of suspicion for ILD in SSc patients presenting with a gold mining history, dyspnoea, cough and bibasal crackles.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2002. / Vita. Includes bibliographical references (leaves 126-140).

published_or_final_version / Medicine / Master / Doctor of Medicine

Scleroderma is a chronic autoimmune connective tissue disease of unknown etiology characterized by excessive fibrosis and is broadly divided into two clinical entities: localized scleroderma (LSc) and systemic sclerosis (SSc). SSc is a multi-system disease resulting in both skin and visceral organ fibrosis. The more benign disorder, LSc is for the most part self-limited with the disease pathology being confined to the skin and subcutaneous tissues. Proposed factors involved in the pathogenesis of these disorders include endothelial cell injury and dysfunction, immunological alterations and inflammatory activation, and abnormal ECM production by activated fibroblasts. However, the initiating mechanisms that leads to these changes remains largely unknown. This thesis examines the hypothesis that the transcriptional expression at the edge and centre of expanding LSc plaques could represent the metabolic changes involved in the different stages of disease. The major finding of this thesis was the identification of two panels of genes that showed significant changes in expression between LSc patients and healthy controls irrespective of whether the sample was taken from a diseased or clinically unaffected area of the patient. The first panel consisted of inflammatory genes including those genes characteristic of the Thl response and those induced by NF-KB. The Thl response was supported by an increased infiltration of CD4+ T cells in the LSc patients. The second panel consisted of a subset of array identified genes (scleroderma Signature) in SSc patients. Of interest, WIF1 was down regulated in both disorders and showed a gradual decrease in expression across the clinically different areas of the LSc patients. Both panels of genes showed the biggest changes of expression at the edge of the plaque suggesting their involvement in the initiating events of the disease. These results suggest that, like SSc, the underlying pathology of LSc is related to systemic changes in genes controlling amongst others, immunological and inflammatory responses. This information not only sheds light on the mechanisms involved in the initiation and progression of scleroderma, but could also contribute to the creation of a diagnostic test for the early detection of sufferers of this rare, but important disease.

Aim. To establish standardised infrared thermography (IRT) within a specialist connective tissue disease unit, assessing its utility: • for the evaluation of Raynaud’s Phenomenon (RP) in clinical rheumatology and research • for the detection of active localised scleroderma (LS) lesions in paediatric patients and to develop improvements in IRT quality assurance for these medical applications. Methods. For the evaluation of RP, a protocol for cold challenge of the feet was developed and validated. IRT was applied with hand cold challenge for the assessment of response to oral vasodilator therapies in two large randomised pilot studies. An infrared thermometer technique was developed, validated against IRT, and subsequently used for the assessment of peripheral vasospasm in a twin study into the heritability of RP. The utility of inspection of thermograms for detecting clinically active LS lesions was established. A protocol was developed incorporating photography, IRT and laser Doppler flowmetry (LDF) for LS assessment, and the normal range of temperature and LD blood flow across several body sites was established in adults and children. The utility of the protocol for assessing LS activity in children was investigated. To develop quality assurance of thermography, the author contributed to the specification and validation of blackbody medical temperature reference sources, and published guidelines for procuring and commissioning a medical thermal imager. Results. Healthy controls had a higher mean toe temperature than RP patients (at baseline 29.2 ± 1.5oC v 24.8 ± 1.5oC [mean ± SD], p < 0.01; t-test). IRT demonstrated improved finger rewarming 10 minutes after cold challenge in primary RP patients 11 treated with fluoxetine compared with those treated with nifedipine (58.8% v 43.1%, p=0.03; t-test). IRT showed no such improvement in finger rewarming over nifedipine in patients treated with losartan. In a hospital setting, an infrared thermometer technique performed similarly to IRT with cold challenge for the detection of RP: the sensitivity of IRT was 83%, whereas for the infrared thermometer it was 89%. The specificity of both instruments was 84%. In a population setting using the infrared thermometer both baseline finger temperature and rewarming after ten minutes were significantly lower for RP subjects than for controls (for baseline: 28.3oC v 30.0oC, p < 0.01, t-test; for rewarming: 4.6oC v 5.3oC, p < 0.05, t-test). Infrared thermometer measurements in monozygotic and dizygotic twin pairs revealed a heritability of 65% for baseline finger temperature, 35% for fall after cold challenge, and 24% for rewarming over ten minutes. In the larger of two published studies on the inspection of thermograms for detecting clinically active LS, sensitivity was 92%, and specificity was 68%. In lesions imaged within 2 years of onset, sensitivity was 81% and specificity 88%. Validation of a protocol combining IRT and LDF measurements revealed that, in adult controls, the mean temperature difference between the two sides of the body was less than 0.5oC at all body regions. Mean differences in contralateral LD flux were less than 40% at all body sites. Variability in LD and IRT readings due to experimental factors was acceptably small in comparison to the physiological differences recorded. Applying the protocol in children with LS, the median relative increase in LD blood flow in clinically active lesions (compared with blood flow in contralateral unaffected skin) was 89% (range -69% to +449%), whereas the median flow increase in clinically inactive lesions was 11% (range -46% to +302%), p < 0.001. Using IRT, the median temperature difference between clinically active lesions and contralateral unaffected skin was 0.5°C (range -0.1°C to +4.1°C), whereas the median temperature difference for clinically inactive plaques was 0.3oC (range -1.9°C to +2.7°C), p=0.024. 12 In hand cold challenge measurements made at the Royal Free Hospital, application of the medical blackbody temperature reference sources reduced the overall uncertainty in temperature readings by a factor of about 4, from typically ±2°C to ±0.5°C. Conclusion. IRT or infrared thermometer data on skin temperature before and after cold challenge affords RP studies an important element of objectivity. RP detected in a population setting exhibits milder vasospasm than RP recruited from hospital patients, and thus the results of research performed at specialist centres may not be translatable to community settings. Inspection of thermograms is an effective method for the detection of clinically active LS, although LDF performed better than IRT using a protocol reliant on objective measurements from small regions of interest. IRT and infrared thermometry were generally less effective at discriminating between healthy and diseased subjects in situations where the temperature difference between groups was small (<2°C). The introduction of temperature reference sources, which reduce uncertainty in radiometric measurements to the order of 0.5°C, would increase the utility of IRT in settings where the temperature change associated with disease is small.

Over the course of the last century mycorrhizal symbiosis has come to be recognized as highly beneficial for the host plant. Increased growth, water relations, nutrient acquisition, amelioration of the effects of metal toxicity, and increased resistance to pathogens are all benefits attributed to plants colonized by mycorrhizal fungi. A large body of literature exists that indicates that mycorrhizal inoculation programs are useful for improving the performance of forest tree seedlings. Commercial mycorrhizal products, many containing the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch (Pt), have emerged from this research and are now being marketed for landscape tree growers. In this study, two experiments were conducted to determine the best protocol for inoculation of landscape trees, and to determine if mycorrhizal inoculation is beneficial to trees growing in modern landscape tree container production systems. The first experiment evaluated mycorrhizal inoculation programs utilizing two inocula types employing various substrates for landscape tree production in containers. Quercus palustris Muenchh. (pin oak) and Q. coccinea Muenchh. (scarlet oak) grown in pine bark, sterilized pine bark, sterilized mineral soil, and sterilized vermiculite-based substrates were inoculated with Pt commercial spore inoculum or Pt vegetative inoculum. The vegetative inoculum was unsuccessful at forming mycorrhizae even though the Pt continued to live in the planting substrates. A higher proportion of pin oak was colonized than scarlet oak in all substrates, and vermiculite was a superior environment for mycorrhizal formation than the other three substrates. The second experiment examined the effectiveness of an indigenous mycorrhizal fungus, Scleroderma bovista Fr., to increase growth and resistance to drought stress of scarlet oak grown in containers in the pot-in-pot growing system. Scleroderma bovista did not affect tree growth, and mycorrhizal trees in containers proved to be more susceptible to drought stress than nonmycorrhizal trees. Leaf water potential was more negative for mycorrhizal trees, and conductance was lower for mycorrhizal trees after a 10 day dry down period. A commercial Pt product was also used as an inoculum in this study and again proved completely ineffective at colonizing scarlet oak in pine bark substrate. / Master of Science