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1.
Stud Health Technol Inform ; 90: 627-31, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-15460769

RESUMO

In 2001, the revised course Software Engineering has been implemented in the Medical Informatics curriculum at the Academic Medical Center, Amsterdam. This 13 weeks, full-time course consists of three parts: internship, theory and project. All parts are provided in problem-oriented manner with special attention for relevant skills such as project management, documentation and presentation. During the internship, students observe how health care professionals at several hospital wards work and how information supply is organized. In the theory part, students study concepts and methods of software engineering by means of case descriptions and self-directed learning. During the project, they apply their acquired knowledge to an observed, clinical information problem and complete several stages of the software engineering process. Evaluation by inquiry showed that, compared to other courses, students spent more time, and distributed their time more evenly, during the whole period of the course. In conjunction with theory, a combination of internship and project in a hospital seems to provide a surplus value compared to a practical in a computer laboratory. The integration of software theory, clinical practice and problem-based approach, contributed to the enthusiastic, intensive and realistic way students learned in this important topic that might be chosen as a future profession.


Assuntos
Hospitais de Ensino/organização & administração , Aprendizagem , Informática Médica/educação , Software , Currículo , Humanos , Internato e Residência , Países Baixos , Avaliação de Programas e Projetos de Saúde
2.
Gastroenterology ; 117(5): 1089-97, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10535871

RESUMO

BACKGROUND & AIMS: Whether the bacterial flora contributes to the pathogenesis of inflammatory bowel disease (IBD) by increased penetration in mucus, increased adherence to epithelial cells, or invasion of the epithelium is unknown. We therefore studied the spatial distribution of bacteria in the mucosa of rectal biopsy specimens from patients with IBD and from controls. METHODS: Rectal biopsy specimens from 19 patients with IBD and from 14 controls were studied by using nonradioactive ribosomal RNA in situ hybridization. Total mucosal surface length examined for each patient was measured, and the number of bacteria visualized was estimated semiquantitatively. RESULTS: No bacteria were observed in biopsy specimens from 10 controls (71%) and 6 IBD patients (32%) (P = 0.04; odds ratio, 5.42; 95% confidence interval, 1.23-23.9). IBD rectal specimens contained significantly more bacteria than control samples (P = 0.004). Bacteria were localized within the mucus layer but did not adhere to the epithelial cells and were not present within the lamina propria. There was no correlation between the numbers of bacteria present and either the degree of inflammation or the use of anti-inflammatory agents or sulfasalazine compounds. CONCLUSIONS: The intestinal mucus in IBD patients is less protective against the endogenous microflora than in controls, resulting in increased association of luminal bacteria with the mucus layer.


Assuntos
Bactérias/isolamento & purificação , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/metabolismo , Muco/microbiologia , Biópsia , Colo/microbiologia , Colo/patologia , Contagem de Colônia Microbiana , Humanos , Íleo/microbiologia , Íleo/patologia , Hibridização In Situ , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucinas/metabolismo , Muco/metabolismo , Reto/microbiologia , Reto/patologia , Valores de Referência , Coloração e Rotulagem
3.
Circulation ; 98(19): 2000-3, 1998 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-9808596

RESUMO

BACKGROUND: The distribution of macrophages and smooth muscle cells (SMCs) within atherosclerotic plaques is highly variable. This is clinically relevant because these cell types have opposite effects on the stability of atherosclerotic plaques. The present study was designed to investigate whether local variations in arterial flow over the plaque surface could relate to differences in the distribution of SMCs and macrophages in plaques. METHODS AND RESULTS: Thirty-three entire carotid plaques were collected at autopsy and marked at their proximal (in relation to the direction of the blood flow) ends, and the cell composition of upstream parts (where high flow and high shear prevail) was compared with that of downstream parts (low flow and low shear stress). Seventy percent of plaques showed more SMCs in their downstream part, and 67% of plaques contained more macrophages in the upstream part. Immunostained macrophage areas were larger in upstream parts (P=0. 011). Immunostained SMC areas were larger in downstream parts (P=0. 031). Rupture sites of 6 of 9 ruptured plaques were in the upstream part. CONCLUSIONS: Significant differences in cell composition between upstream and downstream parts of plaques indicate a role for arterial flow in the distribution of different cell types. The low-flow/low-shear downstream areas of plaques contain significantly more SMCs, which could provide the background for slowly progressive growth at distal ends of plaques. The significantly high number of macrophages in the upstream areas suggests a relationship between high flow/high shear and plaque instability.


Assuntos
Arteriosclerose/patologia , Arteriosclerose/fisiopatologia , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/fisiopatologia , Circulação Cerebrovascular/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Cadáver , Humanos , Imuno-Histoquímica , Inflamação/patologia , Macrófagos/patologia , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia
4.
J Clin Pathol ; 51(9): 662-6, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9930069

RESUMO

AIM: To evaluate similarities and differences between gastric stump cancer and conventional carcinoma in the non-operated stomach. METHODS: 26 stump carcinomas were compared with 24 conventional stomach cancers. Stage, histological type, and demographics were comparable in the two groups. Expression of p53 and p21-Waf1/Cip1 was evaluated by immunohistochemical staining. Helicobacter pylori infection was evaluated by examining haematoxylin-eosin stained slides and immunohistochemistry. Epstein-Barr virus infection was evaluated by RNA in situ hybridisation. RESULTS: Expression of p53 and p21-Waf1/Cip1 was similar in both groups and positive in more than half of the patients. H pylori infection was observed in six stump carcinomas and 17 conventional carcinomas in the intact stomach (p < 0.01). RNA in situ hybridisation (EBER1-ISH) for Epstein-Barr virus was positive in nine stump carcinomas and two carcinomas in the non-operated stomach (p < 0.05). CONCLUSIONS: There appear to be aetiological differences between stump carcinoma and cancer in the intact stomach. Further study of these differences may improve our understanding of gastric carcinogenesis in general.


Assuntos
Infecções por Vírus Epstein-Barr/complicações , Infecções por Helicobacter/complicações , Helicobacter pylori , Neoplasias Gástricas/virologia , Proteína Supressora de Tumor p53/metabolismo , Idoso , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Inibidores Enzimáticos/metabolismo , Feminino , Coto Gástrico , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , RNA Viral/análise , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
5.
Gastroenterology ; 112(2): 561-6, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9024310

RESUMO

The origin and neoplastic potential of gastric epithelial polyps remains an area of great interest, and treatment choices are a topic of controversy. This report describes a patient diagnosed with three concurrent hyperplastic gastric polyps that were studied for genetic alterations. The polyps were investigated for alterations in the K-ras oncogene and the p53 tumor suppressor gene and for p21WAF1/Cip1 and MDM2 protein overexpression. In addition, loss of heterozygosity at several loci that are frequently involved in human cancer was analyzed, microsatellite instability, a hallmark of the "mutator" phenotype, was determined, and Epstein-Barr virus infection was investigated. All separate areas from the three independent polyps harbored the same activating point mutation in codon 12 of the K-ras oncogene, indicating a clonal origin. DNA sequence alterations in p53 were not found, although high p53 protein levels could be shown by immunohistochemistry in areas of carcinoma within the largest polyp. No alterations in any of the other molecular markers were observed. The results strongly favor a clonal origin of the three independent gastric polyps and support the notion that these hyperplastic polyps may carry a risk for malignancy.


Assuntos
Proteínas Nucleares , Pólipos/patologia , Gastropatias/patologia , Idoso , Células Clonais , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Feminino , Genes p53 , Genes ras , Humanos , Hiperplasia , Mutação Puntual , Pólipos/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Fatores de Risco , Gastropatias/genética , Neoplasias Gástricas/etiologia , Proteína Supressora de Tumor p53/metabolismo
6.
In Vitro Cell Dev Biol Anim ; 33(1): 42-9, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9028834

RESUMO

Availability of a standard human melanocyte cell line with unlimited growth potential and otherwise normal melanocytic properties will greatly facilitate research in melanocyte biology and in vitro studies on the etiology of pigmentary disorders and melanoma. Using a retroviral vector, E6 and E7 open reading frames of human papilloma virus type 16 (HPV 16) have been introduced into cultured normal human melanocytes. Cells selected by increased resistance to geneticin conveyed by the vector and expressing E6E7 mRNA have been cloned to ensure genetic homogeneity. Since their establishment as primary cells, cloned PIG1 cells have undergone more than twice the amount of population doublings of senescent parental cells. Moreover, in passage numbers when parental cells had become senescent, proliferation of clonal cells was retained at levels exceeding those of normal human melanocytes in third passage by 100%. Further characterization has revealed that the cells remain dependent on tetradecanoyl phorbol 13-acetate (TPA) for growth and do not proliferate in soft agar nor form tumors in nude mice. The antigenic profile of the cells was slightly altered as compared to parental cells, but was incomparable to that of M14 melanoma cells. Importantly, PIG1 cells contain more melanin pigment than parental cells.


Assuntos
Técnicas de Transferência de Genes , Melanócitos/citologia , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Células Clonais , Humanos , Melaninas/metabolismo , Melanócitos/metabolismo , Camundongos , Camundongos Nus , Proteínas E7 de Papillomavirus , Ploidias , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas
7.
J Neurol Sci ; 142(1-2): 39-44, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8902718

RESUMO

In earlier studies of sporadic amyotrophic lateral sclerosis (ALS), a disease of unknown etiology, the amount of metallothioneins (MTs), a group of small (6-7 kDa) metal-binding proteins, appeared higher in liver, kidney and spinal cord from patients than from non-neurologic controls. Immunohistochemically, the expression of MT in the central nervous system appeared limited to glia. Since the highly conserved MTs isotypes share antigenic epitopes, they could not be distinguished by immunological methods. It thus proved necessary to estimate the expression of each individual MT messenger ribonucleic acid (mRNA) by performing reverse transcriptase polymerase chain reaction (RT-PCR)-mediated analysis of tissue samples. Tissues selected included liver, motor cortex and cervical cord at C6; MT mRNAs analyzed included MT1A, 1B, 1E, 1F, 1G, 2A, and 3. Also, special care was taken to avoid interference by amplification of the 6 MT pseudogenes. Except of MT3, already known as brain-specific, and MT1B which was not expressed in any tissue, mRNA levels of the other MT genes tended to be higher in ALS than in control liver samples, but the differences did not attain statistical significance. In the nervous system, the diverse MT genes were expressed over a greater range in ALS than in controls, but exhibited no change in a consistent direction. At the motor cortex, changes seemed to be less pronounced than at C6. MT3 was expressed in the motor cortex and the cord. The results provide no evidence for either the induction of a specific MT repertoire, or for the inability of glia to express any MT gene in ALS. Because the semi-quantitative RT-PCR technique does not permit detailed comparisons between the subtypes of MT expressed in the various tissues, the question whether a single inductor may be held responsible for the elevation of MT in the ALS liver and nervous system remains open. In conclusion, ALS tissue remains capable of expressing all the major MT genes. MT, present in protoplasmic glia, arises locally and is not secondary to increases of hepatic or renal MT. Because MT3 is also expressed by the normal and ALS spinal cord, it is a central nervous system-specific and not only a brain-specific protein. Thus, the excess of MT in ALS liver seems to be an effect of slower catabolism rather than faster synthesis of protein.


Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Fígado/metabolismo , Metalotioneína/genética , Córtex Motor/metabolismo , Medula Espinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/metabolismo , Primers do DNA , Expressão Gênica/fisiologia , Humanos , Fígado/química , Metalotioneína/biossíntese , Metalotioneína/metabolismo , Pessoa de Meia-Idade , Córtex Motor/química , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Medula Espinal/química
8.
J Neurol Sci ; 140(1-2): 21-9, 1996 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-8866423

RESUMO

The recent discovery of missense mutations in the superoxide dismutase (SOD)-1 gene as a cause of familial amyotrophic lateral sclerosis (ALS) and the ensuing description of transgenic SOD-1 mutant mouse models have focussed scientific interest on free radical scavenging mechanisms in all other familial (FALS) and sporadic (SALS) forms of the disease. We have compared the presence of intracellular cytosolic copper-zinc SOD-1 and mitochondrial manganese SOD-2 in the CNS from FALS and SALS patients and from non-neurological controls by immunohistochemical assessment, in the knowledge that no SOD-1 mutations have been found in any of 18 Dutch ALS pedigrees. ALS specimens from the motor cortex and the spinal cord presented enhanced SOD-2 immunoreactivity, especially of astrocytes and occasionally of neurons. Astrocyte staining appeared to be increased at the cerebral cortical and the spinal cervical and lumbar levels, but was only slightly increased in the thoracic anterior horns and not at all in the brain stem. This indicates that, by the time of death, the disease had burnt out in the brain stem and thoracic cord. Increased staining of neurons was limited to the small lateral and dorsal nuclei of the spinal cord. FALS and SALS cases exhibited the same staining patterns. SOD-1 immunoreactivity did not differ between disease and control specimens. SOD-1 and -2 staining was normal in the ALS cortical, brain stem and spinal motoneurons. This suggests that SALS and non-SOD-1 mutant FALS are not accompanied by loss of SOD-1 or -2 protein. An enzyme-linked immunosorbent assay revealed no differences in SOD-1 and SOD-2 levels between ALS patients and controls. Our major finding of locally increased SOD-2 immunoreactivity of astrocytes in FALS and SALS specimens, probably reflects reactive fibrillary and protoplasmatic gliosis in areas of ongoing degeneration but may also result from an attempt at compensation for free radical injury.


Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Astrócitos/enzimologia , Superóxido Dismutase/análise , Adulto , Idoso , Esclerose Lateral Amiotrófica/patologia , Astrócitos/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/enzimologia , Neurônios/patologia
9.
Br J Cancer ; 74(2): 165-71, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8688317

RESUMO

Inactivation of the p53 tumour-suppressor gene is common in a wide variety of human neoplasms. In the majority of cases, single point mutations in the protein-encoding sequence of p53 lead to positive immunohistochemistry (IHC) for the p53 protein, and are accompanied by loss of the wild-type allele. Recently, the WAF1/Cip1 gene was identified as one of the genes induced by wild-type p53, and increased expression of p21WAF1/Cip1 has been found to reflect the status of the p53 tumour-suppressor pathway. We investigated the inactivation of p53 in a relatively small, but well-characterised, group of 46 colorectal carcinomas that were previously studied for allelic alterations, ras oncogene mutations and DNA aneuploidy. Alterations in p53 were identified by IHC, loss of 17p and DNA sequence analysis of exons 5-8, whereas p21WAF1/Cip1 protein expression was determined by IHC. p53 mutations were identified in 19 of the 46 tumours (41%), whereas positive IHC for p53 was found in 21 of the 46 tumours (46%). Positive IHC for p21WAF1/Cip1 was detected in 16 of 42 cases (38%). We found no relationship between p21WAF1/Cip1 staining and p53 protein expression or p53 mutational status. Inactivating mutations in the p53 gene correlated with LOH at 17p but not with LOH at 5q or 18q, Dukes' stage, tumour grade or DNA ploidy. There was a higher survival rate independent of Dukes' stage in the group with no alterations in p53 compared with those with evidence of dysfunction of p53, but the difference was not statistically significant. We conclude that inactivation of p53 and altered expression of p21WAF1/Cip1 are common in colorectal carcinoma but do not correlate with each other or with the clinical or pathological parameters investigated.


Assuntos
Adenocarcinoma/química , Adenocarcinoma/genética , Neoplasias Colorretais/química , Neoplasias Colorretais/genética , Ciclinas/análise , Genes p53 , Mutação , Adenocarcinoma/patologia , Idoso , Alelos , Biomarcadores Tumorais/análise , Cromossomos Humanos Par 17 , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Genes ras , Heterozigoto , Humanos , Imuno-Histoquímica , Mucosa Intestinal/química , Mucosa Intestinal/metabolismo , Masculino
10.
J Pathol ; 178(3): 264-7, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8778330

RESUMO

Immunohistochemistry (IHC) for intranuclear p53 gene product is a simple, routine alternative to molecular genetic analysis of the p53 gene. Several methods for antigen enhancement are currently in use for IHC. This study evaluates the effect of extreme antigen enhancement for p53, using a monoclonal antibody (DO7) and a polyclonal antibody (CM1). The cases studied were five colorectal carcinomas, two specimens of normal colorectal mucosa, and four colorectal carcinomas with genetic alterations which are expected to preclude p53 gene product expression, namely mutation to a STOP codon in the p53 gene detected by denaturing gradient gel electrophoresis with subsequent sequencing and allelic loss of 17p in the region where p53 is located, detected by restriction fragment length polymorphism analysis. The findings suggest that extreme antigen enhancement may cause false-positive results with a distinct nuclear staining pattern when MAb DO7 is used as a primary antibody. It is concluded that all antigen enhancement methods should be thoroughly tested to evaluate their validity and that there may be a limit to the extent to which antigen enhancement can be applied in IHC for p53 protein.


Assuntos
Anticorpos Monoclonais , Antígenos , Neoplasias Colorretais/genética , Imuno-Histoquímica/métodos , Proteína Supressora de Tumor p53/imunologia , DNA/análise , Eletroforese , Reações Falso-Positivas , Humanos , Mucosa Intestinal/química , Micro-Ondas , Mutação , Proteína Supressora de Tumor p53/genética
11.
J Virol Methods ; 53(1): 37-45, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7635926

RESUMO

A herpes simplex virus (HSV) type 2 specific recombinant plasmid probe designated pH2S3 was constructed from non-HSV-1 crossreactive regions of the HSV-2 genome. DNA in situ hybridization on in vitro reconstructed tissue samples of sheep collagen matrix impregnated with herpes virus-infected human cells was used to demonstrate absence of crossreactivity of the pH2S3 probe with HSV-1 and varicella zoster virus under stringent posthybridization washing conditions. It was demonstrated that DNA in situ hybridization with pH2S3 allows specific detection of HSV-2 in routine formalin-fixed, paraffin-embedded patient material.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/isolamento & purificação , Hibridização In Situ/métodos , Pele/virologia , Animais , Biópsia , Bovinos , Células Cultivadas , Sondas de DNA , DNA Viral/análise , Herpes Simples/diagnóstico , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/isolamento & purificação , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/isolamento & purificação , Humanos , Polietilenoglicóis , Pele/patologia
12.
J Clin Pathol ; 48(3): 218-22, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7730480

RESUMO

AIMS: To determine the potential efficiency of molecular markers specific for neoplastic change--mutations of the K-ras oncogene and the p53 tumour suppressor gene--in diagnosing pancreatic carcinoma. METHODS: Archival cytology samples obtained from 17 patients with established pancreatic carcinoma were assayed for alterations in K-ras and p53. To detect changes in p53 expression, immunocytochemistry with polyclonal antibody CM1 was performed on the archival cytology slides after destaining. Mutations in K-ras codon 12 were then analysed on the scrapings of the same slides using mutant enriched polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism analysis with allele specific oligonucleotide hybridisation for confirmation and characterisation. RESULTS: False negative results were recorded for five of the cytology slides when compared with p53 immunostaining of the surgical resection specimen. These five cases had been stained previously with Giemsa which interacts adversely with the immunostaining in contrast to the Papanicolaou procedure. The K-ras codon 12 mutations followed the well established distribution frequency and spectrum for pancreatic cancer and corresponded with the findings in the resection specimens in all cases. Two scrapings yielded insufficient DNA for PCR. Importantly, for two cases with an inconclusive cytology diagnosis on routine light microscopy, the diagnosis was confirmed by one of the molecular markers. The application of the molecular markers increased the diagnostic accuracy of cytology in this small study from 76 to 89%. CONCLUSIONS: The study indicates that assessment of alterations in the K-ras and p53 genes may be a valuable adjunct to diagnostic cytopathology of the head region of the pancreas, although there are some difficulties which will have to be overcome.


Assuntos
Adenocarcinoma/diagnóstico , Genes ras/genética , Mutação , Neoplasias Pancreáticas/diagnóstico , Proteína Supressora de Tumor p53/análise , Adenocarcinoma/química , Adenocarcinoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Estudos Retrospectivos
13.
Gut ; 36(1): 76-80, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7534253

RESUMO

Colorectal tumorigenesis evolves through a series of molecular genetic changes, providing putative markers for tumour progression. This study investigated the relation between expression of the tumour suppressor gene p53 and splice variants v5 and v6 of the cell adhesion molecule CD44 by immunohistochemistry on tissue samples of early adenomas (n = 12), late adneomas (n = 12), Dukes's A and B carcinomas (n = 21), and Dukes's C and D carcinomas (n = 22) and compared these results with expression of these proteins in normal colonic mucosa (n = 17). A statistically significant trend of increasing expression was seen for both p53 (p < 0.005) and CD44 variant exon v6 (p < 0.0005) in subsequent stages of this tumour progression model. High expression of CD44 v5 was seen in most colorectal neoplasms (83%-96%), independent of stage. A statistically significant correlation was present between p53 expression and expression of variant v6 of CD44 (p < 0.01). Both p53 expression and CD44 v6 expression in adenomas increased with the degree of dysplasia (p < 0.05). The results of this study show that mutant p53 protein and variant v6 of the CD44 glycoprotein are markers of tumour progression in colorectal cancer.


Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Transporte/biossíntese , Neoplasias Colorretais/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Retorno de Linfócitos/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Adenoma/genética , Adenoma/metabolismo , Proteínas de Transporte/genética , Neoplasias Colorretais/genética , Progressão da Doença , Éxons , Expressão Gênica , Genes p53 , Humanos , Receptores de Hialuronatos , Técnicas Imunoenzimáticas , Mutação , Receptores de Superfície Celular/genética , Receptores de Retorno de Linfócitos/genética
14.
Am J Clin Pathol ; 101(6): 684-8, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8209852

RESUMO

Mutations in the p53 tumor suppressor gene are frequently identified in human neoplasms. These mutations may be associated with stabilization and, therefore, with overexpression of the p53 protein product as determined by immunohistochemical staining. Using a new antigen retrieval method and a polyclonal antibody to p53 (CM-1), the authors examined 48 formalin-fixed paraffin-embedded adenocarcinomas of the pancreas for overexpression of the p53 gene product. These 48 carcinomas were obtained from a series of patients with well-documented clinical histories and extensive follow-up. The carcinomas had been analyzed previously for K-ras gene mutations, tumor ploidy, and tumor proliferating index. Specific diffuse nuclear staining for the p53 protein was identified in 19 of the 48 (40%) infiltrating carcinomas examined. Focal or negative staining was seen in the remaining 29 cases (60%). In addition, 17 of the neoplasms contained synchronous in situ carcinomas; two (12%) of these displayed diffuse nuclear staining for the p53 protein. Overexpression of p53 was associated with aneuploidy (P = .05), which had been a poor prognosticator in this series of adenocarcinomas of the pancreas. Although overexpression of p53 appeared to be associated with poor prognosis (hazard ratio, 1.8; P = .07), this was not statistically significant. Overexpression of p53 was not significantly associated with K-ras oncogene mutations or tumor proliferating index. The authors conclude that overexpression of the p53 protein occurs frequently in invasive adenocarcinomas of the pancreas and in some in situ carcinomas, as well.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Núcleo Celular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Análise de Sobrevida
15.
Am J Pathol ; 144(5): 889-95, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-8178941

RESUMO

Mutations in the K-ras oncogene and in the p53 tumor suppressor gene are commonly identified in sporadic cases of pancreatic adenocarcinoma. Although these genes might serve as useful markers for early diagnosis of pancreatic carcinoma in patients at risk for the development of this disease, familial pancreatic carcinomas have not been studied for these mutations. We recently had the opportunity to examine a pancreas prophylactically removed from a patient with a strong family history of pancreatic carcinoma. This gave us the unique opportunity to study the early events in the development of familial adenocarcinoma of the pancreas. Histopathological examination of the pancreas revealed multifocal papillary and nonpapillary mucinous duct hyperplasia. Seven of these foci were microdissected and analyzed for K-ras and p53 mutations. The K-ras mutations were detected by combined mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. Five of the seven duct lesions harbored activating point mutations in codon 12 of K-ras; a G to A transition was found in four and a G to C transversion in one. In contrast, these lesions did not harbor detectable p53 mutations as determined by denaturing gradient gel electrophoresis of exons 5 to 8, nor was there overexpression of the p53 protein as determined by immunohistochemistry. These findings suggest that mutations in K-ras represent an early event in the pathogenesis of pancreatic carcinoma. In addition, monitoring of patients with a strong family history of pancreatic carcinoma for K-ras mutations may identify patients at risk for the development of invasive carcinoma.


Assuntos
Genes ras/genética , Mutação/genética , Ductos Pancreáticos/patologia , Sequência de Bases , Carcinoma/genética , Análise Mutacional de DNA , Feminino , Humanos , Hiperplasia/genética , Hiperplasia/patologia , Hiperplasia/cirurgia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ductos Pancreáticos/cirurgia , Neoplasias Pancreáticas/genética
16.
Pigment Cell Res ; 7(1): 33-43, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8072944

RESUMO

An ex vivo model system was developed to investigate melanocyte migration. Within this model system, melanocytes migrate among other epidermal cells in the epibolic outgrowth of skin explants. This process is initiated by loss of contact inhibition of epidermal cells at the rim of the explants and by locally produced chemotactic factors. Punch biopsies provided explants of reproducible diameter. Optimal culture conditions include medium consisting of Dulbecco's Minimal Essential Medium containing 10% inactivated normal human serum and placement of explants epidermal side up at the air-liquid interphase. Within 7 days, epidermal cells completely surround the explant. Approximately 3 days after the onset of keratinocyte migration, melanocytes distribute themselves within the newly formed epidermis. Throughout the 7-day culture period, melanocytes and keratinocytes show maintenance of subcellular morphology, and the dermo-epidermal junction remains intact. Melanocyte migration was quantified using immunoperoxidase staining in combination with light microscopy and computer-aided image analysis. Preliminary results using the model system to compare migration in control and nonlesional vitiligo skin indicate that no inherent migration defect is responsible for impaired repigmentation of vitiligo lesions. The organotypic culture model system allows for investigations on melanocytes within their environment of autologous epidermal and dermal components, closely resembling in vivo circumstances in human skin.


Assuntos
Melanócitos/citologia , Técnicas de Cultura de Órgãos , Pele/citologia , Adulto , Biópsia , Divisão Celular , Movimento Celular/efeitos dos fármacos , Inibição de Contato , Células Epidérmicas , Humanos , Recém-Nascido , Queratinócitos/citologia , Leucotrieno C4/farmacologia , Melanócitos/efeitos dos fármacos , Microscopia Eletrônica , Vitiligo/patologia
17.
Biotechniques ; 16(1): 148-53, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7639838

RESUMO

A newly developed reagent was tested for its applicability in in situ hybridization and in reversed hybridization of DNA fragments generated by PCR amplification. This Platinum-complex, designated universal linkage system (ULS), equipped, for instance, with biotin or fluorescein as hapten, enables versatile nonenzymatic "one step" labeling of genomic, cloned or amplified DNA. Here we demonstrate direct in situ detection of integrated human papilloma virus (HPV) DNA in cervical carcinoma cells using DNA probes labeled with fluorescein-ULS. In cervical smears the presence of HPV or Chlamydia trachomatis was assessed by PCR. To analyze the amplified DNA, a reversed hybridization assay was developed. Immobilized probes were incubated with amplimers that were labeled post-amplification through the action of the biotinylated (BIO)-ULS complex. This novel type of nonradioactive analysis appeared to be as sensitive as its isotopic or colorimetric equivalents. This labeling procedure is simple, versatile and can be included as a universal hapten linkage system in any PCR test or in situ hybridization assay aiming at the detection and identification of DNA or RNA molecules.


Assuntos
Biotina/análogos & derivados , Chlamydia trachomatis/genética , DNA Viral/análise , Compostos Organoplatínicos/metabolismo , Papillomaviridae/genética , Platina/metabolismo , Biotina/metabolismo , Feminino , Haptenos , Humanos , Hibridização In Situ , Reação em Cadeia da Polimerase , Esfregaço Vaginal
18.
Cancer Res ; 53(20): 4754-6, 1993 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-7691404

RESUMO

Specific CD44 variant glycoproteins are overexpressed at particular stages of colorectal tumor progression. Some variants of the CD44 glycoprotein without exon v6 sequences appear at the earliest stage of tumorigenesis, i.e., in early adenomas. Expression of variants containing exon v6 sequences is largely restricted to the advanced stages of tumor development and in addition is more prevalent and intense in metastatic (Dukes C/D) than in nonmetastatic (Dukes A/B) carcinomas. The observation that CD44 variants containing a protein domain of CD44 that confers full metastatic potential to rat carcinoma and sarcoma cell lines is increasingly expressed during colorectal tumor progression indicates that this domain may have an important role in tumor progression and metastasis in humans. Information on v6 expression, which can be obtained by routine immunohistochemistry, may prove of important prognostic value, particularly in carcinomas (Dukes A and B) that have not yet given rise to detectable metastases.


Assuntos
Antígenos CD/biossíntese , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Expressão Gênica , Variação Genética , Receptores de Retorno de Linfócitos/biossíntese , Adenoma/metabolismo , Adenoma/patologia , Antígenos CD/análise , Biomarcadores Tumorais/análise , Southern Blotting , Colo/citologia , Colo/metabolismo , Neoplasias Colorretais/imunologia , DNA/análise , DNA de Neoplasias/análise , Éxons , Humanos , Receptores de Hialuronatos , Imuno-Histoquímica , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Metástase Neoplásica , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Receptores de Retorno de Linfócitos/análise
19.
Am J Pathol ; 142(2): 381-5, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8434637

RESUMO

With the aid of a newly developed target unmasking fluid (TUF), p53 overexpression was visualized by immunohistochemistry on recent and archival paraffin-embedded tissue samples of colon, stomach, and pancreas neoplasms. Using monoclonal anti-p53 antibody pAb1801 as well as polyclonal antiserum to p53 CM1, TUF-mediated immunohistochemistry was fully concordant for p53 overexpression in paraffin-embedded carcinoma samples compared with freshly frozen tissue from the same tumors. Thus, prognostic and diagnostic assessment of p53 overexpression in malignant tissue, routinely fixed in formalin and embedded in paraffin wax, by TUF-mediated immunohistochemistry with monoclonal and polyclonal antibodies may be adopted as a new tool in diagnostic and research histopathology.


Assuntos
Anticorpos , Expressão Gênica , Genes p53 , Imuno-Histoquímica/métodos , Neoplasias/genética , Congelamento , Humanos , Inclusão em Parafina
20.
Exp Parasitol ; 74(1): 1-10, 1992 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1730264

RESUMO

We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events.


Assuntos
Plasmodium berghei/genética , Animais , Gametogênese/genética , Variação Genética , Cariotipagem , Malária/parasitologia , Camundongos , Mitose/genética , Mutação , Plasmodium berghei/citologia , Plasmodium berghei/crescimento & desenvolvimento , Reprodução Assexuada/genética
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