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Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous group of inherited blistering skin diseases that affect 500,000 individuals worldwide. The clinically more severe recessive dystrophic (RDEB) variant affects ~5% of EB individuals with a prevalence of 8 per one million of the population. RDEB is caused by loss-of-function mutations in the type VII collagen gene, COL7A1, which leads to reduced or absent type VII collagen (C7) and structurally defective anchoring fibrils at the dermo-epidermal junction (DEJ). From a clinical perspective, individuals with RDEB have fragile skin and are susceptible to blistering following minimal trauma, which leads to poor wound healing, scarring, contractures and oesophageal strictures. The major cause of mortality in RDEB is metastatic cutaneous squamous cell carcinoma (SCC). At present, care is mainly supportive and there are no effective treatments for this debilitating disease. The main therapeutic challenge, therefore has been to develop gene, protein, cell and drug therapies that are safe, effective and affordable. The basis of this thesis is to evaluate safety and efficacy of allogeneic cell-based therapies and attempt to elucidate their mechanism of action in wound healing in RDEB. To examine if allogeneic fibroblasts promote healing of chronic wounds in RDEB I intradermally injected allogeneic fibroblasts around the wound margins in an individual with RDEB. I demonstrated that these injections result in Heparin-Binding EGF-like Growth Factor encoding gene (HBEGF) and COL7A1 upregulation followed by C7 production. These led to reduction in wound size by 30% at 8 months post injections. HB-EGF, a member of the EGF family, has been implicated in RDEB-associated SCC. I also assessed whether another growth factor, EGF, which is commercially available for human use and which is related to HB-EGF, could upregulate COL7A1 expression in RDEB epidermal cells. I demonstrated in vitro that 100ng/ml EGF at 90 minutes and 10ng/ml at 15 minutes led to 3-fold and 5-fold COL7A1 upregulation in RDEB keratinocytes and fibroblasts respectively. HB-EGF also led to COL7A1 upregulation in RDB keratinocytes (6-fold) but to a lesser extent in RDEB fibroblasts (2-fold). To evaluate the effects of intradermally injected allogeneic fibroblasts in a larger number of RDEB individuals, I conducted a prospective, randomised, vehicle-controlled, phase II clinical trial. Twenty-six wounds in 11 adults with RDEB were injected. Fourteen wounds received fibroblasts and 12 were injected with vehicle only. I showed that allogeneic fibroblasts are safe and lead to a greater reduction in erosion area compared to vehicle within the first 28 days following treatment with a single set of injections to the wound margins, but not thereafter. Finally, I explored the safety and potential of intravenously administered allogeneic bone marrow-derived mesenchymal stromal cells (BM-MSCs) in children with RDEB in an open-label trial. I showed that the infusions were well-tolerated, with no serious adverse events. Although, there was no increase in C7 deposition, children and their parents reported improved wound healing, reduction in blister numbers and pruritus, increased skin resilience to trauma, and reduced pain during dressing changes. In addition, significant reduction in circulating inflammatory cytokines (IL-10, p < 0.001; IFN-γ, p=0.04 and IL-17A, p=0.03) was observed. Taken together, these data reveal new insights into the mechanisms of action of allogeneic cell-based therapies in RDEB and provide evidence for their efficacy in wound healing and reducing morbidity in the context of clinical trials.

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Dystrophic epidermolysis bullosa results from mutations in COL7A1 that encodes collagen VII, the major component of anchoring fibrils. General paradigms have emerged attributing dominant DEB to heterozygous glycine substitutions and recessive DEB to nonsense, frameshift or splice site mutations on both COL7A1 alleles. Several aspects of the genotype-phenotype heterogeneity encountered in DEB remain unexplained, although genetic, epigenetic and environmental modulators have been implicated. In this thesis, various aspects of DEB were studied, in a bid to refine genotype-phenotype correlation. A detailed analysis of missense GS and non-GS mutations identified 57 novel mutations and was in-keeping with the general established paradigms. Unique clinical entities such as EB-pr and BDN were studied. The role of the matrix metalloproteinase-1 promoter polymorphism, -1607 1G/2G, on disease modification in EB-pr was explored, but was shown to be an unlikely modulator. A large study of BDN, highlighted that intracytoplasmic retention of collagen VII and stellate bodies were not exclusive to BDN and can be associated with various subtypes of DEB, non-EB cases and normal skin. The first case of revertant mosaicism in DEB was studied revealing intragenic cross-over as the underlying mechanism. Finally, intradermal injection of allogeneic fibroblasts was shown to result in increased and sustained expression of COL7A1 possibly through the upregulation of HB-EGF. Recognition of disease modifiers in DEB and refinement of genotype–phenotype correlation will not only further our understanding of DEB but will have implications on diagnosis, counselling and prognosis through patient specific and targeted therapy.

This thesis contributes to knowledge by developing algorithms that automatically detect and quantify structures of clinical interest in reflectance confocal microscopy (RCM) images, captured in-vivo and from excised skin tissue. The first part of the thesis presents an algorithm that detects the dermalepidermal junction (DEJ), characterised by papillae, in cubes of RCM images of in-vivo skin. A cube of images is a number of mosaic images captured at different depths parallel to the skin surface. A classifier, which makes use of texture and anatomical-based features was designed. The anatomical-based features are parameters that quantify the absence and presence of papillae across different images of the cube. The second part of the thesis analyses RCM images of excised tissue collected during Mohs surgery. These tissue samples include basal cell carcinoma (BCC) and non-diseased samples. An algorithm was developed to differentiate between (i) cancerous regions, (ii) regions of inflammation, and (iii) non-diseased regions. A classifier based on texture and nuclei-concentration features was designed. The nuclei concentration in cancerous sites is different from that in nondiseased sites and thus can be used to distinguish the two. The third part of the thesis analyses RCM video sequences of in-vivo skin imaged at the level of the DEJ. The boundaries of superficial skin capillaries can be delineated by visually observing the highly reflective red blood cells (RBCs) passing through the capillaries. An algorithm that automatically detects skin capillaries in RCM video sequences was developed. Additionally, an algorithm that quantifies the velocity of RBCs in cross-sectionally imaged capillaries is devised. The change in total capillary area (per unit frame area), individual capillary parameters and RBC velocity due to incremental ultra-violet radiation (UVR) doses are analysed in both fair and dark skinned volunteers. The work presented in this thesis has the potential to increase the acceptance of RCM in the dermatology clinic, both for diagnosis and for assessing treatment response of skin conditions located at (or above) the DEJ. Additionally, the thesis enhances the potential of using RCM images of excised samples instead of preparing the tissue for histological examinations during surgery.

Dystrophic epidermolysis bullosa results from mutations in COL7A1 that encodes collagen VII, the major component of anchoring fibrils. General paradigms have emerged attributing dominant DEB to heterozygous glycine substitutions and recessive DEB to nonsense, frameshift or splice site mutations on both COL7A1 alleles. Several aspects of the genotype-phenotype heterogeneity encountered in DEB remain unexplained, although genetic, epigenetic and environmental modulators have been implicated. In this thesis, various aspects of DEB were studied, in a bid to refine genotype-phenotype correlation. A detailed analysis of missense GS and non-GS mutations identified 57 novel mutations and was in-keeping with the general established paradigms. Unique clinical entities such as EB-pr and BDN were studied. The role of the matrix metalloproteinase-1 promoter polymorphism, -1607 1G/2G, on disease modification in EB-pr was explored, but was shown to be an unlikely modulator. A large study of BDN, highlighted that intracytoplasmic retention of collagen VII and stellate bodies were not exclusive to BDN and can be associated with various subtypes of DEB, non-EB cases and normal skin. The first case of revertant mosaicism in DEB was studied revealing intragenic cross-over as the underlying mechanism. Finally, intradermal injection of allogeneic fibroblasts was shown to result in increased and sustained expression of COL7A1 possibly through the upregulation of HB-EGF. Recognition of disease modifiers in DEB and refinement of genotype–phenotype correlation will not only further our understanding of DEB but will have implications on diagnosis, counselling and prognosis through patient specific and targeted therapy.

No description available.