Lucy Ghali

Smooth muscle cholinergic denervation hypersensitivity in diverticular disease

BACKGROUND Evidence from clinical and laboratory investigations into the causes of diverticular disease suggests that disturbances in cholinergic activity are important, the effector mechanisms of which have yet to be established. We aimed to investigate the role of smooth muscle and neural cholinergic activity in the pathogenesis of this disease. METHODS Two investigators independently did a blinded immunohistochemical image analysis of localising antibodies to choline acetyltransferase, co-localised with protein gene product (PGP)--a marker of general neural tissue-and smooth muscle muscarinic M3 receptors, on three histological sections of sigmoid colons from ten patients with diverticular disease and ten controls, after resections for rectal tumours. We also did isotonic organ bath experiments to assess muscle strip sensitivities to exogenous acetylcholine. FINDINGS In circular muscle, activity of choline acetyltransferase was lower in patients with diverticular disease than in controls: median percentage surface area of choline acetyltransferase over PGP was 17.5% (range 10.0-37.0) in patients with diverticular disease and 47.0% (29.0-54.0) in controls (p<0.0001). M3 receptors were upregulated in patients with diverticular disease compared with controls: the median surface area was 13.2% (6.0-23.3) in patients with diverticular disease and 2.5% (1.6-3.7) in controls (p<0.0001). The sensitivity to exogenous acetylcholine was increased in patients with diverticular disease (mean -log EC(50) 5.6 [SD 0.3]) compared with controls (4.9 [0.5]; difference 0.7 [95% CI 0.3-1.1], p=0.006). In longitudinal muscle, choline acetyltransferase activity was lower in patients with diverticular disease (median 19.5%, range 12.0-30.0) than in controls (47.0%, 35.0-60.0; p<0.0001), with upregulation of M3 receptors in diverticular disease (diverticular disease 7.8% [1.9-20.4], controls 1.7% [0.8-3.0]; p<0.0001). However, sensitivity to exogenous acetylcholine did not differ between the two groups (diverticular disease mean 5.6% [SD 0.3], controls 5.2% [0.4]; difference 0.4% [95% CI -0.02-0.7], p=0.06). INTERPRETATION Our results suggest that cholinergic denervation hypersensitivity can affect smooth muscle. Upregulation of smooth muscle M3 receptors might account for specific clinical, physiological, and pharmacological abnormalities associated with diverticular disease.

Laser modulation of angiogenic factor production by T-lymphocytes.

In previous investigations, small variations in the energy densities of low level light therapy (LLLT) were found to produce significant differences in the proliferation of resting T‐lymphocytes in vitro. Pulsing these cells with mitogen in addition to laser therapy produced inhibitory effects regardless of the amplitude of the energy density used. In the current study, the effect of LLLT on the production of angiogenic factor(s) by T‐lymphocytes was investigated in vitro.

The E7 Protein of Cutaneous Human Papillomavirus Type 8 Causes Invasion of Human Keratinocytes into the Dermis in Organotypic Cultures of Skin

Human papillomaviruses (HPV) have been implicated in the development of nonmelanoma skin cancer (NMSC). The molecular mechanisms by which these viruses contribute towards NMSC are poorly understood. We have used an in vitro skin-equivalent model generated by transducing primary adult human epidermal keratinocytes with retroviruses expressing HPV genes to investigate the mechanisms of viral transformation. In this model, keratinocytes expressing HPV genes are seeded onto a mesenchyme composed of deepidermalized human dermis that had been repopulated with primary dermal fibroblasts. Expression of the HPV8 E7 gene caused both an enhancement of terminal differentiation and hyperproliferation, but most strikingly, the acquisition of the ability to migrate and invade through the underlying dermis. The basement membrane integrity was disrupted in a time-dependent manner in areas of invading keratinocytes, as evidenced by immunostaining of its protein components collagen types VII, IV, and laminin 5. This was accompanied by the overexpression of extracellular matrix metalloproteinases MMP-1, MMP-8, and MT-1-MMP. These results suggest that the cutaneous HPV type 8 that is frequently found in NMSC of epidermodysplasia verruciformis patients may actively promote an invasive keratinocyte phenotype. These findings also highlight the importance of epithelial-extracellular matrix-mesenchymal interactions that are required to support cell invasion.

Role of HPV E6 proteins in preventing UVB-induced release of pro-apoptotic factors from the mitochondria

Apoptotic elimination of UV-damaged cells from the epidermis is an important step in preventing both the emergence and expansion of cells with carcinogenic potential. A pivotal event in apoptosis is the release of apoptogenic factors from the mitochondria, although the mechanisms by which the different proteins are released are not fully understood. Here we demonstrate that UV radiation induced the mitochondrial to nuclear translocation of apoptosis inducing factor (AIF) in normal skin. The human papillomavirus (HPV) E6 protein prevented release of AIF and other apoptotic factors such as cytochrome c and Omi from mitochondria of UV-damaged primary epidermal keratinocytes and preserved mitochondrial integrity. shRNA silencing of Bak, a target for E6-mediated proteolysis, demonstrated the requirement of Bak for UV-induced AIF release and mitochondrial fragmentation. Furthermore, screening non-melanoma skin cancer biopsies revealed an inverse correlation between HPV status and AIF nuclear translocation. Our results indicate that the E6 activity towards Bak is a key factor that promotes survival of HPV-infected cells that facilitates tumor development.

LEKTI demonstrable by immunohistochemistry of the skin: a potential diagnostic skin test for Netherton syndrome

Background   Netherton syndrome (NS) is a rare autosomal recessive condition characterized by ichthyosiform erythroderma, trichorrhexis invaginata and atopic manifestations. Confirming the diagnosis may be difficult in the early stages. Mutations in the SPINK5 gene which encodes for the serine protease inhibitor LEKTI are associated with NS. These mutations create premature termination codons which result in absent or abnormal expression of LEKTI in patients with NS.

Role for WNT16B in human epidermal keratinocyte proliferation and differentiation

WNT signalling regulates a variety of cell functions including cell fate, polarity, and differentiation via the canonical or β-catenin stabilisation pathway and/or the planar cell polarity or non-canonical pathway. We have previously demonstrated that two isoforms (A and B) from the WNT16 locus have differential expression in various adult human tissues. In this study we show that WNT16B but not WNT16A isoform was upregulated in basal cell carcinomas compared with normal skin. We further investigated the cellular and molecular functions of WNT16B in primary human epidermal keratinocytes and a keratinocyte cell line. Cellular expression of WNT16B neither stabilised β-catenin nor activated the lymphoid enhancer factor or T-cell factor transcriptional reporter in primary keratinocytes. WNT16B activated the Jun-N-terminal kinase cascade suggesting the activation of a non-canonical WNT signalling pathway. Constitutive expression of WNT16B significantly enhanced the rate of cell proliferation and prolonged clonogenicity in primary keratinocytes. Silencing WNT16B by RNA interference reduced keratinocyte proliferation. Furthermore, overexpression of WNT16B induced a hyperproliferation phenotype in an organotypical culture system. This work presents the first evidence that WNT16B activates human keratinocyte proliferation possibly via a β-catenin-independent non-canonical WNT transduction pathway.

Longitudinal muscle shows abnormal relaxation responses to nitric oxide and contains altered levels of NOS1 and elastin in uncomplicated diverticular disease

Objective  Recent evidence challenges the ‘low‐fibre/high‐colonic intraluminal pressure’ hypothesis of diverticular disease (DD) and raises the possibility that other mechanisms are involved. Although bowel wall smooth muscle is known to be hypercontractile in DD, the nature of its relaxation is unknown. The present study investigated colonic smooth muscle responses to nitric oxide, as well as the smooth muscle contents of neural nitric oxide and elastin associated with the disease.

Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours

The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs.

Expression of keratin K2e in cutaneous and oral lesions: association with keratinocyte activation, proliferation, and keratinization.

The cytoskeleton in keratinocytes is a complex of highly homologous structural proteins derived from two families of type I and type II polypeptides. Keratin K2e is a type II polypeptide that is expressed in epidermis late in differentiation. Here we report the influence of keratinocyte activation, proliferation, and keratinization on K2e expression in samples of cutaneous and oral lesions. The normal expression of K2e in the upper spinous and granular layers of interfollicular epidermis is increased in keloid scars but showed distinct down-regulation in psoriasis and hypertrophic scars where keratinocytes are known to undergo activation. Unlike normal and psoriatic skin, K2e expression in hypertrophic and keloid scars began in the deepest suprabasal layer. In cutaneous basal and squamous cell carcinomas, K2e was absent in most tumor islands but the overlying epidermis showed strong expression. No significant K2e expression in nonkeratinized or keratinized oral epithelia, including buccal mucosa, lateral border of tongue and gingiva was detected. In oral lichen planus K2e expression was undetectable, but in benign keratoses of lingual mucosa induction of K2e along with K1 and K10 was observed. In mild-to-moderate oral dysplasia with orthokeratinization, K2e was highly expressed compared with parakeratinized areas but in severe dysplasia as well as in oral squamous cell carcinoma, K2e expression was undetectable. Taken together, the data suggest that K2e expression in skin is sensitive to keratinocyte activation but its up-regulation in oral lesions is a reflection of the degree of orthokeratinization.

HPV8 early genes modulate differentiation and cell cycle of primary human adult keratinocytes

Abstract:  Human papillomaviruses (HPV) have been associated with the development of non‐melanoma skin cancer (NMSC) but the molecular mechanisms of the role of the virus in NMSC development are not clearly understood. Abnormal epithelial differentiation seen in malignant transformation of keratinocytes is associated with changes in keratin expression. The purpose of this study was to investigate the phenotype of primary human adult keratinocytes expressing early genes of HPV8 with specific reference to their differentiation and cell cycle profile to determine whether early genes of HPV8 lead to changes that are consistent with transformation. The expression of HPV8 early genes either individually or simultaneously caused distinct changes in the keratinocyte morphology and induced an abnormal keratin expression pattern, that included simple epithelial (K8, K18, K19), hyperproliferation‐specific (K6, K16), basal‐specific (K14, K15) and differentiation‐specific (K1, K10) keratins. Our results indicate that expression of HPV8 early genes disrupts the normal keratin expression pattern in vitro. Expression of HPV8‐E7 alone caused polyploidy that was associated with decreased expression of p21 and pRb. Expression of individual genes or in combination differentially influenced cell morphology and cell cycle distribution which might be important in HPV8‐induced keratinocyte transformation.