No description available.

No description available.

No description available.

A limited number of studies have investigated the concept of using enteric probiotic bacteria to alter microbial communities in areas other than the gut. The use of enteric probiotics is attractive because they are considered generally as non-pathogenic and safe. The aim of this thesis was to explore the possibility that probiotic bacteria, or material derived from them, have utility for skin in health and disease. Primarily, the thesis investigates whether probiotics can protect skin cells from the effects of the common pathogen, S. aureus, and whether probiotics can accelerate the wound healing process. Furthermore, some of the underlying mechanisms were investigated. In the first study, the potential of probiotics to protect primary human keratinocytes from the effects of S, aureus was investigated. When primary human keratinocytes were exposed to S. aureus, only 25% remained viable following 24h incubation. However, in the presence of 108cfu/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57%. Interestingly, L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes (P=0.006, P=0.01) following the same period of incubation. Keratinocyte survival was enhanced significantly, regardless of whether the probiotic was applied in the viable form, or as cell lysates, 2h before or simultaneously (P=0.005) or 12h after (P=0.01) to S. aureus infection. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion; however, only viable bacteria or the lysate could displace S. aureus (P=0.04 and 0.01 respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid (Chapter 3). Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion. This idea has been confirmed by partial purification of L. rhamnosus GG lysate. Fractionation of the lysate demonstrated that the protective effect of L. rhamnosus GG lysate depends not only on inhibitory substances, but also on anti-adhesion substances present in the lysate (Chapter 4). The L. rhamnosus GG lysate increased the re-epithelialisation rate of model wounds in vitro in a scratch assay(P=0.02) (Chapter 5). Furthermore, L. rhamnosus GG lysate stimulated the re-epithelialisation of ex-vivo skin cultures (Chapter 6). In vitro and ex-vivo proliferation and migration assays demonstrated that L. rhamnosus GG lysate significantly increased keratinocyte proliferation and migration relative to controls; however, the dominant mechanism was migration (Chapter 5). Therefore, the pathways underlying these effects were explored by doing Affymetrix analysis for genes expressed in response to treatment with L. rhamnosus GG lysate. The results highlight that the CXCR2/CXCL2 and FGF7/FGFR2 are up-regulated, which may mediate the acceleration of re-epithelialisation of keratinocytes through stimulation of cell migration under the effect of L. rhamnosus GG lysate. In summary, these data suggest that L. rhamnosus GG and its extract may be a useful method of counteracting S. aureus infection and reducing the toxicity of pathogens. At the same time, specific probiotic lysates may be useful for improving the wound healing process and ultimately reduce the severity of impaired wounds in the community. Thus, the use of safe probiotic bacteria or lysates presents new options for the development of a new treatment that could improve wound healing while simultaneously reducing infection.

After cutaneous injury, wound healing is an essential process that restores barrier and homeostatic function to the skin. Tissue restoration is classically grouped into four phases, involving the dynamic, regulated and sequential interaction of multiple cells types, effector molecules and extracellular matrix components. While most wounds heal in a timely fashion, local and systemic factors can prevent wound resolution resulting in chronic wound formation. Examples include diabetic and venous ulcers, which are costly, prone to recurrence and associated with reduced quality of life for sufferers. Poor therapeutic efficacy has driven the development of new treatment modalities including bioengineered skin substitutes. First developed in the 1980’s, these biomaterials have become increasingly diverse in their structure, cellular content and biomechanical properties. Their evidence base in chronic wound management has increased, though their exact mode of action remains elusive. Furthermore, skin substitute use in acute human wounds has never been studied and it is unclear whether they have a role in this setting. This thesis evaluates a novel acellular dermal skin substitute, known as Decellularised Dermis (DCD), through 2 large human studies involving clinical and laboratory components. In the first phase, a pilot study utilising DCD as part of a management strategy for treatment-resistant chronic wounds resulted in healing rates of 60% after six months with average wound surface area reduction of 87%. Additionally, there was significantly increased angiogenesis 6 weeks after treatment and a statistical association between ulcer duration pre-therapy and likelihood of successful treatment. In the second phase, a prospective cohort study was undertaken involving acute wounds in fifty healthy volunteers. DCD was compared to controls, autografts and collagen-GAG scaffolds. Whole genome micorarrays demonstrated that treatments exerted differential effects upon the type, magnitude and temporality of gene expression with resultant variation in key processes during wound healing including angiogenesis, fibroplasia and scar formation. Concurrently, skin substitute architecture strongly influenced the influx of host cells into biomaterials and dictated reformation of the dermis and vasculature. These data reveal that DCD has significant pro-angiogenic effects in both acute and chronic wound settings. Moreover, there was reduced fibrosis 6 weeks after wounding in DCD-treated wounds compared to controls. A number of potentially significant genes were identified including prokineticin 2 and membrane type-6 matrix metalloproteinase that may underlie these findings and represent potential therapeutic targets. In conclusion, these studies effectively evaluate DCD, demonstrating potential therapeutic roles in chronic wounds and scar reduction through genetic and translational modification of angiogenesis and fibroplasia during wound healing. Furthermore, optical coherence tomography was shown to be an effective diagnostic tool with potential use in assessment of cutaneous morphology and quantitation of tissue fibrosis.

The objectives of this study were threefold: to determine the prevalence of skin diseases among primary school children in the UAE; to assess the ability of the primary health care physicians (PCPs) to diagnose skin diseases; and to plan a dermatological education program for children of the UAE. An epidemiological survey included the clinical examination of selected school children by two quantified dermatologists and the relationships between the prevalence of skin diseases and a range of hygiene and socioeconomic factors were examined.1536 Local schoolchildren aged 6-9 years were included. The prevalence of common skin diseases in the children was 19%. The most common skin diseases included: dermatitis (8.1%), viral diseases (3.8%), and pigmentation disorders (1.7%). Selected skin diseases showed significant associations with a range of socioeconomic and hygiene factors on multivariate analyses. Physicians were better at correctly diagnosing common skin diseases compared with the less frequent, or rare skin diseases. Finally, PCPs with specific training in dermatology showed more ability to diagnose a range of skin diseases compared with those with no relevant training. Based on the results of this study, an improvement in socioeconomic and personal hygiene conditions along with the initiation of pertinent educational programs for children, patients and teachers may reduce the prevalence of some skin disorders. Important aspects of such dermatological health care programs are proposed. In addition, specific dermatological training for PCPs should be considered to increase their ability to diagnose and manage common skin diseases in children.

Psoriasis is a chronic autoimmune skin disease characterised by hyperplasia of epidermal keratinocytes and the accumulation of activated immune cells at sites of the disease. The disease is associated with aberrant activation of phagocytes, T-lymphocytes and the production of pro-inflammatory cytokines and chemokines. This thesis examines the therapeutic efficacy and mechanisms of action of the ginger species Halia Bara, or Zingiber officinale Roscoe var. rubrum (ZOR), on key immunopathogenic mechanisms relevant to psoriasis. In-depth experiments first determined the effect of a ZOR extract in chloroform (HB02) and its fractions on nitric oxide (NO) and prostaglandin E2 (PGE2) production. The results of these experiments showed that HB02 and its fractions efficiently inhibited NO and PGE2 production by activated macrophages. Extensive fractionation and characterisation experiments succeeded in identifying two compounds 6-shogaol (6S) and 1-dehydro-6-gingerdione (GD) with potent inhibitory effects on NO and PGE2. These effects were comparable to dexamethasone and indomethacin. Studies on the effects of HB02, its fractions and compounds showed inhibitory effects on the level of mRNA for iNOS, TNFα, IL-12p40 and IL-23p19 in pre-treatment experiments of macrophages. Studies of cell migration showed that the fractions and compounds from ZOR inhibited the migration of polymorphonuclear neutrophils (PMNs) through human vascular endothelial cells (HUVEC) by influencing CD11b expression and CD62L shedding. Further studies showed that the ZOR samples also inhibited the activation of CD8+ cytotoxic T-lymphocytes and reduced CD25 and CD69 expression. Furthermore, an in vitro model of epidermal inflammation showed that ZOR directly inhibits keratinocyte proliferation and the production of IL-20 and IL-8, both key psoriasis-promoting cytokines. The studies reported in this thesis provide experimental evidence for potent anti-inflammatory properties of ZOR and for potential mechanisms of action in ameliorating psoriasis.

Background: Atopic Eczema (AE) is a common skin disease that results from a complex interplay between genetic and environmental factors. It may be associated with other atopic phenotypes including; asthma, hayfever and food allergy. The onset, typically in early childhood, can affect any part of the body but often occurs in the flexures of the elbows and knees. Aim and hypothesis: The aim of this thesis was to investigate the genetics of AE in the Bangladeshi families of East London to identify possible positive association with AE or other atopic diseases. This involved investigating previously associated genes and also novel genes mapping to regions showing suggestive linkage from a microsatellite genome scan and data from a haplotype tagging SNP based Illumina based Array genotyping. Methods: A total of 70 families (n=384) individuals were recruited via the Paediatric dermatology Clinic at the Royal London Hospital. Three genes; FOXP3, SPINK5 and TNC were assessed for genetic association with AE and other atopic phenotypes using a combination of techniques, such as PCR, dHPLC, sequencing, and Taqman SNP assays. FBAT software was used for statistical analysis. Results and future work: Suggestive associations with AE and other allergic disorders were identified in all genes examined however, none of these results remained significant (p-value<0.05) after correcting for multiple testing. As part of this project, several novel genes have been identified including TNC, JAK3 and KYNU. To follow up these promising findings, replication studies could be conducted in other populations with AE in particular by large case-control analyses. In addition, with the development of high throughput SNP genotyping and, in particular, exome sequencing, the larger Bangladeshi families with multiple AE affected could be investigated to identify AE-associated disease mutations

Background: Cutaneous squamous cell carcinoma (SCC) is the second most common cancer to affect populations of European descent. It is potentially fatal and causes substantial morbidity. Up to 70% of SCC arise from clinically identifiable precancerous lesions, known as actinic keratoses (AK), permitting early diagnosis and intervention. Keratinocyte Intra-epidermal Neoplasia (KIN) are the histological representation of AK. Immunosuppressed organ transplant recipients have 60 – 200 fold higher rates of SCC that are characteristically multiple, aggressive, recurrent and have higher metastatic tendency. The molecular pathogenesis of SCC involves the accumulation of multiple genetic and epigenetic aberrations, but in spite of the magnitude of the problem, the cellular basis for SCC has received little scientific attention compared with other epithelial cancers. Methodology: Skin samples representing sequential keratinocyte carcinogenesis (from non sun-exposed, sun-exposed and AK/KIN skin) were collected from 53 individuals (31 immunosuppressed and 22 immunocompetent), 30 of whom underwent an additional biopsy after prospective treatment with one of three commercially available topical agents. Genome-wide single nucleotide polymorphism (SNP) profiling was performed on all samples and transcriptional profiling was performed on 7 series from each treatment group. Results: The mean number of SNP changes was 3.57 and 3.48 in NSE and SE skin, respectively, increasing to 6.93 in KIN. Several recurrent areas of loss or gain were identified in KIN, specifically deletion at 3p, 9p, 12q, 16q, 18q and X and amplification of 8q, 9p and 17p. Expression profiling identified 428 genes (false discovery rate 0.05) with altered expression in KIN skin compared to paired normal skin. Of these genes, 31% displayed concordant change with regions of copy number change. Several gene networks of interest were identified including F- and -actin, NFB, PPAR and TGF suggesting that they may be key pathways in KIN evolution. Several candidate genes in KIN skin continue to be dysregulated in SCC, some of which, such as ANG, S100A9, ACVR2A, FHL1 and SFN resolve after topical chemoprevention and others, WIF1, CCL27 and LEPR persist. Conclusions: This is the first systematic, in-depth, in vivo study of molecular events characterising KIN and provides a detailed profile of the critical events contributing to malignant progression in keratinocyte neoplasia and the molecular effects of widely used topical chemopreventive agents Together, these data provide clinically relevant insights into cutaneous squamous tumour biology and identify biomarkers with diagnostic, prognostic and therapeutic potential. Acknowledgements

Desmosomes are intercellular junctions that connect intermediate filaments (IFs) of adjacent cells within tissues, generating a large and mechanically resilient network. Desmosomes are prominent in epithelia and the myocardium. Their importance for maintenance of the strength and flexibility of these tissues is underlined by mutations in desmosomal genes which compromise skin or heart and in some instances both. This thesis is focused on two obligate plaque proteins of the desmosome, desmoplakin (DP) and plakoglobin (PG), and their role in disease of skin and heart. A novel homozygous nonsense mutation, p.S24X, in the gene encoding PG, JUP, was identified in three non-consanguineous Argentinean patients, resulting in skin fragility, palmoplantar keratoderma (PPK) and woolly hair with no symptoms of cardiomyopathy. p.S24X mRNA was readily detected and an alternative AUG codon translates an N-terminally truncated PG, which is expressed in patient and parental skin samples at barely detectable levels. At the same time another novel homozygous mutation, c.468G>A, was identified in JUP causing a very similar phenotype in two Kuwaiti siblings and resulting also in very low levels of PG in the skin. These results suggest that PG is essential for maintenance of skin integrity, but not for normal heart development in children. The gene encoding DP, DSP, produces two alternative splice variants, DPI and DPII. A novel DP alternative splice isoform, DPIb, is described. As shown by RT-PCR, DSPIb is expressed in several epithelial and cardiovascular tissues and is the only DSP isoform detected in the aorta. Western blotting proteins from HaCaT and K1 cells identified this novel isoform, which has been previously missed due to its similarity in size to DPII. DPIb may help compensate defects in patients with DSPI-specific mutations. The molecular mechanisms of three different DSP mutations leading to skin disease or skin disease associated with cardiomyopathy were investigated. Mutations causing DPI/DPII haploinsufficiency and DPI knock-out lead to decreased expression levels of the desmosomal proteins DSC2, DSC3 and PKP1 in an experimental system using HaCaT keratinocytes. Expression of a mutant DSPI construct harbouring a dominant 30 bp insertion results in the formation of large DPI aggregates at the cell membrane of HaCaT cells. The spectrum of DSP and JUP mutations which cause different clinical phenotypes is discussed.