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1.
Head Neck ; 40(6): 1109-1119, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29522268

RESUMO

BACKGROUND: In head and neck squamous cell carcinoma (HNSCC), the occurrence of concurrent lung malignancies poses a significant diagnostic challenge because metastatic HNSCC is difficult to discern from second primary lung squamous cell carcinoma (SCC). However, this differentiation is crucial because the recommended treatments for metastatic HNSCC and second primary lung SCC differ profoundly. METHODS: We analyzed the origin of lung tumors in 32 patients with HNSCC using human papillomavirus (HPV) typing and targeted next generation sequencing of all coding exons of tumor protein 53 (TP53). RESULTS: Lung tumors were clearly identified as HNSCC metastases or second primary tumors in 29 patients, thus revealing that 16 patients had received incorrect diagnoses based on clinical and morphological data alone. CONCLUSION: The HPV typing and mutation analysis of all TP53 coding exons is a valuable diagnostic tool in patients with HNSCC and concurrent lung SCC, which can help to ensure that patients receive the most suitable treatment.


Assuntos
Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias Pulmonares/etiologia , Segunda Neoplasia Primária/diagnóstico , Papillomaviridae/isolamento & purificação , Carcinoma de Células Escamosas de Cabeça e Pescoço/etiologia , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Segunda Neoplasia Primária/etiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
2.
J Mol Diagn ; 20(3): 344-354, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29471115

RESUMO

Myelodysplastic syndromes are hematological neoplasias in which immunohistologic examination of bone marrow trephines is important for a definite diagnosis. Unequivocal distinction from reactive bone marrow changes is, however, sometimes difficult. Because neoplastic clones in myelodysplastic syndrome carry mutations in recurrent genes, mutation detection by targeted next-generation sequencing may be a useful support for differential diagnosis. To elucidate the accuracy of this approach in the clinical diagnostic setting, we analyzed single and consecutive bone marrow trephines processed for immunohistologic examination from 145 patients by targeted next-generation sequencing of 12 genes recurrently mutated in myelodysplastic syndromes. Of 110 patients with immunohistologic unequivocal diagnosis, 41 of 47 with myelodysplastic syndrome carried mutations. In 14 consecutive samples available from these patients, remissions were accompanied by loss of mutations and ongoing disease with persisting mutations. Of 35 samples with indefinite immunohistologic appearance, 22 developed clinical unequivocal myelodysplastic syndrome in the further course, and 19 carried mutations already in the initial biopsy, which persisted in consecutive samples available from 13 patients. No mutation was detected in any initial and consecutive sample of 13 patients with indefinite immunohistologic appearance without clinical unequivocal myelodysplastic syndrome in the further course. We conclude that targeted next-generation sequencing is an accurate tool for differential diagnosis of myelodysplastic syndrome in the clinical diagnostic setting.


Assuntos
Medula Óssea/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Células Clonais , Diagnóstico Diferencial , Humanos , Mutação/genética , Taxa de Mutação , Síndromes Mielodisplásicas/patologia
3.
Am J Pathol ; 182(4): 1205-18, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23462508

RESUMO

Despite recent advances in understanding the relevance of cell adhesion-related signaling in the pathogenesis of ischemic cardiomyopathy (ICM) in animal models, substantial questions remain unanswered in the human setting. We have previously shown that the neural cell adhesion molecule CD56 [neural cell adhesion molecule (NCAM1)] is specifically overexpressed in ICM; it was the aim of the current study to further elucidate the role of CD56 in the pathogenesis of human ICM. We used quantitative real-time PCR and IHC in human ICM and a rat model of coronary obstruction to demonstrate that CD56(140kD), the only extraneuronally expressed NCAM1 isoform with a cytoplasmic protein domain capable of inducing intracellular signaling, is the only up-regulated CD56 isoform in failing cardiomyocytes in human ICM in vivo. In subsequent analyses of the cellular effects of CD56(140kD) overexpression in the development of ICM using differential whole transcriptome expression analyses and functional in vitro cardiomyocyte cell culture assays, we further show that the up-regulation of CD56(140kD) is associated with profound gene expression changes, increased apoptosis, and reduced Ca(2+) signaling in failing human cardiomyocytes. Because apoptosis and Ca(2+)-related sarcomeric dysfunction are molecular hallmarks of ICM in humans, our results provide strong evidence that CD56(140kD) up-regulation plays a pivotal role in the pathogenesis of ICM and may be a target for future immunotherapeutic strategies in the treatment of this common and often fatal disease.


Assuntos
Antígeno CD56/metabolismo , Cardiomiopatias/patologia , Isquemia Miocárdica/patologia , Animais , Apoptose , Antígeno CD56/genética , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatias/complicações , Cardiomiopatias/genética , Proliferação de Células , Modelos Animais de Doenças , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Peso Molecular , Proteínas Mutantes/metabolismo , Isquemia Miocárdica/complicações , Isquemia Miocárdica/genética , Miocárdio/metabolismo , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
6.
Arch Anim Nutr ; 63(1): 26-38, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19271549

RESUMO

Rumen adaptation plays an important role in the productive cycle of dairy cattle. In this study, the time course of functional rumen epithelium adaptation after a change from hay feeding (ad libitum) to a mixed hay/concentrate diet was monitored by measuring Na+ transport rates in Ussing chamber experiments. A total of 18 sheep were subjected to different periods of mixed hay/concentrate feeding ranging from 0 weeks (control; hay ad libitum) to 12 weeks (800 g hay plus 800 g concentrate per day in two equal portions). For each animal, the net absorption of sodium was measured following the mixed hay/concentrate feeding period. Net Na transport, Jnet, significantly rose from 2.15 +/- 0.43 (control) to 3.73 +/- 1.02 microeq x cm(-2) x h(-1) after one week of mixed hay/ concentrate diet, reached peak levels of 4.55 +/- 0.50 microEq x cm(-2) x h(-1) after four weeks and levelled out at 3.92 +/- 0.36 microeq x cm(-2) x h(-1) after 12 weeks of mixed feeding. Thus, 73% of functional adaptation occurred during the first week after diet change. This is in apparent contrast to findings that morphological adaptation takes approximately six weeks to reach peak levels. Hence, early functional adaptation to a mixed hay/concentrate diet is characterised by enhanced Na absorption rates per epithelial cell. Absorption rates are likely to be further enhanced by proliferative effects on the rumen epithelium (number and size of papillae) when concentrate diets are fed over longer periods of time. Early functional adaptation without surface area enlargement of the rumen epithelium appears to be the first step in coping with altered fermentation rates following diet change.


Assuntos
Adaptação Fisiológica , Transporte Biológico/fisiologia , Rúmen/fisiologia , Ovinos/fisiologia , Sódio/metabolismo , Absorção/fisiologia , Ração Animal , Fenômenos Fisiológicos da Nutrição Animal , Animais , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Distribuição Aleatória , Rúmen/metabolismo , Ovinos/metabolismo , Fatores de Tempo
7.
Am J Pathol ; 174(4): 1160-71, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19246644

RESUMO

Alternative splicing of transcripts from many cancer-associated genes is believed to play a major role in carcinogenesis as well as in tumor progression. Alternative splicing of one such gene, the neural cell adhesion molecule CD56 (NCAM), impacts the progression, inadequate therapeutic response, and reduced total survival of patients who suffer from numerous malignant neoplasms. Although previous investigations have determined that CD56 exists in three major isoforms (CD56(120kD), CD56(140kD), and CD56(180kD)) with individual structural and functional properties, neither the expression profiles nor the functional relevance of these isoforms in malignant tumors have been consistently investigated. Using new quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) strategies and novel CD56 isoform-specific antibodies, CD56(140kD) was shown to be exclusively expressed in a number of highly malignant CD56(+) neoplasms and was associated with the progression of CD56(+) precursor lesions of unclear malignant potential. Moreover, only CD56(140kD) induced antiapoptotic/proliferative pathways and specifically phosphorylated calcium-dependent kinases that are relevant for tumorigenesis. We conclude, therefore, that the specific detection of CD56 isoforms will help to elucidate their individual functions in the pathogenesis and progression of malignant neoplasms and may have a positive impact on the development of CD56-based immunotherapeutic strategies.


Assuntos
Antígeno CD56/biossíntese , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/genética , Anticorpos , Especificidade de Anticorpos , Apoptose/fisiologia , Biomarcadores Tumorais/análise , Western Blotting , Antígeno CD56/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
10.
APMIS ; 115(6): 687-700, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17550376

RESUMO

Transforming growth factor beta (TGF-ss) is able to inhibit proliferation of epithelial cells and is involved in the carcinogenesis of human mammary tumours. Three latent transforming growth factor-beta binding proteins (LTBP-1, -3 and -4) are involved in TGF-beta function. The aim of the study was to analyze the expression profiles of TGF-beta 1 and 2 and LTBP-4 in human mammary carcinoma cell lines as well as in human mammary tumours. Expression analysis was performed at the transcription and protein level under in vivo and in vitro conditions. LTBP-4 expression was quantitatively analysed in human carcinomas of the mammary gland and in healthy mammary tissues of the same patients. Downregulation of LTBP-4 in all investigated human mammary tumours compared to normal tissues could be demonstrated. Results also revealed that protein levels of TGF-beta 1 are downregulated and of TGF-beta 2 are upregulated in human mammary carcinoma cell lines compared to primary (normal) human mammary epithelial cells. LTBP-4 reduction in neoplasms leads to a possible decrease of TGF-beta 1 extracellular deposition with reduced TGF-beta 1 bioavailability. TGF-beta 2 was upregulated, which indicates a possible compensatory mechanism. This study demonstrated a possible functional role of LTBP-4 for TGF-beta bioavailability with respect to carcinogenesis of human mammary tumours in vivo and in vitro.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a TGF-beta Latente/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Adenocarcinoma/genética , Neoplasias da Mama/genética , Regulação para Baixo , Humanos , Imuno-Histoquímica , Proteínas de Ligação a TGF-beta Latente/imunologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Neoplásico/química , Fator de Crescimento Transformador beta/imunologia , Células Tumorais Cultivadas
11.
Am J Physiol Gastrointest Liver Physiol ; 291(6): G1171-9, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16825706

RESUMO

In this study, the existence and functional activity of a vacuolar-type H(+)-ATPase (vH(+)-ATPase) was explored in primary cultures of sheep ruminal epithelial cells (REC). The mRNA transcripts of the E and B subunits of vH(+)-ATPase were detectable in RNA from REC samples by RT-PCR. Immunoblotting of REC protein extractions with antibodies directed against the B subunit of yeast vH(+)-ATPase revealed a protein band of the expected size (60 kDa). Using the fluorescent indicator BCECF and selective inhibitors (foliomycin, HOE 694, S3226), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger (NHE) subtype 1 and 3 activity to the regulation of intracellular pH (pH(i)) was determined in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium containing 20 mM of the short-chain fatty acid butyrate as well as after reduction of the extracellular Cl(-) concentration ([Cl(-)](e)) from 136 to 36 mM. The initial pH(i) of REC was 7.4 +/- 0.1 in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium and 7.0 +/- 0.1 after acid loading with butyrate. Selective inhibition of the vH(+)-ATPase with foliomycin decreased pH(i) by 0.19 +/- 0.03 pH units. On the basis of the observed decreases in pH(i) resulting from inhibition of vH(+)-ATPase as well as of subtypes 1 and 3 of NHE, vH(+)-ATPase activity appears to account for approximately 30% of H(+) extrusion, whereas the activities of NHE subtypes 3 and 1 account for 20 and 50% of H(+) extrusion, respectively. Lowering of [Cl(-)](e) induced a pH(i) decrease (-0.51 +/- 0.03 pH units) and impaired pH(i) recovery from butyrate-induced acid load. Moreover, reduction of [Cl(-)](e) abolished the inhibitory effect of foliomycin and markedly reduced the HOE 694- and S3226-sensitive components of pH(i), indicating a role of Cl(-) in the function of these H(+) extrusion mechanisms. We conclude that a vH(+)-ATPase is expressed in ovine REC and plays a considerable role in the pH(i) regulation of these cells.


Assuntos
Células Epiteliais/enzimologia , Mucosa Gástrica/enzimologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Rúmen/enzimologia , Ovinos/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Dados de Sequência Molecular , Distribuição Tecidual
12.
Parasitol Res ; 99(3): 253-61, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16541260

RESUMO

Analyses were made on the adenosine transporter-1 gene in Trypanosoma brucei (TbAT1), encoding a P2-like nucleoside transporter, from T. brucei brucei field stocks to investigate a possible link between the presence of mutations in this gene and isometamidium resistance. We have analysed the gene from 11 isometamidium-sensitive field stocks isolated from cattle in Uganda, two sensitive reference clones and two resistant reference clones. A sequence alignment showed that the isometamidium-sensitive T. b. brucei contained the wild-type sequence patterns. In contrast, the isometamidium-resistant T. b. brucei stocks showed the mutant-type sequence patterns with six point mutations that had previously been reported in a laboratory-derived arsenical-resistant T. brucei strain. To analyse the restriction fragment length polymorphism pattern of a fragment of TbAT1 (nucleotides 430-1108), the 677-bp polymerase chain reaction products from eight of the isometamidium-sensitive and two of the isometamidium-resistant T. b. brucei were subjected to digestion with Sfa NI. The results revealed two different banding patterns: the digest resulted in fragment sizes of 566 and 111 bp in the case of TbAT1 from isometamidium-sensitive stocks, whereas it produced fragment sizes of 435 and 242 bp in the case of TbAT1 from isometamidium-resistant stocks. Thus, the isometamidium-sensitive and isometamidium-resistant T. b. brucei could be successfully distinguished by digestion with the restriction endonuclease Sfa NI.


Assuntos
DNA de Protozoário/genética , Resistência a Medicamentos/genética , Fenantridinas/farmacologia , Reação em Cadeia da Polimerase/métodos , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Substituição de Aminoácidos/genética , Animais , Bovinos , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Mutação de Sentido Incorreto , Proteínas de Transporte de Nucleosídeos/genética , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Trypanosoma brucei brucei/classificação , Trypanosoma brucei brucei/isolamento & purificação , Tripanossomíase Bovina/parasitologia
13.
Am J Physiol Gastrointest Liver Physiol ; 290(1): G56-65, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16109844

RESUMO

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018+/-0.009 in a Na+-free medium to 0.73+/-0.3 mM.l cells-1.min-1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e=24 mM; maximal velocity=11 mM.l cells-1.min-1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375+/-105 microM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Magnésio/metabolismo , Rúmen/citologia , Sódio/farmacologia , Animais , Antiporters/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Células Cultivadas , Cobalto/farmacologia , Imipramina/farmacologia , Compostos Organometálicos/farmacologia , Ovinos , Sódio/metabolismo , Fatores de Tempo
14.
J Comp Physiol B ; 175(8): 575-91, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16177895

RESUMO

The effects of luminal hyperosmolarity on Na and Cl transport were studied in rumen epithelium of sheep. An increase of luminal osmotic pressure with mannitol (350 and 450 mosm/l) caused a significant increase of tissue conductance, G (T), which is linearly correlated with flux rates of (51)Cr-EDTA and indicates an increase of passive permeability. Studies with microelectrodes revealed, that an increase of the osmotic pressure caused a significant increase of the conductance of the shunt pathway from 1.23 +/- 0.10 (control) to 1.92 +/- 0.14 mS cm(-2) (450 mosm/l) without a change of fractional resistance. Hyperosmolarity significantly increased J (sm) and reduced J (net) Na. The effect of hyperosmolarity on J (ms) Na is explained by two independent and opposed effects: increase of passive permeability and inhibition of the Na(+)/H(+) exchanger. Hypertonic buffer solution induced a decrease of the intracellular pH (pH(i)) of isolated ruminal cells, which is consistent with an inhibition of Na(+)/H(+) exchange, probably isoform NHE-3, because NHE-3-mRNA was detectable in rumen epithelium. These data are in contrast to previous reports and reveal a disturbed Na transport and an impaired barrier function of the rumen epithelium, which predisposes translocation of rumen endotoxins and penetration of bacteria.


Assuntos
Mucosa Intestinal/fisiologia , Pressão Osmótica , Rúmen/fisiologia , Ovinos/fisiologia , Sódio/metabolismo , Animais , Células Cultivadas , Cloretos/metabolismo , Cromo/metabolismo , AMP Cíclico/metabolismo , Ácido Edético/metabolismo , Eletrofisiologia , Feminino , Concentração de Íons de Hidrogênio , Mucosa Intestinal/efeitos dos fármacos , Masculino , Manitol/farmacologia , Microeletrodos , Concentração Osmolar , Permeabilidade/efeitos dos fármacos , Rúmen/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/fisiologia , Teofilina/farmacologia
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