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Background Human T cell Lymphotropic Virus Type I (HTLV-I) associated infective dermatitis, first described by Sweet in Jamaican children, is a pattern of eczema characterized by exudation, crusting around the nostrils, ears and scalp with eventual appearance of a generalized fine papular rash. More recently LeGranade and co-workers have proposed major and minor criteria in establishing the diagnosis of HTLV-I associated infective dermatitis (HAID). HTLV-I has been aetiologically linked to Adult T cell leukaemia/lymphoma (ATLL) and tropical spastic paraparesis (TSP). HAID is not only a marker of childhood infection with HTLV-I but may be a harbinger of more serious HTLV-I associated diseases later on in life such as ATLL or TSP. The pathogenesis of HAID is poorly understood so are the histopathological features of this entity. The effects of co-infection with human immunodeficiency virus- 1 (HIV-1) are inconclusive. HAID is described in Sub Saharan Africa, Senegal but no data is published on this entity in Southern Africa, characterizing the clinical, laboratory features and the histopathology of this entity. Aims and Objectives 1) To describe the clinical and histological features of HTLV-I associated infective dermatitis in KZN, South Africa 2) To determine the virological characteristics of HTLV-I in KZN, South Africa 3) To assess for HTLV-I / HIV co-infection Methods This was a prospective study of all patients with HAID who presented to King Edward VIII hospital (KEH), outpatient department over a period of 42 months. These were patients who fulfilled the clinical criteria of HAID. Enrolled patients were subjected to a confirmatory HTLV-I serology testing. Demographic data was obtained from all HTLV-I seropositive patients. Their clinical examination included dermatological, neurological and pathological examination. A blood count, immunoglobulin levels, serum protein electrophoresis measuring albumin levels and globulin fractions were measured. For bacteriological assessment skin swabs were taken from the affected sites with stool samples examined for parasites, ova and cysts. The HIV-1 status together with HIV-1 viral load were determined on those enrolled. The CD4 count, CD8 counts and CD4/CD8 ratio were also calculated. Skin biopsies were taken for histological examination. PCR for HTLV subtyping was performed on a subset of the cohort. Results Demography Of the 60 patients recruited, 33 fulfilled criteria for HAID. The majority of patients fell between age categories of 6 to lOyears. The male to female ratio was 1:1. There were more females in the adult group than there were within the childhood group. All of the patients in our cohort were African. Clinical features The lesions were erythematous, scaly, exudative, and crusted in all cases. The distribution of lesions was as follows: scalp (77.4%), retroauricular areas (71%), the axilla (65%) and paranasal areas (58%) were the sites more commonly affected. Nasal crusting was not a significant feature in this series. Bacteriology Culture was positive for Staphylococcus aureus (S. aureus) in 90%, with streptococcal group of organisms found in 68% of the skin swabs taken from the lesional skin. Haematological Our patients were mildly anaemic as has been shown in previous studies. They had a mean Hb of 11.5g/dl. In 12 of the 14 patients tested, the erythrocyte sedimentation rate (ESR) was elevated. Serum protein electrophoresis and levels of Immunoglobulin A, G and M were raised. The mean CD4 count in the entire group was elevated at 1730 cells/fil, CD8 was 1299 cells/ul Histopathology The major histological findings were as follows: 38% demonstrated a superficial and deep perivascular inflammatory infiltrate, 28% had a superficial and deep perivascular inflammatory infiltrate together with a lichenoid dermatitis, 12.9% had features of superficial and deep inflammatory infiltrate with an interface dermatitis, 6.4% revealed features of seborrhoeic dermatitis. Genotyping Our patients were infected with the strains belonging to the Cosmopolitan, A Subtype (HTLV-Ia). Complications Complications were low in this series with the commonest being scabies in 6(18.1%), corneal opacities in 3(8.6%), 2(6 %) with HAM/TSP. No parasitic worm infestations were isolated. HIV/HTLV-I co-infection Of the 33 patients, 9 (30 %) were co-infected with HIV. The mean viral load in this group was 52 000 copies/ml. Their mean CD4 count was also elevated at 1505cells/^il with a CD8 of 1704 cells/Mi and a CD4/CD8 ratio of 1.15. Discussion Thirty three of the 60 patients enrolled met the diagnosis for HAID according to the established criteria. The mean age in this series was 17 years (range: 8 months-46 years)however; almost a third (30.3%) were children under 12 years, reinforcing the entity as a childhood infective condition. There was an equal male female distribution in the childhood group and a female predominance in the adult group. Clinically patients presented with infected erythematous, scaly lesions mainly on the scalp, neck and post- auricular area. The clinical features were in keeping with other series worldwide. The complication rate was low in our cohort. S. aureus was the predominant organism in both anterior nares and lesional skin. The most common histological pattern was superficial and deep perivascular inflammatory infiltrate. The subtype in our series was the Cosmopolitan Subtype A (HTLV-Ia) as opposed to subtype B in Japan. We share with Brazil a common subtype. A subset of our patients (30%) was co-infected with HIV. The CD4 cell count in this subgroup was lower than the entire group but this was not statistically significant. The histological patterns found in this subgroup infected with HIV were similar to the rest of the group except for a more intense eosinophilic infiltrate in these skin biopsy specimens. Conclusion HTLV-I associated infective dermatitis is distinct entity which affects the African population of KwaZulu Natal, South Africa. It is predominantly a disease of childhood with an equal female to male ratio in children. The clinical features are an exudative, erythematous scaly rash most commonly found involving the scalp, axillae, paranasal and retroauricular areas. HTLV-I positivity is essential for the diagnosis; the Cosmopolitan Subtype A is commonest in South Africa. The commonest histological pattern is a superficial and deep perivascular infiltrate in 38%. A subset, 30%, was co-infected with HIV. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2008.

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The purpose of this study was to investigate ROCK2 activation in squamous cell carcinogenesis and assess its co-operation with rasHa and fos oncogene activation together with loss of PTEN mediated AKT regulation. The analysis of ROCK deregulation with these genes in the MAP Kinase and PI3K pathways, two of the most commonly deregulated signalling systems, employed a well-characterised, transgenic mouse skin model of multi-stage carcinogenesis. A major goal was to study co-operation between these genes in the conversion of benign tumours to malignancy and investigate subsequent progression to aggressive carcinomas, given these are the most significant clinical stages of carcinogenesis from a patient’s viewpoint; and also investigated effects of ROCK2 deregulation on the processes of normal epidermal differentiation. ROCK2 is an effector protein of RhoA, which is a member of the ras superfamily and ROCK2 activation has been associated with tumour progression via an increase in tissue stiffness mediated by changes in actomyosin cytoskeleton leading to increased cellular motility. Thus, ROCK2 expression is commonly associated with the later events in cancer. Furthermore, there are many studies investigating ROCK2 in cancer given that it may be a useful therapeutic target in being downstream of oncogenic ras signalling. Yet, relatively few studies have explored the possibilities of a definite link that confirms the co-operation status of ROCK2 activation with ras/MAPK/fos and /or PTEN/PI3K/AKT pathways in SCC aetiology. Thus, questions exist as to exactly when does ROCK2 activation become causal; and what are the collaborating genes involved in the mechanism that drive the early or late stage events in carcinogenesis. To begin to answer these questions, inducible ROCK2 activation has been introduced into a well-characterised transgenic mouse skin carcinogenesis model that expressed a combination of ras and fos activation, driven by a modified human keratin 1 vector (HK1). Thus, exclusive epidermal expression of activated rasHa and fos oncogenes, in proliferative basal layers, gave hyperplasia and papillomatogenesis; but with no evidence of spontaneous malignant conversion. This stability of phenotype is thus ideal to assess roles for multiple transgene co-operations in the development of benign tumours and their conversion to malignancy. Hence, ROCK transgenic mice that expressed a conditionally active, 4-hydroxytamoxifen (4-HT)-regulated of human ROCK2 transgene were crossed with mice expressing activated rasHa and /or fos exclusively in epidermal transit amplifying keratinocytes (HK1.ras, HK1.fos). Inducible PTEN tumour suppressor gene mutation via exon 5 ablation (K14.Cre/D5PTENflx) and thus loss of AKT regulation was also incorporated into this model. This was achieved via deletion of exon 5 employing the RU486-mediated cre/loxP system, driven by keratin K14 promoter expression in basal layer keratinocytes. Therefore, to facilitate this investigation, a new and unpublished inducible ROCK2 system was employed in order to target the identical keratinocytes as PTEN loss. This new transgenic line of lsl.ROCKer transgenic mice employed the same 4-HT inducible ROCKer transgene but now driven by a generic CAG promoter following cre mediated ablation of the stop cassette once treated with RU486. In bi-genic K14.ROCKer/HK1.ras1205 mice, synergism between ROCK2 activation and (wound-promotion) sensitive HK1.ras1205 line showed direct co-operation and achieved malignant conversion of benign papillomas to well-differentiated squamous cell carcinoma (wdSCCs) histotypes (12 weeks of 4-HT treatment). This placed ROCK2 activity as the causal event driving malignant conversion, but in the absence of a wound promotion stimulus (loss of ear tag), papillomas did not convert. The correct papilloma context was required for ROCK to become causal proved to be the case on employing the wound insensitive HK1.ras1276 line. Here, K14.ROCKer/HK1.ras1276 mice failed to exhibit any papillomas and required the constitutive promotion stimulus from additional fos activation. Interestingly, following cessation of 4-HT, two intriguing observations were recorded. Firstly, that once bi-genic ROCK/ras1205 achieved malignancy, exogenous ROCKer expression appeared to show no involvement once squamous cell carcinomas (SCCs) progressed to poorly differentiated squamous cell carcinomas (pdSCCs), given the elevated expression of endogenous ROCK upon malignant progression. Secondly, the rapid growth of papilloma appeared upon cessation of 4-HT with highly intense p21 expression indicated the requirement of exogenous ROCK2 for malignant conversion and the possibility of a papilloma inhibition by 4-HT anti-cancer activity. Another major novel finding demonstrated ROCK2 activation could act as an initiator in co-operation with fos activation. Direct co-operation between ROCK2 and fos activation produced benign squamous papillomas yet, whereas ROCK2 activation alone induced hyperplasia as did fos activation alone at this time point, given papilloma formation in HK1.fos mice occur over 4-5 months. However, unlike deregulation of MAP Kinase signalling in bi-genic ROCK/ras1205 mice, in bi-genic ROCK/fos mice, no malignant conversion was observed due to high levels of compensatory p53/p21 expression. Thus, this bi-genic K14.cre/lsl.ROCKer/HK1.fos model suggests the requirement of additional mutation event for malignant conversion. An unexpected result appeared in bi-genic ROCK/Δ5PTENflx co-operation experiments where K14.cre/lsl.ROCKer/Δ5PTENflx cohorts exhibited epidermal hyperplasia with folded papillomatous appearance to the epidermis, but without papillomatogenesis even after seven month of period. This either indicates a weak co-operation between ROCK2 and Δ5PTENflx which may be due to the unexpected low levels of p-AKT from a compensatory increased p21 expression; and additional events needed to fill in the oncogenic gap in this bi-genic ROCK/Δ5PTENflx model, or may possibly highlight redundancy in the oncogenic hits provided by ROCK and PTEN. This latter suggest similar links exist between their normal roles in the epidermis which may be accountable for the alterations observed in keratinocyte differentiation. In both in vitro and in vivo experiments, K14.cre/lsl.ROCKer/Δ5PTENflx cohorts showed alterations in epidermal differentiation via anomalous K1 and low levels of K6 expression. Interestingly, activated ROCK2 appeared to induce or accelerate differentiation activity in K14.cre/lsl.ROCKer keratinocytes via increased K1 (early differentiation marker) and reduced K6 (proliferation marker) expression profiles. These results were consistent with in vivo data where K6 was expressed in low levels in K14.cre/lsl.ROCKer hyperplasia histotypes. In contrast, in K14.cre/lsl.ROCKer/Δ5PTENflx keratinocytes, inactivation of PTEN-mediated AKT activity may be accountable for restored keratin K6 and anomalous keratin K1 expression; as keratin K1 was expressed in a similar fashion of normal keratinocytes in K14.cre/lsl.ROCKer/fos keratinocytes. Interestingly, all tri-genic cohorts: K14.cre/lsl.ROCKer/ras1276/fos, K14.cre/lsl.ROCKer/fos/Δ5PTENflx and K14.cre/lsl.ROCKer/ras1276/Δ5PTENflx synergisms exhibited malignant conversion and /or malignant progression in all animals highlighting a novel role of ROCK2 activation. Further, the stage-specific consequences in each model in this study were shown to be influenced by p53/p21 status, where typically p53 expression disappeared in late stage papillomas yet, p21 expression persisted. This demonstrated the importance of compensatory p53/p21 expression in modulating tumour pathogenesis in all these models. Given that this study incorporated PTEN mutation, the influence of AKT activity was investigated in the SCC progression of tri-genic ROCK/ras1276/PTENflx and ROCK/fos/PTENflx cohorts; revealing a crucial antagonism between p21 and AKT. However, this study revealed that the malignancy in tri-genic ROCK/ras1276/fos cohorts was not influenced by p-AKT expression, and as this tri-genic model achieved wdSCCs only. This suggests that as the roles of ROCK in altering cytoskeletal organisation leading to increase in tissue stiffness are overlaid onto both MAP Kinase and AKT deregulation, the outcome is a very aggressive pdSCC. Thus, suggesting ROCK signalling to be a potential therapeutic target for ras/MAPK/fos pathway in carcinogenesis. Overall, this study showed the involvement of ROCK2 activation in the initiation stage for papillomatogenesis with fos oncogene, and demonstrated ROCK2 as a converter again and also in malignant progression with ras/fos/Δ5PTENflx mutations. This indicates the link of ROCK2 signalling with both MAPK and PI3K pathways, thus targeting ROCK2 would aid in development of cancer therapy.

The human epidermis is a self-renewing stratified epithelial tissue that forms the outermost protective layer of the skin. The epidermis is comprised of a number of cell types, the most abundant of which are keratinocytes. Normal function of the epidermis requires that keratinocyte proliferation, differentiation, and apoptosis be precisely regulated and a failure to regulate these processes is a feature of many skin diseases. Although the precise mechanism by which epidermal homeostasis is regulated is still far from clear, much progress has been made in the characterisation of signaling pathways involved in normal epidermal function. A key group of signaling proteins that have been clearly implicated in epidermal function are the Rho family of small GTP-binding proteins. This thesis focuses on one member of the family, RhoE/Rnd3, and the analysis of the role it plays in the regulation of proliferation, differentiation, apoptosis and cell-cell adhesion in the epidermis. Use of RNA interference to specifically ‘knock-down’ expression of RhoE has led to the discovery of a novel role for RhoE in regulation of cell-cell adhesion and apoptosis. Loss of RhoE expression resulted in keratinocytes developing resistance to apoptosis mediated via either the intrinsic or extrinsic pathways. RhoE depletion was also associated with increased expression of desmosomal proteins and increased numbers of desmosomes. Resistance to apoptosis was shown to be a function of desmosome-mediated cell-cell adhesion and a component of demosomes – plakoglobin – was shown to play a key role in RhoE-mediated resistance to apoptosis.

Thesis (M.S.)--Rochester Institute of Technology, 2009. / Typescript. Includes bibliographical references (p. 97-105).