A mutation in the mucosal keratin K4 is associated with oral white sponge nevus.

White sponge nevus (WSN) is a benign autosomal dominant disorder which affects non-cornifying stratified squamous epithelia (MIM 193900) (ref. 1). Phenotypically it presents as white 'spongy' plaques (oral leukokeratoses), most commonly in the mouth but also reported in the esophagus and anogenital mucosa. Histologically, the plaques show evidence of hyperproliferation, acanthosis and tonofilament aggregation. These types of pathogenic changes are characteristic of many of the epidermal keratin disorders. Keratins are expressed in pairs by epithelial cells in a tissue and cell specific manner. The major differentiation specific keratins of the buccal mucosa, nasal, esophageal and anogenital epithelia are K4 and K13 (ref. 7). The tissue distribution and nature of the lesions in patients affected by WSN suggested that mutations in K4 and/or K13 might be responsible for this disorder. We have now confirmed this hypothesis and report here a three base-pair (bp) deletion in the helix initiation peptide of K4 in affected members from two families with this condition.

Keratin 16 and keratin 17 mutations cause pachyonychia congenita.

Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC.

The relationship between hyperproliferation and epidermal thickening in a mouse model for BCIE.

Epidermal thickening is a phenomenon common to many genodermatoses but little is known about the underlying causes. We have recently created a mouse model for the human skin disease bullous congenital ichthyosiform erythroderma by gene targeting. Mice heterozygous for a truncated keratin 10 gene exhibit acanthosis and hyperkeratosis as seen in the human disease. The degree of epidermal thickening is highly variable, offering a novel opportunity to investigate how epidermal homeostasis is modulated in keratin disorders by comparing epidermis from different body regions. We have performed bromodeoxyuridine labeling experiments and detected proliferation antigens by immunohistochemical means to compare proliferation in the epidermis of wild-type and heterozygous mice. These results have been compared with the expression of epidermal differentiation markers and of the "hyperproliferation associated" keratins K6 and K16. These experiments indicated that hyperproliferation is only partly responsible for the morphologic changes and that other mechanisms such as decreased desquamation are likely to be involved.

Leucocyte populations in ectopic tubal pregnancy.

Leucocytes at the ectopic implantation site in 10 cases of early tubal pregnancy were characterised with a series of monoclonal antibodies using an indirect immunoperoxidase technique on cryostat sections. Most were HLA-DR positive macrophages, and there were a small number of mature T lymphocytes (UCHT1 and Dako-T1 positive cells). These results were compared with those reported in normal first trimester intrauterine pregnancies, and the contributions of the various leucocyte types to successful implantation at both the ectopic and intrauterine sites were assessed.

Immunohistochemical characterization of stromal leucocytes in nonpregnant human endometrium.

Stromal leucocytes in normal premenopausal human endometrium were characterised by an indirect immunoperoxidase technique employing a panel of monoclonal antibodies. T cells were scanty in proliferative endometrium but increased in frequency in the secretory phase of the menstrual cycle. An additional population of phenotypically unusual lymphocytes (CD2-positive, CD3-negative) was detected in the stratum functionalis in mid- and late secretory phase endometrium, particularly in areas exhibiting pseudodecidual change. The distribution of these unusual lymphocytes mirrored that of the so-called "endometrial stromal granulocytes," which have recently been shown to be granulated lymphocytes. Macrophages were common throughout the menstrual cycle. B lymphocytes were detected in lymphoid aggregates in the basalis but rarely in the functionalis.

Investigation of the expression of amnion antigens by spiral arteries in human utero-placental tissues.

Two monoclonal antibodies raised against human amnion, GB3 and GB5, were used in an indirect immunoperoxidase method to investigate the expression of amnion antigens by spiral arteries in pregnant and nonpregnant uterine tissues. GB3 showed focal reactivity with occasional spiral arteries in the placental bed throughout pregnancy, but no GB3-staining was observed in nonpregnant endometrium. In contrast, GB5 showed bandlike circumferential reactivity with spiral arteries at all gestational ages examined. GB5-positivity showed no relation to the presence of endovascular or perivascular trophoblast. In nonpregnant endometrium, GB5 labeled rare spiral arteries. However, in a premenstrual specimen showing pseudodecidual change, there was circumferential reactivity with GB5 resembling that in pregnancy. The reaction patterns of GB3 or GB5 were not similar to those for two other basement-membrane components, fibronectin and type IV collagen. The results suggest that expression of the GB5 antigen may in part be regulated by hormones.

Ionizing radiation induces a dramatic persistence of p53 protein accumulation and DNA damage signaling in mutant p53 zebrafish.

Mutant p53 proteins accumulate to high levels in human tumors and in preneoplastic lesions in the skin and fallopian tube. However examination of tissues from mice and fish that are homozygous for mutant p53 surprisingly showed that the protein was present only at low levels except in the tumors that arose in these animals. The mutant protein did accumulate, however, following treatment with ionizing radiation in the same tissues in which the wild-type protein is induced. Here we study in detail the accumulation of mutant and wild-type p53 proteins following ionizing radiation in zebrafish embryos. We found that the mutant protein was induced by lower levels of radiation and reached higher levels than the wild-type protein. Morpholino knockdown of the zebrafish homologs of Mdm2 and Mdm4 caused dramatic accumulation of mutant p53 protein. The most remarkable results were observed by examining p53 protein levels over an extended time course. Mutant p53 protein increased and persisted for days after irradiation and this was accompanied by persistent elevation of phosphorylated H2AX (γH2AX), implying that the resolution of DNA damage signaling in these embryos is severely compromised by mutations in p53. Thus mutation in p53 results in an exaggerated and persistent damage response, which could in turn drive the process of cancer development as high levels of mutant p53 can act as an oncoprotein to drive invasion and metastasis.

Assessment of p53 protein expression in normal, benign, and malignant oral mucosa.

Recent studies have shown the accumulation of high levels of p53 protein to be associated with malignant disease, within a range of tissues. This paper assesses p53 expression in oral mucosal disease. Biopsies were obtained from a range of oral disorders which included normal, benign, premalignant, and malignant oral tissue. In addition, oral smears were obtained from a limited number of patients with biopsy-proven oral cancer. Expression of the p53 protein was assessed using the polyclonal antibody CM1, together with a standard immunoperoxidase technique. A total of 37 oral cancers were assessed, of which 20 were found to express the p53 protein (54 per cent of cases). The p53 protein was not identified in normal, benign, or premalignant oral mucosa (54 cases). The identification of p53 within biopsies of oral mucosal lesions would appear to correlate with oral malignancy.

Keratin K6irs is specific to the inner root sheath of hair follicles in mice and humans.

BACKGROUND: Keratins are a multigene family of intermediate filament proteins that are differentially expressed in specific epithelial tissues. To date, no type II keratins specific for the inner root sheath of the human hair follicle have been identified. OBJECTIVES: To characterize a novel type II keratin in mice and humans. METHODS: Gene sequences were aligned and compared by BLAST analysis. Genomic DNA and mRNA sequences were amplified by polymerase chain reaction (PCR) and confirmed by direct sequencing. Gene expression was analysed by reverse transcription (RT)-PCR in mouse and human tissues. A rabbit polyclonal antiserum was raised against a C-terminal peptide derived from the mouse K6irs protein. Protein expression in murine tissues was examined by immunoblotting and immunofluorescence. RESULTS: Analysis of human expressed sequence tag (EST) data generated by the Human Genome Project revealed a fragment of a novel cytokeratin mRNA with characteristic amino acid substitutions in the 2B domain. No further human ESTs were found in the database; however, the complete human gene was identified in the draft genome sequence and several mouse ESTs were identified, allowing assembly of the murine mRNA. Both species' mRNA sequences and the human gene were confirmed experimentally by PCR and direct sequencing. The human gene spans more than 16 kb of genomic DNA and is located in the type II keratin cluster on chromosome 12q. A comprehensive immunohistochemical survey of expression in the adult mouse by immunofluorescence revealed that this novel keratin is expressed only in the inner root sheath of the hair follicle. Immunoblotting of murine epidermal keratin extracts revealed that this protein is specific to the anagen phase of the hair cycle, as one would expect of an inner root sheath marker. In humans, expression of this keratin was confirmed by RT-PCR using mRNA derived from plucked anagen hairs and epidermal biopsy material. By this means, strong expression was detected in human hair follicles from scalp and eyebrow. Expression was also readily detected in human palmoplantar epidermis; however, no expression was detected in face skin despite the presence of fine hairs histologically. CONCLUSIONS: This new keratin, designated K6irs, is a valuable histological marker for the inner root sheath of hair follicles in mice and humans. In addition, this keratin represents a new candidate gene for inherited structural hair defects such as loose anagen syndrome.

K15 expression implies lateral differentiation within stratified epithelial basal cells.

Keratins are intermediate filament proteins whose expression in epithelial tissues is closely linked to their differentiated state. The greatest complexity of this expression is seen in the epidermis and associated structures. The critical basal (proliferative) cell layer expresses the major keratin pair, K5 and K14, but it also expresses an additional type I keratin, K15, about which far less is known. We have compared the expression of K15 with K14 in normal, pathological, and tissue culture contexts; distinct differences in their expression patterns have been observed that imply different regulation and function for these two genes. K15 appears to be preferentially expressed in stable or slowly turning over basal cells. In steady-state epidermis, K15 is present in higher amounts in basal cells of thin skin but in lower amounts in the rapidly turning over thick plantar skin. Although remaining high in basal cell carcinomas (noninvasive) it is suppressed in squamous cell carcinomas (which frequently metastasize). Wounding-stimulated epidermis loses K15 expression, whereas K14 is unchanged. In cultured keratinocytes, K15 levels are suppressed until the culture stratifies, whereas K14 is constitutively expressed throughout. Therefore, unlike K14, which appears to be a fundamental component of all keratinocytes, K15 expression appears to be more tightly coupled to a mature basal keratinocyte phenotype.