RESUMO
Ehlers-Danlos syndrome (EDS) type III is a inherited connective tissue disorders characterized by extensibility of the skin, hypermobility of the joints, chronic pain, tissue fragility, easy bruising, and delayed wound healing with result of atrophic scars. The patients report commonly a history of recurrent dislocations of the shoulders and knees after low-impact trauma, chronic joint pain, and early osteoarthritis, which lead to diagnosis. The pathogenesis of this condition is unknown, and the diagnosis is generally made in adult age, based only on clinical criteria. In this report, we describe a case of a 50-year-old woman with a 30-year history of recurrent dislocations and atrophic scars. We performed diagnosis of EDS type III after a complete clinical and instrumental evaluation, comprising of histological and electron microscopic studies, that highlighted collagen abnormalities.
Assuntos
Derme/ultraestrutura , Síndrome de Ehlers-Danlos/diagnóstico , Colágenos Fibrilares/ultraestrutura , Microscopia Eletrônica de Transmissão , Biópsia , Síndrome de Ehlers-Danlos/complicações , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patologia , Feminino , Humanos , Luxações Articulares/etiologia , Instabilidade Articular/etiologia , Pessoa de Meia-Idade , Valor Preditivo dos Testes , RecidivaRESUMO
Several studies have suggested a possible role for HPV in the pathogenesis of the breast cancer. We investigated the presence of the HPV DNA in breast cancers and non malignant disease breast tissues by the use of a standard HPV detection method (INNO-Lipa HPV), in order to detect HPV DNA in metastatic nodes, to investigate a possible cervical HPV co-infection, and to evaluate the E6/E7 mRNA expression in HPV DNA positive breast cancer tissues. The rate of HPV infection was significantly higher in the cancer group than in controls (9/31 vs. 0/12, p = 0.04). One out of eight metastatic axillary nodes was positive for HPV infection; 2/3 of the positive HPV breast cancer patients were co-infected at the cervical site. The role of the virus in breast oncogenesis is still unclear, since our analysis failed in demonstrating the expression of viral E6 and E7 in positive HPV positive breast tumor tissues.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Fibroadenoma/metabolismo , Papiloma/metabolismo , Adulto , Idoso , Neoplasias da Mama/virologia , Carcinoma Ductal de Mama/virologia , Carcinoma Lobular/virologia , DNA Viral/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroadenoma/virologia , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Papillomavirus Humano 31/isolamento & purificação , Papillomavirus Humano 6/isolamento & purificação , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/metabolismo , Papiloma/virologia , Proteínas E7 de Papillomavirus/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Since the introduction of the cytological screening programs, a significant reduction in the incidence of cervical cancer has been achieved. Almost all of these cancers are related to high-risk (HR) Human Papillomavirus (HPV) cervical infections. However, the natural history of HPV infection seems to be different in younger patients, resulting in a higher rate of regression. There is, therefore, the need to identify HPV-related biomarkers in order to enhance the effectiveness of screening of high-risk cytological lesions, in particular in women over 35 years of age. This study aims to evaluate the prognostic value of the HR HPV E6 and E7 mRNA expression in women with intraepithelial lesions of the cervix, older or younger than 35 years of age. One hundred and eighty-four HR HPV DNA positive patients with a low squamous intraepithelial lesion (LSIL) were tested for mRNA expressions, included in an observational study, and evaluated at follow-up with standard cytology up to 24 months from the mRNA test. The frequency of HSIL/LSIL cytology in the older cohort of mRNA positive patients was significantly higher compared to mRNA-negative patients, both at 1 and 2 years of follow-up (Chi-square: p 0.007 and p 0.009), but this difference was not found in the younger cohort. According to our results, the E6/E7 mRNA test could be a biomarker for viral activity, useful in identifying patients at higher risk of abnormal cytology, and in implementing the management of HR HPV DNA-positive women over 35 years of age.
Assuntos
Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , RNA Mensageiro/análise , RNA Viral/análise , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Proteínas do Envelope Viral/genética , Adolescente , Adulto , Fatores Etários , Idoso , Distribuição de Qui-Quadrado , DNA Viral/análise , Feminino , Humanos , Itália , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Infecções por Papillomavirus/complicações , Valor Preditivo dos Testes , Prognóstico , Kit de Reagentes para Diagnóstico , Medição de Risco , Fatores de Risco , Fatores de Tempo , Neoplasias do Colo do Útero/patologia , Esfregaço Vaginal , Adulto Jovem , Displasia do Colo do Útero/patologiaRESUMO
Anetoderma is a benign condition characterized by round or oval macular lesions with focal loss of dermal elastic tissue resulting in localized areas of flaccid or herniated saclike skin. Often, the anetoderma is associated with immuno-mediated pathogenetic mechanism. In this article, we describe the association between anetoderma and autoimmune diseases, by underlining the role and the action of macrophages as a possible etiopathogenesis.
Assuntos
Anetodermia/imunologia , Doenças Autoimunes/imunologia , Autoimunidade , Macrófagos/imunologia , Pele/imunologia , Idoso , Anemia Hemolítica Autoimune/complicações , Anemia Hemolítica Autoimune/imunologia , Anetodermia/patologia , Doenças Autoimunes/complicações , Biópsia , Feminino , Humanos , Macrófagos/patologia , Pessoa de Meia-Idade , Esclerose Múltipla/complicações , Esclerose Múltipla/imunologia , Miastenia Gravis/complicações , Miastenia Gravis/imunologia , Fatores de Risco , Pele/patologiaRESUMO
We investigated the expression and distribution of keratinocyte growth factor (KGF) (FGF-7) and its receptor (KGFR) during reepithelialization of human skin. KGF mRNA levels increased rapidly by 8-10-fold and remained elevated for several days. In contrast, KGFR transcript levels decreased early but were significantly elevated by 8-9 d. A KGF-immunoglobulin G fusion protein (KGF-HFc), which specifically and sensitively detects the KGFR, localized the receptor to differentiating keratinocytes of control epidermis, but revealed a striking decrease in receptor protein expression during the intermediate period of reepithelization. Suramin, which blocked KGF binding and stripped already bound KGF from its receptor, failed to unmask KGFRs in tissue sections from the intermediate phase of wound repair. The absence of KGFR protein despite increased KGFR transcript levels implies functional receptor downregulation in the presence of increased KGF. This temporal modulation of KGF and KGFRs provides strong evidence for the functional involvement of KGF in human skin reepithelialization.
Assuntos
Epiderme/fisiologia , Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/biossíntese , Receptores de Fatores de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento/biossíntese , Cicatrização/genética , Adulto , Diferenciação Celular , Regulação para Baixo , Epiderme/efeitos dos fármacos , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Imunoglobulinas , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Proteínas Recombinantes de Fusão/biossíntese , Transplante de Pele , Suramina/farmacologiaRESUMO
BACKGROUND: Cutaneous pigmentation is regulated by a complex melanogenic network in which both keratinocytes and fibroblasts synthesize growth factors and cytokines. Solar lentigo (SL) is characterized by hyperpigmented lesions occurring on photodamaged skin areas. Despite the association of SL to ultraviolet (UV) exposure, the mechanisms underlying the development of these spots are not completely defined. OBJECTIVES: To analyse the involvement of the fibroblast-derived growth factors, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) in SL hyperpigmentation; to evaluate whether the photoageing process occurring in fibroblasts could be responsible for the altered expression of these cytokines; and to investigate a new possible role of KGF in regulating pigmentation through the specific induction of melanogenic cytokines by keratinocytes. METHODS: We performed immunohistochemical analysis of HGF, KGF and SCF on SL biopsies. We analysed the mRNA expression of these cytokines using an in vitro model of photoageing induced on fibroblasts. Finally, we evaluated the effects of KGF on the expression of melanogenic cytokines at the mRNA and protein levels on keratinocytes. RESULTS: We found positive staining for HGF, KGF and SCF in the upper dermis of SL lesions and a significant induction of the three cytokines in photoaged fibroblasts. We also demonstrated the contribution of KGF to pigmentation, showing its ability specifically to modulate the expression of SCF in keratinocytes. CONCLUSIONS: Fibroblasts may be persistently activated by UV exposure to release melanogenic growth factors; this inducible cytokine network acts both directly and indirectly through keratinocytes and may contribute to the hyperpigmentation of SL.
Assuntos
Fator 7 de Crescimento de Fibroblastos/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Hiperpigmentação/metabolismo , Lentigo/metabolismo , Fator de Células-Tronco/metabolismo , Luz Solar/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Biópsia , Western Blotting , Feminino , Humanos , Hiperpigmentação/etiologia , Imuno-Histoquímica , Lentigo/etiologia , Masculino , Pessoa de Meia-Idade , Transtornos de Fotossensibilidade/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismo , Envelhecimento da Pele/fisiologiaRESUMO
We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.
Assuntos
Herpesvirus Humano 8/química , Proteínas Virais , Linfócitos B/virologia , Linhagem Celular , Núcleo Celular , Humanos , Membrana Nuclear , Proteínas Nucleares , Fases de Leitura Aberta , RNA Viral/análise , Homologia de Sequência , Proteínas Virais/análise , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.
Assuntos
Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Sindbis virus/metabolismo , Proteínas Virais/metabolismo , Animais , Membrana Celular/análise , Células Cultivadas , Embrião de Galinha , Cricetinae , Fibroblastos/ultraestrutura , Técnica de Fratura por Congelamento , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Rim , Mesocricetus , Sindbis virus/crescimento & desenvolvimento , Proteínas do Envelope Viral/metabolismo , Proteínas Estruturais ViraisRESUMO
Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin-associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces. Label is sparse over the protoplasmic faces. These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face. Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freeze-fracture. This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton.
Assuntos
Membrana Eritrocítica/análise , Eritrócitos/análise , Glicoforinas/análise , Sialoglicoproteínas/análise , Membrana Eritrocítica/ultraestrutura , Técnica de Fratura por Congelamento , Histocitoquímica , Humanos , Receptores de Concanavalina A/análise , Receptores Mitogênicos/análiseRESUMO
We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes. In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments: one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues.
Assuntos
Compartimento Celular , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Membranas Intracelulares/ultraestrutura , Animais , Concanavalina A , Técnica de Fratura por Congelamento , Complexo de Golgi/ultraestrutura , Lectinas , Lisossomos/ultraestrutura , Microcorpos/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Ratos , Aglutininas do Germe de TrigoRESUMO
The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membrane is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin. The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.
Assuntos
Ilhotas Pancreáticas/análise , Pâncreas/análise , Receptores de Concanavalina A/análise , Receptores Mitogênicos/análise , Animais , Membrana Celular/análise , Retículo Endoplasmático/análise , Técnica de Fratura por Congelamento , Histocitoquímica , Ilhotas Pancreáticas/ultraestrutura , Membrana Nuclear/análise , Pâncreas/ultraestrutura , RatosRESUMO
Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold-conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane.
Assuntos
Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Técnica de Fratura por Congelamento , Glicoproteínas/biossíntese , Proteínas de Membrana/biossíntese , Microscopia Eletrônica , Membrana Nuclear/ultraestrutura , Sindbis virus/genética , Proteínas Virais/biossínteseRESUMO
We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the Golgi complex.
Assuntos
Glicoproteínas/farmacocinética , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas do Envelope Viral/farmacocinética , Animais , Transporte Biológico , Membrana Celular/metabolismo , Imunofluorescência , Imuno-Histoquímica , Microscopia Eletrônica , Células Vero , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Applications of the new fracture-labeling techniques for the observation of cytochemical labels on platinum-carbon replicas are described. Frozen cells, embedded in a cross-linked protein matrix, and frozen tissues are fractured with a scalpel under liquid nitrogen, thawed, labeled, dehydrated by the critical point drying method, and replicated. This method allows direct, high-resolution, two-dimensional chemical and immunological characterization of the cellular membranes in situ, as well as detection of sites within cross-fractured cytoplasm and extracellular matrix.
Assuntos
Células/ultraestrutura , Técnica de Fratura por Congelamento/métodos , Animais , Antígenos/análise , Grânulos Citoplasmáticos/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Eritrócitos/ultraestrutura , Humanos , Indicadores e Reagentes , Membranas Intracelulares/ultraestrutura , Leucócitos/ultraestrutura , Microscopia Eletrônica/métodos , Pâncreas/ultraestrutura , Adeno-Hipófise/ultraestrutura , Platina , RatosRESUMO
EBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.
Assuntos
Autofagossomos/virologia , Autofagia , Herpesvirus Humano 4/fisiologia , Mitocôndrias/metabolismo , Monócitos/virologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/genética , Autofagossomos/metabolismo , Autofagia/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Diferenciação Celular/genética , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-4/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Monócitos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , RNA Interferente Pequeno , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
CONCLUSION: Distribution of the receptor for epidermal growth factor (EGF-R) and of the receptor for the keratinocyte growth factor (KGF-R) in cholesteatoma was found to differ in analogy with other epithelial tissues and accordingly to epidermal differentiation and intensity of paracrine stimulation. Moreover, both EGF-R and KGF-R expression was increased, suggesting a fair correlation with aggressiveness and recurrence rate of this pathology. OBJECTIVES: To obtain information on the biological behaviour of cholesteatoma by assessing the expression and localization of EGF-R and KGF-R and correlating their tissue distribution with that of cytokeratins as a marker of differentiation. MATERIALS AND METHODS: Cholesteatoma tissue was taken during tympanoplasty surgery and processed for indirect immunofluorescence. Murine monoclonal antibodies were tested for the different growth factor receptors and pancytokeratins analysed. Fluorescence intensity signal was measured on randomly captured digital images, using FISH 2000/HI software, with a pseudocolours generation module. RESULTS: EGF-R was mostly expressed at the level of keratinocytes of the basal layer, while KGF-R signal was mainly distributed on the spinous and granular suprabasal layers that were also highly positive for cytokeratins. Significant correlation between the immunofluorescence signals was found for KGF-R and cytokeratins only, demonstrating that KGF-R expression is increased in more differentiated areas of the cholesteatoma tissue, while EGF-R is associated with proliferative and migratory portions of the lesion.
Assuntos
Colesteatoma da Orelha Média/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/biossíntese , Anticorpos Monoclonais , Biomarcadores/metabolismo , Colesteatoma da Orelha Média/patologia , Receptores ErbB/biossíntese , Receptores ErbB/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Queratinócitos/metabolismo , Microscopia de Fluorescência , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/imunologiaRESUMO
The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.