RESUMO
BACKGROUND: Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients. OBJECTIVES: To investigate the effects of heterozygous nonsense mutations in desmoplakin (Dp) and desmoglein (Dsg) 1 which cause the autosomal dominant disorder striate palmoplantar keratoderma (SPPK), focusing on changes in desmosome structure and composition and the associated keratin intermediate filament (KIF) network in palm skin, and in cultured keratinocytes generated from the same site. METHODS: We analysed palm and nonpalm skin sections from four SPPK patients with Dp mutations and one patient with a Dsg1 mutation with respect to tissue and subcellular morphologies, and correlated the in vivo and in vitro findings. RESULTS: Using electron microscopy, we found abnormalities of desmosomes and cell-cell adhesion in the suprabasal layers in the epidermis from patients with both Dsg1- and Dp-associated SPPK. These changes were more advanced in skin from patients with Dp mutations. Both Dp and Dsg1 mutations were accompanied by significantly reduced numbers of desmosomes in the suprabasal layers, while decreased desmosome size was evident only in Dsg1-associated SPPK. Confocal microscopy analysis showed marked differences in the expression of keratins and of desmosome components, both between the two types of SPPK, and between SPPK and normal skin. The expression of keratins K5, K14 and K10 was reduced in Dsg1-associated SPPK skin, whereas perinuclear aggregation of keratin filaments was more evident in Dp-associated SPPK. In both types of SPPK upregulation of K16 was pronounced and involucrin labelling was abnormal. CONCLUSIONS: Mutations in Dp and Dsg1 genes causing SPPK may be associated with perturbations in epidermal differentiation accompanied by a marked disruption of several components of the epidermal scaffold including desmosomes and the KIF network.
Assuntos
Caderinas/genética , Códon sem Sentido , Proteínas do Citoesqueleto/genética , Desmossomos/ultraestrutura , Ceratodermia Palmar e Plantar/genética , Adulto , Idoso , Caderinas/metabolismo , Adesão Celular/genética , Diferenciação Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Desmogleína 1 , Desmogleínas , Desmoplaquinas , Desmossomos/genética , Epiderme/metabolismo , Epiderme/ultraestrutura , Humanos , Queratinas/metabolismo , Ceratodermia Palmar e Plantar/metabolismo , Ceratodermia Palmar e Plantar/patologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Precursores de Proteínas/metabolismoRESUMO
Desmosomes are major cell adhesion junctions, particularly prominent in the epidermis and cardiac tissue and are important for the rigidity and strength of the cells. The desmosome consists of several proteins, of which desmoplakin is the most abundant. Here, we describe the first recessive human mutation, 7901delG, in the desmoplakin gene which causes a generalized striate keratoderma particularly affecting the palmoplantar epidermis, woolly hair and a dilated left ventricular cardiomyopathy. A number of the patients with this syndromic disorder suffer heart failure in their teenage years, resulting in early morbidity. All tested affected members of three families from Ecuador were homozygous for this mutation which produces a premature stop codon leading to a truncated desmoplakin protein missing the C domain of the tail region. Histology of the skin revealed large intercellular spaces and clustering of desmosomes at the infrequent sites of keratinocyte adhesion. Immunohistochemistry of skin from the patients showed a perinuclear localization of keratin in suprabasal keratinocytes, suggesting a collapsed intermediate filament network. This study demonstrates the importance of desmoplakin in the attachment of intermediate filaments to the desmosome. In contrast to null DESMOPLAKIN: mice which die in early development, the truncated protein due to the homozygous 7901delG mutation in humans is not embryonic lethal. This suggests that the tail domain of desmoplakin is not required for establishing tissue architecture during development.
Assuntos
Cardiomiopatia Dilatada/genética , Proteínas do Citoesqueleto/metabolismo , Genes Recessivos/genética , Filamentos Intermediários/metabolismo , Ceratodermia Palmar e Plantar/genética , Mutação/genética , Sequência de Aminoácidos , Cardiomiopatia Dilatada/fisiopatologia , Adesão Celular , Criança , Cromatografia Líquida de Alta Pressão , Códon de Terminação/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Desmoplaquinas , Equador , Epiderme/patologia , Feminino , Haplótipos/genética , Ventrículos do Coração/fisiopatologia , Homozigoto , Humanos , Imuno-Histoquímica , Queratinócitos/patologia , Ceratodermia Palmar e Plantar/patologia , Masculino , Dados de Sequência Molecular , LinhagemRESUMO
Diffuse non-epidermolytic palmoplantar keratoderma (NEPPK) belongs to the heterogeneous group of skin diseases characterized by thickening of the stratum corneum of the palms and soles (1). This autosomal dominant PPK is characterized by a diffuse pattern of palmar and plantar hyperkeratosis giving the affected areas a thickened yellowish appearance with a marked erythematous edge. Linkage of diffuse NEPPK to chromosome 12q11-q13 has been demonstrated in two independent reports (2, 3). In this study, we describe detailed haplotyping with microsatellite markers mapping to this chromosomal region in three diffuse NEPPK pedigrees from the south of England. Fine mapping of a previously identified recombination event and the identification of a common disease haplotype segregating in the three pedigrees places the diffuse NEPPK locus proximal to the type II keratin gene cluster.
Assuntos
Cromossomos Humanos Par 12/genética , Queratinas/genética , Ceratodermia Palmar e Plantar Difusa/genética , Mapeamento Cromossômico , Feminino , Ligação Genética , Haplótipos , Humanos , Ceratodermia Palmar e Plantar Difusa/patologia , Masculino , Repetições de Microssatélites , Família Multigênica , LinhagemRESUMO
Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.
Assuntos
Actinas/metabolismo , Antígenos de Superfície/metabolismo , Desmossomos/metabolismo , Integrinas/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Linhagem Celular Transformada , Desmossomos/ultraestrutura , Humanos , Imuno-Histoquímica , Integrina alfa6beta4 , Queratinócitos/ultraestrutura , Plectina , Ligação Proteica , TransfecçãoRESUMO
Desmosomes are highly organized intercellular adhesive junctions that are particularly prominent in epidermis and other tissues experiencing mechanical stress. Desmoplakin, a constitutive component of the desmosomal plaque, is the most abundant protein present in such junctions and plays a critical role in linking the intermediate filament network to the plasma membrane in these tissues. Here we report the first mutation in the gene encoding desmoplakin. The identified mutation, resulting in a null allele and haploinsufficiency, was observed in genomic DNA from a kindred with the dominantly inherited skin disorder, striate palmoplantar keratoderma. Affected individuals had a linear pattern of skin thickening on the fingers and palms and circumscribed areas of skin thickening on the soles. Affected skin demonstrated loosening of intercellular connections, disruption of desmosome-keratin intermediate filament interactions and a proportion of rudimentary desmosomal structures. The disorder mapped to chromosome 6p21 with a maximum lod score of 10.67. The mutation was a heterozygous C-->T transition in exon 4 of the desmoplakin gene and predicted a premature termination codon in the N-terminal region of the peptide. This is the first reported mutation of desmo-plakin and also the first inherited skin disorder in which haploinsufficiency of a structural component has been implicated. It identifies dosage of desmoplakin as critical in maintaining epidermal integrity.
Assuntos
Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/patologia , Mutação Puntual , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 6/genética , DNA/genética , Primers do DNA/genética , Desmoplaquinas , Desmossomos/metabolismo , Desmossomos/patologia , Feminino , Heterozigoto , Humanos , Ceratodermia Palmar e Plantar/metabolismo , Escore Lod , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Linhagem , FenótipoRESUMO
AIMS: To establish the structural changes that occur in deep surgical wounds engrafted with allogeneic sheets, their time course and inter-relation. METHODS: Deep surgical wounds following shave excision of tattoos (down to deep dermis/subcutaneous fat) were treated with sheets of sex mismatched allogeneic keratinocytes in 19 patients and then biopsied weekly until wound healing was complete. More superficial surgical wounds--that is, 20 standard skin graft donor sites, were biopsied at seven to 10 days (all healed) following application of keratinocyte allografts. All biopsy specimens were examined with a large panel of monoclonal antibodies to keratins, envelope proteins, basement membrane components, and to extracellular matrix components. RESULTS: The hyperproliferative keratin pair K6/16 was expressed in all wounds, for up to six weeks in keratinocyte grafted deep wounds, and up to six months in split thickness skin grafted wounds. CONCLUSIONS: Keratins 6 and 16 have not been detected in normal skin, although the relevant mRNA has. This raises the possibility of regulation at a post-transcriptional level allowing a rapid response to injury with cytoskeletal changes that may aid cell migration. This keratin pair offers the most sensitive marker for altered epidermis following wounding.
Assuntos
Procedimentos Cirúrgicos Dermatológicos , Queratinócitos/transplante , Transplante de Pele , Tatuagem , Cicatrização/fisiologia , Membrana Basal/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Queratinas/metabolismo , Masculino , Mucinas/metabolismo , Período Pós-Operatório , Precursores de Proteínas/metabolismo , Pele/metabolismoRESUMO
Keratinocyte differentiation in psoriasis was examined using a panel of monospecific monoclonal antibodies to keratins (K), including two recently developed monoclonal antibodies raised to carboxy terminal peptides of K6 (LL020) and K16 (LL025). Keratinocytes from normal skin, untreated psoriatic plaques and non-lesional psoriatic skin, were cultured using multiple in vitro systems. Time-lapse cinephotography was used to measure the intermitotic time of normal and psoriatic keratinocytes in both low calcium-defined and serum-containing media. The intermitotic time did not differ significantly between psoriatic and normal keratinocytes. Keratin expression of psoriatic and normal keratinocytes in vitro was examined by both gel electrophoresis and immunocytochemistry. K6, K16 and K17 were detected suprabasally in all culture systems in vitro, but only in interfollicular psoriatic epidermis in vivo, and not in normal skin. Small subpopulations of keratinocytes expressed simple epithelial keratins K7, K8, K18 and K19 in cultures on plastic substrates, but these keratins were absent in skin equivalents of normal or psoriatic skin. No psoriasis-specific pattern of differentiation was found in vitro. As the K6 peptide antibody reacted with basal cells of normal skin, probably due to K5 cross-reactivity, K16 expression determined by LL025 was found to be the most sensitive indicator of the psoriatic state of differentiation, and this antibody is recommended for future work on psoriasis. K17 had a distinct pattern of tissue distribution in normal skin: K17, but not K16, was present in basal myoepithelial cells in sweat glands, and the deep outer root sheath, but K17 distribution paralleled that of K16 in suprabasal psoriatic epidermis. As keratins K6, K16 and K17 are expressed in keratinocyte hyperproliferation, when high levels of certain cytokines are also expressed, the role of growth factors and regulatory nuclear transcription factors in the control of K6, K16 and K17 expression in psoriasis requires further study, in order to provide insight into the relationship between proliferation and differentiation.
Assuntos
Queratinócitos/química , Queratinas/análise , Psoríase/patologia , Pele/citologia , Biomarcadores/análise , Diferenciação Celular , Divisão Celular , Células Cultivadas , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas Imunoenzimáticas , Queratinócitos/patologia , FotografaçãoRESUMO
Keratins K6 and K16 are expressed in suprabasal interfollicular epidermis in wound healing and other pathological conditions associated with hyperproliferation, such as psoriasis and are induced when keratinocytes are cultured in vitro. However, these keratins are also constitutively expressed in normal suprabasal mucosal and palmoplantar keratinocytes. Mutations in keratins have been reported in the basal keratin pair K5 and K14 in epidermolysis bullosa simplex and in suprabasal epidermal keratins K1, K2 and K10 in epidermolytic ichthyoses. Two families with autosomal dominant disorder of focal non epidermolytic palmoplantar keratoderma, have oral mucosal and follicular lesions in addition to the palmoplantar hyperkeratosis. Previous studies have shown linkage in these families to the type I keratin gene cluster at 17q12-q21 and this report shows that the cDNA of affected members of both families have novel heterozygous mutations in the expressed keratin 16 gene. These mutations (R10C and N8S) lie in the helix initiation motif of the 1A domain. These mutations do not appear to cause epidermolysis on light or electron microscopy, which may reflect differences in function, assembly or interaction of the 'hyperproliferative' or 'mucoregenerative' keratins from other major types of keratins. The mutations reported here are the first to describe the molecular pathology of focal non epidermolytic palmoplantar keratoderma.
Assuntos
Queratinas/genética , Ceratodermia Palmar e Plantar/genética , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Sequência de Bases , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 17 , Análise Mutacional de DNA , Primers do DNA , Desoxirribonucleases de Sítio Específico do Tipo II , Feminino , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Ceratodermia Palmar e Plantar/patologia , Masculino , Dados de Sequência Molecular , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Família Multigênica , Linhagem , Reação em Cadeia da Polimerase , Pele/metabolismo , Pele/patologia , Pele/ultraestruturaRESUMO
Suction blister injury is an experimental model for the investigation of the possible derangement of dermal/epidermal interaction in injury. An extensive fibre network can be stained in skin using antisera to the pan-neuronal marker protein gene product 9.5 (PGP) and the sensory neuropeptide calcitonin gene-related peptide (CGRP), while microvessels are identifiable with antisera to the endothelial marker von Willebrand's factor (vWf) and the peptide endothelin (ET). To investigate the possible involvement of superficial cutaneous innervation and microcirculation during the repair process in injury, human skin biopsies taken at different times after suction blister injury were investigated by quantitative immunohistochemistry. Neural and endothelial changes were seen in both edge and blister areas. PGP- and CGRP-immunoreactive nerves showed an increase in both areas compared with control skin up to 6 h after injury, followed by a decrease which lasted until 72 h. This was followed by a gradual increase of both nerve types starting from the blister edge and lasting up to 8 days after injury when the values were similar to controls. Similarly, in the blister area of the skin, vWF-immunoreactive capillaries showed statistically significant increases between 0 and 6 h after injury, followed by a decrease at 12 and 18 h which was maintained up to 72 h. ET-1 immunoreactivity showed a similar, although more variable, pattern of changes. At the blister edge from 23 h onwards, both vWf and ET-1 immunoreactivities showed a second increase from the edge of the blister spreading towards the centre of the blister ahead of the nerve increase. This lasted up to 8 days, when vWf immunoreactivity showed a statistically significant increase compared with control skin. Generally the numerical increase observed at early time points was accompanied by a strong staining intensity, which reverted to a normal staining intensity at later time points. These results suggest that there is a reactive process for all immunoreactive elements in the early stages of injury, followed by changes indicative of neuronal and endothelial damage (depletion phase) and the subsequent repair process. The repair started with re-epidermalization from the blister edge, followed by revascularization and, lastly, by reinnervation of the tissue. These results indicate a close relationship between epidermis, blood vessels, and nerve fibres during the healing process which follows suction blister injury.
Assuntos
Peptídeos/metabolismo , Pele/lesões , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Epiderme/patologia , Imunofluorescência , Humanos , Microcirculação/metabolismo , Microcirculação/patologia , Neurônios/metabolismo , Pele/irrigação sanguínea , Pele/inervação , Pele/patologia , Sucção , Tioléster Hidrolases/metabolismo , Ubiquitina Tiolesterase , Fator de von Willebrand/metabolismoRESUMO
The profile of keratin expression in benign warts from various cutaneous and mucosal sites along with dysplastic warts and squamous cell carcinomas has been examined using a panel of monospecific antibodies to epithelial keratins. Viral warts and verrucous keratoses from immunosuppressed renal transplant recipients show a spectrum of squamous atypia from benign lesions, from minimal changes to full thickness dysplasia. Changes associated with malignancy include loss of differentiation-specific keratins 1 and 10 together with expansion of basal cell epitopes and inappropriate expression of simple epithelial keratins 8, 18, and 19 in advanced squamous cell carcinoma. This late expression of keratins 8 and 18 contrasts with early expression of keratin 17 in all dysplastic lesions examined. Keratin 17 is found suprabasally in hyperproliferative lesions, including benign warts, but marked basal plus suprabasal expression is seen increasingly in malignantly transformed epidermis. These findings were not specific to immunosuppression, as shown by identical findings in control squamous cell carcinoma from nonimmunosuppressed individuals. Keratin 17 expression may prove prognostically helpful when assessing dysplasia in epidermal tumors.
Assuntos
Biomarcadores/análise , Transformação Celular Neoplásica/patologia , Queratinas/análise , Dermatopatias/patologia , Neoplasias Cutâneas/química , Neoplasias Cutâneas/patologia , Pele/química , Verrugas/patologia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Transformação Celular Viral , Condiloma Acuminado/metabolismo , Condiloma Acuminado/patologia , Epitélio/química , Epitélio/metabolismo , Epitélio/patologia , Feminino , Humanos , Imuno-Histoquímica , Queratinas/metabolismo , Queratinas/fisiologia , Pele/metabolismo , Pele/patologia , Dermatopatias/metabolismo , Neoplasias Cutâneas/metabolismo , Verrugas/metabolismoRESUMO
Monospecific antibodies to individual keratin polypeptides can be used to examine the tissue and cellular coexpression of members of keratin pairs. Monospecific monoclonal and polyclonal antibodies have been raised to keratins 1 and 10 using both crude cytoskeletal extracts and synthetic peptides. The tissue distribution of these keratins has been determined against a panel of freshly frozen normal tissues from humans, rodents and pigs. Epidermal expression has been examined in psoriatic plaques, and healing wounds, as examples of epidermal hyperproliferation. Cultured keratinocytes in monolayer (low calcium), stratified (high calcium), and complex cultures, transformed keratinocytes, and tumour cell lines, have been examined for the in vitro expression of these keratins. The sensitivity and precise localization of reactivity with these monospecific antibodies gives a highly accurate picture of individual cell expression. There is confirmation of coexpression of keratins 1 and 10 in epidermal and mucosal sites, and with keratin 16 in hyperproliferative states. These monospecific antibodies provide an important means of examining keratin expression in epidermal tumours and keratinizing disorders, and of seeking keratin mutations in cell lines and in skin diseases.
Assuntos
Biomarcadores/análise , Queratinas/metabolismo , Pele/citologia , Adulto , Animais , Anticorpos Monoclonais , Diferenciação Celular , Divisão Celular , Linhagem Celular , Células Cultivadas , Células Epidérmicas , Epiderme/lesões , Epiderme/metabolismo , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Queratinócitos/metabolismo , Queratinas/genética , Ceratose/genética , Ceratose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/metabolismo , Psoríase/genética , Psoríase/metabolismo , Pele/lesões , Pele/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Suínos , CicatrizaçãoAssuntos
Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/patologia , Queratinócitos/patologia , Neoplasias Cutâneas/patologia , Carcinoma Basocelular/química , Carcinoma de Células Escamosas/química , Humanos , Queratinas/análise , Valores de Referência , Neoplasias Cutâneas/químicaRESUMO
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.
Assuntos
Membrana Basal/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Anticorpos Monoclonais , Antígenos/metabolismo , Membrana Basal/imunologia , Vasos Sanguíneos/metabolismo , Carcinoma de Células Escamosas/irrigação sanguínea , Epitopos , Granuloma/metabolismo , Humanos , Boca/irrigação sanguínea , Doenças da Boca/metabolismo , Neoplasias Bucais/irrigação sanguínea , Neovascularização Patológica/metabolismo , Supuração , Distribuição Tecidual , Úlcera/metabolismoRESUMO
A 135-kD conA-binding glycoprotein isolated from pig epidermis was previously localized to the surface of basal cells in stratified epithelia using affinity-purified antibodies. Preembedding immunoperoxidase electron microscopy has now shown that this glycoprotein is concentrated on the lateral surfaces of basal cells but is not detectable on those surfaces adjacent to the basement membrane indicating a role in cell-cell rather than cell-substrate interactions. The basal cell glycoprotein was shown to resemble the beta 1 subunit of the integrin family following the generation of a specific monoclonal antibody (M5.25). The epidermal glycoprotein recognized by M5.25 and by antibodies against the beta 1 fibronectin receptor from human placenta co-migrated on SDS gels under both reducing and non-reducing conditions. Its response to disulphide reducing agents was characteristic of beta 1 integrin subunits. In addition, the basal cell glycoprotein was shown to bind to the 120-kD cell-binding fragment of fibronectin in a RGD-dependent manner. It was readily detected by immunoblotting whole cell lysates of cultured pig keratinocytes suggesting increased expression in cultured cells compared to fresh epithelial tissue. The results suggest that beta 1 integrin subunits may be involved in cell-cell interactions between basal keratinocytes in pig epidermis and that these receptors are lost from the cell surface during terminal differentiation. Thus modulation of beta 1 integrin subunit expression may play an important role in regulating differentiation in pig epidermis.
Assuntos
Moléculas de Adesão Celular/análise , Glicoproteínas/química , Integrinas/análise , Pele/citologia , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Moléculas de Adesão Celular/química , Imunofluorescência , Integrinas/química , Pele/química , SuínosRESUMO
Skin grafts can be produced in the laboratory from simple sheets of cultured keratinocytes (keratinocyte grafts) or in combination with different mixtures of connective tissue components (composite culture grafts). Autologous keratinocyte grafts have been used most extensively in patients with major body surface burns and have proved life saving. Further attention to clinical factors has improved graft take to 50-60% in optimal circumstances although there is some short term instability of the graft. Keratinocyte autografts can also be used to treat other chronic wounds such as leg ulcers, and surgical excisions. Keratinocyte allografts do not survive transplantation but have effects on wound healing by the release of growth factors and matrix components. Composite grafts have been little used in clinical practice and there are inherent problems with the stability of the matrix components in the presence of high levels of wound collagenases, but banks of allogenic skin grafts may provide temporary cover in burns patients. The roles and clinical indications for keratinocyte grafting are now becoming clear following wider clinical experience.
Assuntos
Queratinócitos/transplante , Transplante de Pele , Células Cultivadas , Citocinas/fisiologia , Técnicas Citológicas , Humanos , Queratinócitos/fisiologia , Transplante de Pele/fisiologia , Transplante Autólogo , Transplante Homólogo , CicatrizaçãoRESUMO
In the course of immunohistochemical studies it has become apparent that there is a distinct phenotype of keratin expression that is shared by basal epithelial cells in a variety of different tissues. A basal cell can be defined as a cell in contact with a basal lamina but with no free luminal surface; this distinguishes it from a simple epithelial cell, which has a free luminal surface as well as basal lamina contact, and from stratifying suprabasal keratinocytes, which have neither basal lamina contact nor free luminal surface. All basal cells, whether they are in glandular ductal or secretory epithelia, or in stratified squamous epithelia, express the keratin pair K5 and K14. In this paper we describe monoclonal and polyclonal antibodies that are monospecific for both keratins 14 and 5 or are specific for denaturation-sensitive epitopes unique to basal cells, including five new monoclonal antibodies: LL001 and LL002 (to keratin 14), 2.1.D7 (to keratins 5, 6 and 8), and LH6 and LH8 (conformation-specific basal cell markers). These antibodies have been used to monitor the distribution of the basal cell phenotype and to demonstrate the expression of keratins 5 and 14 in this cell type, in both stratified epithelia and mixed epithelial glands. The consistent association of this keratin pair with basal cells suggests a possible specific function for these keratin in reinforcing epithelia under physical stress, whilst expression of these keratins may conflict with the differentiated functions of most simple epithelial cells.
Assuntos
Anticorpos , Células Epiteliais , Queratinas/análise , Sequência de Aminoácidos , Anticorpos Monoclonais , Especificidade de Anticorpos , Células Cultivadas , Colágeno/análise , Epitélio/química , Humanos , Técnicas Imunoenzimáticas , Queratinócitos/química , Dados de Sequência Molecular , Coloração e RotulagemRESUMO
Expression of hCG and its free subunits by non-trophoblastic tumours is well recognised. Previously we reported hCG secretion by normal and malignant bladder epithelial cells in vitro. Here we examined culture medium from 83 different cell lines derived mainly from common epithelial tumours. Thirty-two of the cell lines were found to secrete hCG-like material into their culture media. Partial immunochemical characterisation showed that of these only choriocarcinoma and fetal tissue cell lines produced intact hCG and alpha subunit. The remaining 28 hCG-expressing epithelial cell lines, which are of mucosal origin, only secreted free beta subunit. Expression of free beta hCG by non-trophoblastic nonendocrine cells would appear to be especially characteristic of mucosal epithelia from the genitourinary and oral/respiratory tracts. Furthermore, this phenomenon may be characteristic of epithelium with transitional and/or squamous cell-like properties.
Assuntos
Carcinoma/metabolismo , Gonadotropina Coriônica/metabolismo , Linhagem Celular , Epitélio/metabolismo , Feminino , Humanos , Masculino , Células Tumorais Cultivadas/metabolismoRESUMO
The type I keratin 19 is unusual in its tissue distribution in that under normal circumstances it does not seem to be restricted, as the other keratins are, to expression in either stratified or simple epithelia. In addition to the previously reported distribution of keratin 19 in human tissues, we have observed keratin 19 in epidermal basal cells, in a defined region of the hair follicle, and in nipple epidermis. We noticed that expression of keratin 19 appears to be especially characteristic of regions of labile or variable cellular differentiation as indicated by the presence of multiple keratin phenotypes in close proximity to each other. Using a monoclonal antibody recognizing keratin 19 (LP2K) to screen a human placenta cDNA expression library, we have isolated, cloned, and sequenced cDNA coding for full-length human keratin 19, as confirmed by its reactivity with several other known anti-keratin 19 monoclonal antibodies and by the near identity of its sequence with that of the bovine keratin 19 homologue. This similarity extends to both proteins being truncated at the C-terminal end to only 13 amino acids beyond the rod domain. Although the amino acid homology over the N-terminal and helical rod domains is particularly high, the human and bovine proteins diverge substantially over the short C-terminal domain, which suggests that this region has no conserved function. Comparison with other type I keratins indicates that the closest evolutionary neighbors of keratin 19 are keratinocyte keratins, probably 13 and 14, and not the simple epithelial keratin 18. Assessing the histochemistry and sequence data together, we propose that the cell may use this apparently deficient keratin as a "neutral" keratin. While unimpaired in its ability to polymerize (keeping the cell integrated into the epithelial sheet via filament-desmosome networks), keratin 19 expression does not irrevocably commit a cell to any one of the local differentiation options. Such predicted differentiational flexibility may also imply vulnerability to transformation.
Assuntos
Queratinas/análise , Sequência de Aminoácidos , Anticorpos Monoclonais , Células Epidérmicas , Humanos , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
The murine monoclonal antibody LH 7:2, which reacts with the basement membrane of stratified squamous epithelia including epidermis, has been characterized biochemically and shown to bind to part of the type VII collagen molecule. Immunoblotting reveals that the antibody binding site lies in the non-helical carboxy terminal region of the type VII collagen dimer and immunoelectron microscopy shows that the epitope is within the lamina densa of the basement membrane. Loss of LH 7:2 binding in the hereditary blistering disease recessive dystrophic epidermolysis bullosa suggests that inadequate synthesis or excessive breakdown of type VII collagen may form the biologic basis for the disease.
Assuntos
Membrana Basal/metabolismo , Colágeno/metabolismo , Epiderme/metabolismo , Genes Recessivos , Dermatopatias Vesiculobolhosas/metabolismo , Colágeno/classificação , Epiderme/ultraestrutura , Imunofluorescência , Humanos , Técnicas Imunológicas , Microscopia Eletrônica , Dermatopatias Vesiculobolhosas/genéticaRESUMO
Cultured keratinocytes were used as allografts to treat 51 patients with chronic venous ulceration or rheumatoid ulcers unresponsive to all previous conventional treatments including split skin grafts. Although early epithelialization could be seen in the centre of some ulcers, a major effect appeared to be healing from the previously indolent edge. This treatment appears to provide some clinical benefit in healing of chronic ulceration.