RESUMO
Summary: The recent European Union and Italian regulations in the matter of in vivo test could strongly impact on current diagnostic approach, increasing the usage of in vitro tests in daily clinical practice. We evaluated 506 patients with both skin prick test and a microarray system (ImmunoCAP ISAC 112). The overall evaluation between ImmunoCAP® ISAC vs SPT showed a moderate agreement (k=0.509, 95% C.I. 0.480-0.540, SE: 0.016) considering both aeroallergens and food allergens. When we considered the concordant results (double-positive plus double-negatives), the agreement ranged from 69% to 80% for pollen allergens, between 74% and 76% for dust mites, and between 74% and 93% for animal epithelia. In the case of food allergens, the accordance was pretty lower, accounting values ranging from 67% to 86%. ISAC testing identified from 22% to 26% more cases than SPTs in peach and nuts hyper-sensitivity. In 2.8% of the control group, the ISAC-test failed to detect an allergy sensitization caused by dust mite, shrimp, Anisakis, or seed storage proteins. Multiplex testing is more than a promising tool for more precise and comprehensive profiling of allergic patients and can be considered as a second-line approach, after the anamnesis, in the diagnosis of allergic diseases.
Assuntos
Hipersensibilidade/diagnóstico , Análise Serial de Proteínas/métodos , Testes Cutâneos/métodos , Adolescente , Adulto , Idoso , Alérgenos/imunologia , Testes Diagnósticos de Rotina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
Summary: The families of seed storage proteins, together with profilins, oil-bodies-associated oleosins, and pathogenesis-related (PR) proteins like PR-10 (Bet v 1-like), PR-12 (defensins) and PR-14 (non-specific lipid transfer protein), are the main causes of IgE sensitization to tree nuts, legumes and seeds. All these allergens, with the exclusion of profilins and of PR-10, are heat-stable and possibly responsible for fatal or almost fatal adverse reactions to such foods. In this short review, we will discuss the relationship and amino acid identities among some of the seed storage homologue molecules identified to date from tree nuts, seeds and legumes.
Assuntos
Alérgenos/genética , Antígenos de Plantas/genética , Hipersensibilidade a Noz/imunologia , Proteínas de Armazenamento de Sementes/genética , Homologia de Sequência de Aminoácidos , Alérgenos/imunologia , Antígenos de Plantas/imunologia , Fabaceae/imunologia , Humanos , Imunoglobulina E/imunologia , Nozes/imunologia , Proteínas de Armazenamento de Sementes/imunologia , Sementes/imunologiaRESUMO
Summary: Background. The development of recombinant technology supported the allergy diagnostic work-up in the daily clinical practice, representing a useful tool for epidemiological studies. Methods. An atlas of the IgE sensitization profiles found throughout Italy was prepared from a nationwide, multicenter, cross-sectional study. Results. 6052 unselected consecutive individuals, belonging to North-West [NW], North-East [NE], Centre [C], South [S], and Islands subset [Is] were evaluated by means of the ImmunoCAP ISAC test. The top-ranked sensitizations found were Cup a 1 in [C] (58.1%) and [S] (53.6%), Phl p 1 in the North (from 46.1% to 49%), and Cyn d 1 in [Is] (44.2%). High frequency of house dust mite group 2 molecules sensitization was found in [C] (36.9%) and [S] Italy (40.8%), whilst low level of reactivity was recorded in [NW] (20%). Pellitory hypersensitivity was mainly found in [C], [S], and [Is], whilst ragweed Amb a 1 sensitivity was particularly found in [NW] Italy. IgE recognition of PR-10, Profilin, and nsLTP was mutually exclusive in 69.1% of cases, PR-10 reactivity mostly occurring in [NE], Profilin in [NW], and nsLTP molecules recognition mainly recorded in [C] and [S]. Conclusions. Divergent IgE sensitization patterns were found along Italy, possibly linked to the distinct geographical locations, indicating multiplex system IgE analysis as a reliable approach for epidemiological evaluation even in small geographical areas.
Assuntos
Hipersensibilidade/epidemiologia , Imunização/estatística & dados numéricos , Imunoglobulina E/metabolismo , Adolescente , Adulto , Alérgenos/genética , Alérgenos/imunologia , Estudos Transversais , Testes Diagnósticos de Rotina , Feminino , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Adulto JovemRESUMO
BACKGROUND: Aplasia cutis congenita (ACC) has been associated with all clinical forms of inherited epidermolysis bullosa (EB), including dominant and recessive dystrophic EB (DDEB and RDEB). To date, only a few patients with DEB specifically combined with ACC have been described and genotyped and almost all cases represent dominant forms of the condition. OBJECTIVES: The aim of this study was to describe new mutations of COL7A1 in patients with DEB and ACC and investigate possible genotype-phenotype correlations. METHODS: Twenty-two patients with DEB and ACC were included among the 123 patients with DEB whose COL7A1 mutations have been identified in the Reference Centre in Nice. RESULTS: Seven patients presented a severe generalized RDEB phenotype (RDEB-sev-gen), while the other 15 suffered from milder phenotypes. We identified 28 mutations in COL7A1, of which nine are novel. Patients with severe phenotypes have mostly mutations leading to premature termination codon (PTC) and/or splice-site or missense mutations. Patients with the milder phenotypes have mostly glycine or arginine substitutions associated or not with other types of mutations. All amino acid substitutions fell within the carboxyl portion of the triple helix domain (THD) of collagen VII, close to the THD interruptions. CONCLUSIONS: Our findings suggest that ACC is a frequent manifestation in patients with DEB irrespective of the severity of the disease, and is due to leg rubbing in utero. In children with a moderate form of DEB with no or moderate skin fragility, a glycine substitution near the THD interruption domain of the collagen VII leading to thermolabile protein could explain this phenomenon.
Assuntos
Substituição de Aminoácidos/genética , Colágeno Tipo VII/genética , Displasia Ectodérmica/genética , Epidermólise Bolhosa Distrófica/genética , Mutação/genética , Criança , Feminino , Genótipo , Humanos , Masculino , FenótipoAssuntos
Alérgenos/imunologia , Hipersensibilidade Alimentar/epidemiologia , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade/epidemiologia , Hipersensibilidade/imunologia , Proteínas de Plantas/imunologia , Profilinas/imunologia , Reações Cruzadas , Humanos , Itália/epidemiologia , Pólen/imunologiaRESUMO
BACKGROUND: Genetic mutations in the plectin gene (PLEC) cause autosomal recessive forms of epidermolysis bullosa simplex (EBS) associated with either muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). Phenotype-genotype analysis has suggested that EBS-MD is due mostly to genetic mutations affecting the central rod domain of plectin, and EBS-PA to mutations outside this domain. OBJECTIVES: This study aimed to describe new phenotypes of patients with EBS-MD and EBS-PA, to identify novel PLEC mutations and to establish genotype-phenotype correlations. METHODS: Seven patients with a suspicion of EBS linked to PLEC mutations were included. A standardized clinical questionnaire was sent to the physicians in charge of each patient. Immunofluorescence studies of skin biopsies followed by molecular analysis of PLEC were performed in all patients. RESULTS: We report the first case of nonlethal EBS-PA improving with age, the first multisystemic involvement in a patient with lethal EBS-PA, and the first patients with EBS-MD with involvement of either the bladder or oesophagus. Eleven novel PLEC mutations are also reported. CONCLUSIONS: Our results confirm that EBS-PA is linked to mutations in the distal exons 1-30 and 32 of PLEC. Long-term survival is possible, with skin improvement, but a delayed onset of MD is probable. While EBS-MD is linked to PLEC mutations in all exons, in most cases one of the mutations affects exon 31. The precocity of MD seems to be linked to the type and localization of the PLEC mutation(s), but no correlation with mucosal involvement has been found.
Assuntos
Epidermólise Bolhosa Simples/genética , Mutação/genética , Plectina/genética , Adulto , Criança , Epidermólise Bolhosa Simples/complicações , Obstrução da Saída Gástrica/complicações , Genótipo , Humanos , Lactente , Recém-Nascido , Distrofias Musculares/complicações , Fenótipo , Piloro/anormalidadesRESUMO
Pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB), is a rare inherited disorder characterized by pyloric stenosis and blistering of the skin as primary manifestations. We demonstrate that in one PA-JEB patient the disease resulted from two distinct mutations in the beta 4 integrin gene alleles. The paternal mutation consists of a one base pair deletion causing a shift in the open reading frame, and a downstream premature termination codon. The maternal mutation occurs in a donor splice site, and results in in-frame exon skipping involving the cytoplasmic domain of the polypeptide. Our results implicate mutations in the beta 4 integrin gene in some forms of PA-JEB.
Assuntos
Epidermólise Bolhosa Juncional/genética , Integrinas/genética , Mutação Puntual , Estenose Pilórica/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Evolução Fatal , Feminino , Expressão Gênica , Humanos , Recém-Nascido , Integrina alfa6 , Integrina beta4 , Integrinas/biossíntese , Queratinócitos/metabolismo , Masculino , Dados de Sequência Molecular , Linhagem , RNA/análiseAssuntos
Antibacterianos/administração & dosagem , Fármacos Dermatológicos/administração & dosagem , Epidermólise Bolhosa Simples/tratamento farmacológico , Eritromicina/administração & dosagem , Dermatopatias Bacterianas/tratamento farmacológico , Administração Oral , Criança , Pré-Escolar , Epidermólise Bolhosa Simples/complicações , Feminino , Humanos , Lactente , Masculino , Dermatopatias Bacterianas/complicações , Infecções Cutâneas Estafilocócicas/complicações , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Staphylococcus aureus , Infecções Estreptocócicas/complicações , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus , Resultado do TratamentoRESUMO
BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life-threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing. OBJECTIVES: To conduct the first mutational analysis of COL7A1 in a Spanish cohort, to assess mutation consequences at protein/mRNA level and to establish genotype-phenotype correlations. METHODS: Forty-nine Spanish patients with DEB were studied. Antigen mapping was performed on patient skin biopsies. COL7A1 mutation screening in genomic DNA was performed by polymerase chain reaction (PCR) and direct sequencing. Mutation consequences were determined by reverse transcriptase-PCR. RESULTS: Eight patients belonged to three unrelated families with dominant DEB. Forty-one were affected with recessive DEB (RDEB). Specifically, 27 displayed the severe generalized subtype, eight the other generalized subtype and six a localized phenotype (two pretibial, three acral and one inversa). Thirty-five mutations were identified, 20 of which are novel. The pathogenic mutation c.6527insC accounted for 46.3% of Spanish RDEB alleles. A consistent genotype-phenotype correlation was established. CONCLUSIONS: Although the COL7A1 database indicates that most DEB mutations are family specific, the pathogenic mutation c.6527insC was highly recurrent in our cohort. This level of recurrence for a single genetic defect has never previously been reported for COL7A1. Our findings are essential to the clinicians caring for patients with DEB in Spain and in the large population of Spanish descendants in Latin America. They also provide geneticists a molecular clue for a priority mutation screening strategy.
Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA/métodos , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lactente , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Espanha , Adulto JovemRESUMO
Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the gamma2 chain that removes the NH(2)-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the gamma2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the gamma2 chain short arm, we expressed mutated gamma2 cDNAs into immortalized gamma2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the gamma2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the gamma2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.
Assuntos
Queratinócitos/citologia , Queratinócitos/metabolismo , Laminina/genética , Laminina/metabolismo , Membrana Basal/metabolismo , Sítios de Ligação/genética , Adesão Celular/fisiologia , Linhagem Celular Transformada , Movimento Celular/fisiologia , Primers do DNA , DNA Complementar , Epidermólise Bolhosa/metabolismo , Epidermólise Bolhosa/patologia , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica/fisiologia , Humanos , Laminina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/fisiologia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
P27kip is a key inhibitory protein of the cell-cycle progression, which is rapidly downregulated in early G1 phase by a post-translational mechanism involving the proteosomal degradation. In this study, using a wounding model that induces cell-cycle entry of human dermal fibroblasts, we demonstrate that p27mRNA is downregulated when cells progress into the G1 phase, and then it returns to its basal level when cells approach the S phase. By using a quantitative polymerase chain reaction screening we identified inhibitors of differentiation (Id3), a bHLH transcriptional repressor, as a candidate mediator accounting for p27 mRNA decrease. Id3 silencing, using an small interfering RNA approach, reversed the injury mediated p27 downregulation demonstrating that Id3 is involved in the transcriptional repression of p27. Reporter gene experiments and a chromatin immunoprecipitation assay showed that Id3 likely exerts its repressive action through ELK1 inhibition. By inhibiting early p27 downregulation, Id3 depletion blocked (i) the G1-phase progression as assessed by the inhibition of pRb phosphorylation and p130 degradation and (ii) the G1/S transition as observed by the inhibition of cyclin A induction, demonstrating that p27 mRNA decrease is required for cell proliferation. Apart from its effect on the early p27 diminution, Id3 appears also involved in the control of the steady-state level of p27 at the G1/S boundary. In conclusion, this study identifies a novel mechanism of p27 regulation which besides p27 protein degradation also implicates a transcriptional mechanism mediated by Id3.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Fase G1/fisiologia , Regulação da Expressão Gênica , Proteínas Inibidoras de Diferenciação/fisiologia , Proteínas de Neoplasias/fisiologia , RNA Mensageiro/metabolismo , Fase S/fisiologia , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Derme/lesões , Derme/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Immunoblotting , Luciferases , Camundongos , Células NIH 3T3 , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Cicatrização/fisiologiaRESUMO
INTRODUCTION: Dystrophic epidermolysis bullosa is a hereditary heterogeneous blistering disease. Clinical examination and additional tests are not always sufficient to identify the subtype or mode of transmission. We describe a case of de novo dominant inherited dystrophic epidermolysis bullosa localised strictly to the knees. CASE REPORT: A 3-year-old boy presented symmetrical lesions on the anterior aspect of the knees since starting to walk. No nail, dental or mucous dystrophy was observed and the parents presented no clinical abnormalities. Optical microscopy, electron microscopy and immunofluorescence analysis suggested dystrophic epidermolysis bullosa. The genealogical tree allowed no distinction between the dominant de novo and mitis recessive forms. Genetic analysis identified a missense G 1776W mutation at exon 61 of gene COL 7A1 in the child's DNA but not the parents'. DISCUSSION: Dystrophic epidermolysis bullosa may present in generalized or localized forms and the disease may be inherited in either autosomal dominant or recessive mode. Genetic analysis shows mutations in COL 7A1. While the clinical features often allow different types to be distinguished when the parents do not have the disease (with the recessive forms being more severe), genetic analysis is essential to confirm the mode of inheritance. In the dominant forms, and more recently in recessive cases, glycine substitutions have been implicated, although the precise role of glycine substitution has yet to be clarified. Localised involvement of the skin alone, as seen in our case report, is very rare. CONCLUSION: Genetic analysis is important for genetic counselling and determination of risk of recurrence.
Assuntos
Colágeno Tipo VII/genética , Colágeno/genética , Epidermólise Bolhosa Distrófica/genética , Substituição de Aminoácidos , Pré-Escolar , Diagnóstico Diferencial , Epidermólise Bolhosa Distrófica/diagnóstico , Epidermólise Bolhosa Distrófica/patologia , Éxons , Feminino , Humanos , Imunofenotipagem , Masculino , Linhagem , Pele/patologia , Pele/ultraestruturaRESUMO
The alpha6 integrin subunit participates in the formation of both alpha6beta1 and alpha6beta4 laminin receptors, which have been reported to play an important role in cell adhesion and migration and in morphogenesis. In squamous epithelia, the alpha6beta4 heterodimer is the crucial component for the assembly and stability of hemidesmosomes. These anchoring structures are ultrastructurally abnormal in patients affected with junctional epidermolysis bullosa with pyloric atresia (PA-JEB), a recessively inherited blistering disease of skin and mucosae characterized by an altered immunoreactivity with antibodies specific to integrin alpha6beta4. In this report, we describe the first mutation in the alpha6 integrin gene in a PA-JEB patient presenting with generalized skin blistering, aplasia cutis, and defective expression of integrin alpha6beta4. The mutation (791delC) is a homozygous deletion of a single base (C) leading to a frameshift and a premature termination codon that results in a complete absence of alpha6 polypeptide. We also describe the DNA-based prenatal exclusion of the disease in this family at risk for recurrence of PA-JEB. Our results demonstrate that, despite the widespread distribution of the alpha6 integrin subunit, lack of expression of the alpha6 integrin chain is compatible with fetal development, and results in a phenotype indistinguishable from that caused by mutations in the beta4 chain, which is expressed in a more limited number of tissues.
Assuntos
Antígenos de Superfície/genética , Epidermólise Bolhosa Juncional/genética , Mutação da Fase de Leitura , Integrinas/genética , Piloro/anormalidades , Sequência de Bases , Northern Blotting , Códon , Epidermólise Bolhosa Juncional/patologia , Feminino , Imunofluorescência , Deleção de Genes , Homozigoto , Humanos , Recém-Nascido , Integrina alfa6beta4 , Queratinócitos/química , Queratinócitos/patologia , Microscopia Eletrônica , Gravidez , RNA Mensageiro/análise , Pele/química , Pele/patologiaRESUMO
Previous studies have correlated the Herlitz junctional epidermolysis bullosa (H-JEB) to an altered expression of the basement membrane component nicein/kalinin. This heterotrimeric glycoprotein appears to be present in H-JEB tissues in an abnormal form, because a number of antibodies specific to the protein either do not react with or weakly stain the epidermal basement membranes of most of the patients. With cDNA probes encoding each subunit of nicein and polyclonal antibodies raised against bacterial fusion polypeptides corresponding to the individual chains of the protein, we have molecularly analyzed the expression of nicein in H-JEB tissues and cultured keratinocytes. By immunohistochemistry, Northern blot, and protein analysis, we show a defective synthesis of one of the nicein subunits in six cases of H-JEB from five different consanguineous families. In two patients, the disease correlates with an impaired synthesis of the nicein B2 (nic B2) chain, in three others with that of the B1 (nic B1) chain, and in a sixth patient with that of the heavy A (nic A) chain. In this report, we thus demonstrate that H-JEB is a genetically heterogeneous disease and we provide strong evidence that the genes of nicein are the candidates for this genodermatosis.
Assuntos
Moléculas de Adesão Celular/biossíntese , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/metabolismo , Expressão Gênica , Queratinócitos/metabolismo , Pele/metabolismo , Northern Blotting , Moléculas de Adesão Celular/análise , Células Cultivadas , Epidermólise Bolhosa Juncional/patologia , Feto , Imunofluorescência , Humanos , Recém-Nascido , Substâncias Macromoleculares , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Valores de Referência , CalininaRESUMO
Plectin is a widely expressed cytomatrix component involved in the attachment of the cytoskeleton to the plasma membrane. We have recently reported that the skin and muscles of three patients affected by epidermolysis bullosa simplex with muscular dystrophy (MD-EBS), a genetic disorder characterized by skin blistering associated with muscle involvement, are not reactive with antibodies specific to plectin. We demonstrated that in the skin, lack of plectin leads to failure of keratin filaments to connect to the plasma membrane via the hemidesmosomes, whereas in the muscle the deficient expression of the molecule correlates with an aberrant localization of desmin in the muscle fibers. In this study we demonstrate that in a MD-EBS kindred with two affected members, the disease results from a homozygous nonsense mutation in the plectin (PLEC1) gene leading to a premature stop codon (CGA to TGA) and decay of the aberrant plectin messenger RNA. The segregation of the mutated allele implicates the mutation in the pathology of the disorder. These results confirm the critical role of plectin in providing cell resistance to mechanical stresses both in the skin and the muscle.
Assuntos
Códon sem Sentido/genética , Epidermólise Bolhosa Simples/genética , Proteínas de Filamentos Intermediários/genética , Distrofias Musculares/complicações , Alelos , Northern Blotting , Códon sem Sentido/fisiologia , Códon de Terminação/genética , Desmina/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/metabolismo , Linhagem , Plectina , Mutação Puntual , Pele/metabolismoRESUMO
Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.
Assuntos
Epidermólise Bolhosa Simples/metabolismo , Proteínas de Filamentos Intermediários/análise , Distrofias Musculares/metabolismo , Adulto , Células Cultivadas , Desmina/análise , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Músculos/química , Plectina , Pele/química , Pele/ultraestruturaRESUMO
The histogenesis of neuroendocrine carcinomas of the skin is still controversial. To determine the degree of neural differentiation of these neoplasias, we studied the expression of intermediate filament proteins in tumoral tissues. Expressions of peripherin, the neurofilament protein NF-L, vimentin, and cytokeratin 8 were analyzed by immunohistochemical methods on 12 human primary tumors and 3 tumor xenografts on nude mice. Peripherin was detected in 10 primary tumors by immunofluorescence. The protein and the corresponding messenger RNA were identified by two-dimensional gel electrophoresis and Northern analysis in extracts of an immunofluorescence-negative tumor. Peripherin, NF-L, and cytokeratin 8 were detected in tumoral cells, whereas vimentin was found exclusively in the stroma. The histological and ultrastructural properties of the original cells of neuroendocrine carcinomas of the skin, as well as coexpression of peripherin, cytokeratin 8, and neurofilament polypeptides, were preserved in tumor xenografts and their primary cultures in vitro. These results bring new elements to the knowledge of the biology of neuroendocrine carcinomas of the skin and indicate that peripherin constitutes a marker for tumor identification.
Assuntos
Carcinoma de Célula de Merkel/química , Proteínas de Filamentos Intermediários/análise , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Neuropeptídeos/análise , Neoplasias Cutâneas/química , Animais , Humanos , Proteínas de Filamentos Intermediários/fisiologia , Queratinas/análise , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neuropeptídeos/fisiologia , Periferinas , Transplante Heterólogo , Células Tumorais Cultivadas , Vimentina/análiseRESUMO
The E2 open reading frame of bovine papillomavirus 1 (BPV1) encodes both positive and negative transcriptional regulatory factors. The full-length E2 gene polypeptide is a strong transcriptional transactivator that acts on enhancers within the papillomavirus long control region (LCR), and two shorter E2 proteins function as transcription repressors. A vaccinia recombinant virus harboring the full length E2 coding sequence of BPV1 directs the synthesis of a 48 kD phosphoprotein with specific DNA binding activity. We show that in BPV1-transformed cells the full-length transactivator is a phosphoprotein, whereas truncated E2 proteins were not detectably phosphorylated.
Assuntos
Papillomavirus Bovino 1/genética , Genes Virais , Papillomaviridae/genética , Transativadores/genética , Aminoácidos/análise , Animais , Sequência de Bases , Papillomavirus Bovino 1/metabolismo , Linhagem Celular , Células Cultivadas , Clonagem Molecular , DNA Viral/genética , DNA Viral/metabolismo , Imunofluorescência , Expressão Gênica , Dados de Sequência Molecular , Fosfoproteínas/análise , Fosfoproteínas/biossíntese , Mapeamento por Restrição , Transativadores/metabolismoRESUMO
The rat cell line FR3T3 was transformed with the retroviral oncogenes v-myc or v-src, with the DNA tumor viruses SV40 or bovine papilloma virus strain 1 (BPV-1) or with the 69% transforming region of BPV-1. The transformants were compared with the uncloned parental line for their susceptibility to the lytic effect and to the replication of MVMp, an autonomous parvovirus. Expression of v-myc and v-src proteins and of SV40 large T antigen correlated with a greater cell susceptibility to MVMp-induced killing. Thus, the expression of both cytoplasmic and nuclear oncogene products may sensitize rat fibroblasts to MVMp. In contrast, cell lines transformed by BPV-1, including highly tumorigenic and tumor-derived clones, were on the average as resistant as the parental cell line to MVMp infection. A similar resistance to MVMp-induced killing was displayed by BPV-1-transformed NIH3T3 cells. However, supertransformation of one of the BPV-1-transformants by the human EJ-Harvey ras-1 oncogene, known to sensitize FR3T3 and NIH3T3 cells, correlated with an increase in susceptibility to MVMp. Therefore, the failure of BPV-1 transformation to sensitize murine cells to parvoviral attack may be ascribed to the tumor virus rather than to the cells undergoing transformation. Hence, cell sensitization to MVMp appears to be oncogene-specific and cannot be taken as an absolute correlative with neoplastic transformation.