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Biomedical subjects

I M Leigh

Publications and source records attributed to I M Leigh.

At least 19 recordsLinked to original sources

Abnormal expression of wild type p53 protein in normal cells of a cancer family patient.

Mutations in the p53 gene are the commonest specific genetic change in human cancer. In normal tissues, p53 protein is present in such low quantities that it is not readily detectable by immunochemical techniques. However, in many tumour cells large amounts of p53 protein accumulate and can be seen by simple immunohistochemical staining; this is generally attributed to the accumulation of stabilised, mutant protein. We have found a mother and daughter, who both have a history of breast cancer, who show strong immunohistochemical staining of p53 in most of their normal epithelial and mesenchymal cells. Their family has a history of multiple cancers developing at an early age. Detailed protein analysis and gene sequencing of material obtained from cultured cells, grown from a skin biopsy taken from the daughter, suggest that her cells contained large quantities of normal (unmutated) p53. We suggest that this phenotype defines a new inherited cancer susceptibility syndrome that is distinct from the germ-line mutations in p53 found in some Li-Fraumeni families. This new syndrome affects p53 tumour suppressor function through an indirect mechanism that stabilises normal p53. It remains to be established whether this mechanism also contributes to the accumulation of p53 in sporadic cancers.

Adult

A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.

In the hereditary blistering condition epidermolysis bullosa simplex, the skin blisters on trauma following rupture of epidermal basal cells. Clinical variations range from severely incapacitating, especially in early childhood, to mild forms that may not even present clinically. Dowling-Meara epidermolysis bullosa simplex is characterized by clusters of epidermal blisters and keratin clumping in the cytoplasm; recent reports describe potentially causal mutations in keratin 14 (refs 2, 3). Here we describe a 'complementary' mutation at the other end of the other keratin expressed by these cells (K5, coexpressed with K14), a change from a Glu to a Gly in the helix termination peptide, detected by altered antibody binding and confirmed by sequencing using the polymerase chain reaction. The two conserved helix boundary peptides are predicted to be essential for filament assembly, and the requirement for two complementary (type I and type II) keratins is absolute. Epidermolysis bullosa simplex diseases demonstrate the function of the keratin cytoskeleton in resisting compaction stresses which otherwise lead to cell lysis.

Amino Acid Sequence

Replication of varicella zoster virus in primary human keratinocytes.

The ability of varicella zoster virus (VZV) to infect and replicate in human keratinocytes in culture was examined. Primary human keratinocytes derived from the abdomen, breast, and foreskin were plated as monolayers and infected by co-cultivation with VZV infected fibroblasts (MRC-5 cells). Replication and spread of the virus was assayed by plaque assay and immunofluorescence of infected cells using a VZV specific monoclonal antibody. Although all three types of keratinocytes tested were capable of supporting productive VZV infection, the keratinocytes showed a 1.5 to 2 log reduction in virus yield as compared to infection of monolayer cultures of MRC-5 cells. Results from immunofluorescence studies and plaque assays indicate a slower rate of cell-to-cell spread of the virus. Testing of an anti-VZV compound in this novel assay system demonstrated an interesting sensitivity compared to that observed in conventional assay systems.

Antigens, Viral

A novel lamina lucida component of epithelial and endothelial basement membranes detected by LH39 monoclonal antibody.

The murine monoclonal antibody, LH39 was characterized in this study and appeared to bind to a novel basement membrane epitope. This antigen was expressed in the epithelial basement membrane of human tissue derived from all three germ cell layers and in basement membranes surrounding small blood vessels within the stroma of all organs examined. LH39 antigen could be first detected in fetal skin at the dermo-epidermal junction at 7 weeks estimated gestational age but was not present in the dermal vasculature until 16 weeks. When tested against tissue from a range of lower mammalian species, LH39 antigen appeared to be primate-specific. The epithelial basement membrane zone in organotypical cultures, where there is de novo synthesis of basement membrane components, contained abundant LH39 antigen in contrast to other basement membrane components, type IV collagen, laminin, and type VII collagen. Ultrastructural localization of LH39 epitope, using immunogold electron microscopy on unfixed freshly frozen tissue, was to the lamina lucida. No cross-reactivity could be detected between LH39 and laminin, fibronectin, and collagens I, III, IV, and V using the ELISA assay. In vitro studies with a range of proteolytic enzymes suggested that the antigen was non-collagenous in nature. LH39 precipitated a polypeptide with a molecular weight of 185 kD from extracts of metabolically labelled cultured keratinocytes, and polypeptides of 185 and 200 kD from the culture medium. The tissue distribution of LH39 antigen suggested that it may be an epitope within anchoring filaments. Potential applications of this antibody include the study of benign and malignant human vascular disorders, diseases and tumours associated with angiogenesis, epithelial neoplasms, and conditions of tissue regeneration and repair, such as wound healing.

Adult

The distribution of LH39 basement membrane epitope in the tumour stroma of oral squamous cell carcinomas.

LH39 monoclonal antibody detects a novel component of epithelial and endothelial basement membranes. The expression of LH39 antigen was investigated by immunohistochemistry in 55 oral squamous cell carcinomas and compared with 15 pyogenic granulomas of skin and oral mucosa, 20 non-specific oral ulcers, and 20 specimens of normal oral mucosa. The distribution of this basement membrane epitope was compared with that of other basement membrane components, type IV collagen, and laminin. LH39 monoclonal antibody labelled basement membrane surrounding small blood vessels in normal human organs. In oral squamous cell carcinomas, in contrast to the other basement membrane antigens, the LH39 epitope was not detectable in vessels within histologically recognizable tumour stroma. Neovascularization is known to attend malignant neoplasms and this finding was interpreted as absence of LH39 antigen within newly formed vessels. In support of this hypothesis, LH39 immunoreactivity was absent in newly formed blood vessels within pyogenic granulomas and the granulation tissue within ulcers. As increasing neovascularization is reported to correlate with a rising rate of metastasis, assessment of tumour angiogenesis may be of value in selecting patients for initial aggressive therapy.

Antibodies, Monoclonal

Lamina densa malformation involved in histogenesis of primary localized cutaneous amyloidosis.

Skin lesions of lichenoid amyloidosis and macular amyloidosis were immunohistochemically investigated using five monoclonal antibodies against basement membrane zone (BMZ) components. A hemidesmosomal component did not contribute to amyloid deposits, but components of the lamina densa and anchoring fibrils were associated with amyloid deposits in the uppermost dermis. Immunoelectron microscopy revealed that these BMZ components were not only aggregated in the BMZ and dermis, but were also involved in the individual amyloid islets. The lamina densa was disrupted in the interface areas just above the amyloid deposits, where cytoplasm of the basal cells directly faced the aggregate of amyloid filaments. Aggregates of some BMZ components were continuous to the amyloid islets from the lamina densa area. These findings suggest that a lamina densa malformation is involved in amyloid production in the interface of the BMZ, and support the secretion theory rather than the fibrillar body theory of amyloidogenesis in these types of primary localized cutaneous amyloidosis.

Amyloid

Selective involvement of keratins K1 and K10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma).

Aggregation of tonofilaments within epidermal keratinocytes is a characteristic histologic feature of epidermolytic hyperkeratosis including the generalized form known as bullous congenital ichthyosiform erythroderma. The histologic distribution and the keratin composition of the altered tonofilaments were investigated to determine whether the aggregation was specific to any particular keratin(s). Skin samples from seven patients and one mid-trimester fetus with generalized epidermolytic hyperkeratosis, and from one patient with a localized or "nevoid" form of epidermolytic hyperkeratosis, were analyzed by using various microscopical and immunocytochemical methods. A conjunctival sample and cultured epidermal keratinocytes from one patient with generalized epidermolytic hyperkeratosis were also examined by electron microscopy and immunocytochemistry. Ultrastructurally, tonofilament aggregates were distributed within the suprabasal stratified epithelial cell layers of the epidermis, of the infundibular part of outer root sheaths, and of the sebaceous ducts and sweat ducts, selectively following the known distribution pattern of keratins K1 and K10. The abnormal tonofilaments were not found in any other cutaneous epithelia, in conjunctival epithelium, or in cultured keratinocytes, where K1 and K10 are absent or only minimally expressed. Immunoelectron microscopy showed that among the keratins detected in suprabasal epidermolytic hyperkeratosis epidermis (K1/K5/K10/K14/K16), the aggregated tonofilaments predominantly expressed K1 and K10 rather than other keratins. These results suggest that the keratin filament abnormality in epidermolytic hyperkeratosis principally involves K1 and K10 and raise the question whether epidermolytic hyperkeratosis might be primarily a disorder of one or both of these keratins.

Adolescent

Ontogenesis of the basement membrane zone after grafting cultured human epithelium: a morphologic and immunoelectron microscopic study.

Sheets of cultured epithelial cells have been successfully used as autografts for the permanent coverage of patients with full-thickness burns and as allografts to stimulate the healing of chronic skin ulcers. The basement membrane zone (BMZ), composed of lamina lucida, lamina densa, and anchoring fibrils, plays a pivotal role in the firm adherence of the epidermis to the dermis. The present study describes the ultrastructural development during various stages of wound healing after resurfacing different wound areas by cultured epithelial grafts. For this purpose, biopsies were obtained from five patients 5 d to 4.5 years after resurfacing full-thickness burns with cultured autografts, and from five patients at various stages after treatment of excised tattoos and chronic skin ulcers with cultured allografts. One biopsy was taken from a spontaneously healed burn wound 30 years post-injury. Ultrathin sections were prepared for transmission and immunoelectron microscopy, using monoclonal antibodies against type IV and VII collagen. Findings were compared to controls of age- and site-matched normal skin. Eleven days after grafting, the first BMZ features had regenerated, including lamina lucida, a discontinuous lamina densa, hemidesmosomes, and sparse anchoring fibrils. The process of de novo synthesis of BMZ components had begun, and within 4 to 5 weeks complete reformation of BMZ was observed, including normal distribution of anchoring fibrils. Immunolabeling of type VII collagen was first observed upon the lower part of lamina densa at day 11 and steadily increased, reaching normal values 5 weeks after grafting. In contrast, gold deposition of type IV collagen upon lamina densa was strongly increased at day 19 compared to normal. This high expression reduced a little at 5 weeks, but remained high up to 30 years after injury. Long-term burn scars exhibited pseudopodia-like extensions of all basal cells, abundant anchoring fibrils, and an increased amount of arching anchoring fibrils. These features might compensate for the lack of proper rete ridges.

Adolescent

Intracellular expression of type VII collagen during wound healing in severe recessive dystrophic epidermolysis bullosa and normal human skin.

The ability of keratinocytes to synthesize basement membrane components in vivo during wound healing in normal human skin and in severe recessive dystrophic epidermolysis bullosa (RDEB) was investigated. Indirect immunofluorescence using anti-type VII collagen (VIIc, recognizing the globular non-helical component of the molecule), anti-type IV collagen, anti-laminin and bullous pemphigoid antisera, was performed on biopsies of intact skin and of healing skin taken between 7 and 14 days after dermatome injury (upper to mid-dermal wounding) in eight patients with severe RDEB and in seven normal subjects. Baseline anti-type VIIc immunofluorescence showed completely absent staining of the epidermis, dermis and dermo-epidermal junction in severe RDEB samples, and bright linear dermo-epidermal junction fluorescence in normal human skin. In 5/5 normal human skin samples taken 9-12 days post-wounding, some type VIIc expression was noted within basal cells as well as in a continuous or interrupted linear distribution at the basement membrane zone. In all the severe RDEB biopsies sampled between days 10 and 13 (5/5), anti-VIIc fluorescence was also seen with varying intensity within basal and lowermost suprabasal cells, and in one day 14 sample at the dermo-epidermal junction. Low levels of intracellular type IVc were seen in both groups, but only in those samples taken 7-9 days after injury; later biopsies showed only continuous dermo-epidermal junction staining. Linear basement membrane zone labelling with laminin and bullous pemphigoid antisera was seen in all samples in both sets of subjects, even at day 7, but there was no detectable intracellular antisera staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent

Keratin expression in basal cell carcinomas.

The keratin phenotype of 15 cases of basal cell carcinoma was assayed immunohistochemically using a panel of monospecific antibodies to single keratin polypeptides. Whilst tumour tissue strongly expressed primary keratins 5 and 14 (normally synthesized in basal keratinocytes) no expression of secondary keratins 1 and 10 (characteristic of skin-type differentiation) was detected. Keratin 17, characteristic of the outer hair root sheath, was strongly expressed in all tumours. Keratin 19 was also normally expressed in parts of the hair follicle and was detected in four cases. The 'high cell turnover' keratin 16 was frequently induced in the overlying epidermis, but was rare within tumour tissue. No expression of simple epithelial keratins 8 and 18 was detected. Whilst the keratin phenotype of tumour cells is similar to that of basal cells within part of the hair root sheath, in keeping with suggestions of a follicular origin for basal cell carcinomas, the findings are also compatible with an origin from interfollicular pluripotent stem cells differentiating towards follicular structures.

Animals

Immunohistochemical localization of basement membrane type VII collagen and laminin in neoplasms of the head and neck.

The distribution pattern of the basement membrane components type VII collagen and laminin, was studied immunohistochemically in normal human head and neck tissues and in a series of benign and malignant tumours from the same site. Using monoclonal antibodies, a basement membrane containing type VII collagen and laminin could be demonstrated beneath the epithelial cell layer in 16 normal head and neck tissues from different localizations. Unlike type VII collagen, laminin was also abundantly present around blood vessels and muscle fibres. With respect to 42 squamous cell carcinomas studied, type VII collagen and laminin were present in basement membranes surrounding small and large tumour fields, independent of the tumour grade. Type VII collagen was demonstrated in the cytoplasm of tumour cells in 36% of the cases, while the antibody to laminin displayed a basement membrane staining pattern mainly. Both antibodies showed a staining gradient in more than half of the cases, with strong staining in the centre of the tumour and weakening of the staining towards the tumour periphery. In a series of 22 salivary gland tumours consisting of 19 pleomorphic adenomas and three adenoid cystic carcinomas, the distribution pattern of type VII collagen and laminin was very heterogeneous. Laminin was present in 17 and type VII collagen in 10 of 19 cases of pleomorphic adenoma, mostly scattered throughout the tumour fields. In the tumours positive for type VII collagen areas with little or no positivity were also found. A correlation between type VII collagen positivity and the presence of basal cell keratin 14 positivity was noticed in the majority of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane

Laminin and type VII collagen distribution in different types of human lung carcinoma: correlation with expression of keratins 14, 16, 17 and 18.

The expression patterns of basement membrane components and keratin intermediate filament proteins were studied in normal human bronchial epithelium and 56 lung carcinomas using monoclonal antibodies to laminin, type VII collagen and the individual keratins 14, 16, 17 and 18. In normal lung, laminin and type VII collagen were present between the epithelium and the lamina propria of bronchi and bronchioles. Keratin 14 was expressed in the basal cells, keratin 17 in the basal and some suprabasal cells and keratin 18 in the columnar cells of the bronchi and bronchioles. Keratin 16 was not present in normal bronchial epithelium. Laminin was found in all subtypes of lung carcinoma, but type VII collagen was present only in squamous cell carcinomas, where it showed a reduction in expression with decreasing differentiation. Type VII collagen was not identified in adenocarcinomas, small cell carcinomas or carcinoids. Antibodies to basal cell keratins 14 and 17 also displayed positivity only in squamous cell carcinomas, although no correlation with the degree of differentiation could be observed. Keratin 16 appeared to be a marker of the squamous phenotype, rather than of hyperproliferation. The keratin 18 marker for columnar epithelial cells showed a reaction pattern opposite to that of the basal cell keratins, being extensively present in adenocarcinomas, small cell carcinomas and carcinoids, with less expression in squamous cell carcinomas. This study shows a correlation between the presence of type VII collagen and the basal cell keratins 14 and 17, and a negative correlation between these components and keratin 18. These findings are likely to be useful in identifying lung cancer subtypes.

Adenocarcinoma

Keratin expression in normal skin and epidermal neoplasms demonstrated by a panel of monoclonal antibodies.

The tissue labelling of a panel of monoclonal antikeratin antibodies (LL001, LL002, LL003, LP2K, BA17, LP34, CAM5.2, and LH1) recognising keratins 1, 5, 8, 10, 14, 18, and 19 were investigated in frozen and formalin-fixed normal skin. Antibodies LL001, LL003, BA17, LP34, CAM5.2, and LH1 were found to be reactive in formalin-fixed material and were used to study 23 basal cell carcinomas, 8 squamous cell carcinomas, 5 keratoacanthomas, 5 Bowen's disease, and 6 clear cell acanthomas. All these tumours demonstrated a loss of keratin 10 expression as demonstrated by loss of labelling with LH1. Keratin 14 expression, as demonstrated by LL001, was reduced but present in all the tumours except squamous cell carcinomas and keratoacanthomas where increased labelling was observed in the more differentiated areas of these tumours. Simple epithelial keratin expression was demonstrated by positive labelling with CAM5.2 and keratin 19 by BA17 in a third of basal cell carcinomas and squamous cell carcinomas. Three of the five keratoacanthomas labelled with BA17, indicating the presence of keratin 19 in these lesions. These results support the concept that keratin expression is a phenotypic marker of the state of differentiation or malignant transformation and that patterns of keratin expression are not specific to any particular premalignant or malignant disorder.

Antibodies, Monoclonal

Cytokeratin profiles in dyskeratosis congenita: an immunocytochemical investigation of lingual hyperkeratosis.

In dyskeratosis congenita, the hyperkeratotic lesions affecting the mucous membranes have a propensity to undergo malignant change. Unfortunately there is presently no reliable method for predicting such an outcome. Oral mucosal biopsies were obtained from a case of dyskeratosis congenita and keratin expression identified using a panel of antikeratin antibodies. Marked changes were found from keratin profiles established for normal oral mucosa. In particular coexpression of three separate type 1 keratins (K16, K10 and K13) were observed in the ventral tongue lesion. The keratin pattern found in the tissue biopsies examined is suggestive of an unusually immature or disturbed state of tissue differentiation, and as such may be indicative of future malignant change.

Child

Extensive variant of cutaneous amyloidosis: report of a case with electron-microscopic and immunohistochemical studies of the basement membrane zone at sites of amyloid production.

A 60-year-old Japanese female developed widespread lichenoid eruptions with pigmentation, which initially appeared in preceding erythematous skin lesions due to dermatomyositis. Thioflavine T and Dylon stainings, electron microscopy and immunohistochemistry revealed that thick amyloid deposits were present in the papillary dermis particularly beneath the epidermis. Autopsy showed no evidence of systemic amyloidosis. Electron microscopy of the lesional skin disclosed the disturbance of lamina densa formation in the epidermal basement membrane zone (BMZ). There was disruption and dissociation of the lamina densa from the basal cell, and a lamina-densa-like substance was found in the amyloid deposits. Immunofluorescence and immunoelectron microscopy showed that type IV and VII collagens, LDA-1 antigen (a noncollagenous component of the BMZ) and laminin were distributed in irregular thick deposits along the BMZ and were also present within the amyloid itself. These findings indicate that morphological and immunohistochemical abnormalities of the lamina densa may be involved in amyloid production at the interface of the epidermis and dermis, at least in this case.

Amyloidosis

Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.

We have examined integrin expression during the remodeling of the epidermis that takes place during wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. By immunofluorescence microscopy, we found that the same integrin subunits were expressed during wound healing as in normal epidermis with very little change in the relative intensity or distribution of staining at the leading edge of the migrating epidermis. However, at the time of wound closure, when the epidermis is still hyperproliferative, alpha 2, alpha 3, alpha 6, and beta 1 were no longer confined to the basal layer, as in normal epidermis, but were also found in all the living suprabasal cell layers, coexpressed with the terminal differentiation markers involucrin, keratin 10, and keratin 16. Strong suprabasal staining for alpha v was also found in one specimen. beta 4, which normally forms a heterodimer with alpha 6, and alpha 5 remained predominantly basal. Three of the integrin ligands, fibronectin, type IV collagen, and laminin, remained largely confined to the basement membrane zone and dermis. By 14 d after wounding, the integrins were once more restricted to the basal layer. Suprabasal integrin expression was also observed in involved psoriatic lesions. Thus, in two situations in which the epidermis is hyperproliferative, there is a failure to downregulate integrin expression on initiation of terminal differentiation. The functional consequences of this aberrant integrin expression remain to be explored.

Blister