RESUMEN
Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.
Asunto(s)
Nervio Accesorio/anatomía & histología , Caballos/anatomía & histología , Nervios Periféricos/anatomía & histología , Nervio Peroneo/anatomía & histología , Nervio Accesorio/química , Animales , Enfermedades de los Caballos/patología , Fibras Nerviosas Mielínicas/fisiología , Fibras Nerviosas Mielínicas/ultraestructura , Enfermedades Neuromusculares/patología , Enfermedades Neuromusculares/veterinaria , Nervios Periféricos/química , Nervio Peroneo/químicaRESUMEN
OBJECTIVES: To examine COX2 expression and its relation to angiogenesis, Ki67 and Bcl2 expression in Barrett's cancer. METHODS: Specimens from 48 R0-resected Barrett's adenocarcinoma were immunostained for cyclooxygenase 2 (COX2), CD 31 and alpha-sm actin to discriminate between mature and immature vessels, Mib-1 and Bcl2. COX2 staining, angiogenesis, Ki67 expression and Bcl2 expression were also measured. RESULTS: COX2 expression was increased in 25 of 48 cases. There was no significant correlation between COX2 expression and age, sex and tumor differentiation. A significant association was found between lymph node positive cases and elevated COX2 expression (p=0.008). The percentage of Ki67 positive cancer cells was 43.8% (range 15.4-67.5%) in the low COX2 group and 57.8% (range 12.0-84.6%) in the high COX2 group. The difference was statistically significant (p=0.046). The median neovascularisation coefficient in the low COX2 group was 11.68 (range 8.22-43.64) and 25.47 (range 8-38.3) in the high COX2 group. The difference was statistically significant (p=0.012). A significant difference in survival was observed between patients in the COX2 low category when compared with the COX2 high category (log-rank test p=0.013). CONCLUSIONS: Elevated COX2 expression is associated with lymph-node metastases and reduced survival in Barrett's cancer. This appears to be related to the induction of angiogenesis and proliferation.
Asunto(s)
Esófago de Barrett/genética , Esófago de Barrett/fisiopatología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/fisiopatología , Perfilación de la Expresión Génica , Neovascularización Patológica , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proliferación Celular , Ciclooxigenasa 2 , Humanos , Inmunohistoquímica , Metástasis Linfática , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/genética , Análisis de SupervivenciaRESUMEN
AIMS: We investigated VEGF expression and neovascularisation in the metaplasia-dysplasia-carcinoma sequence of Barrett's esophagus and 47 shades of adenocarcinoma. METHOD: Slides of 27 cases of Barrett's metaplasia and high grade dysplasia were immunostained for VEGF, CD 31 and alpha-sm actin to discriminate between mature and immature vessels. VEGF stained slides were quantitatively evaluated measuring optical density with a computer based program. The neovascularisation coefficient was estimated with an interactive analytic computer program. RESULTS: The median VEGF expression increased from metaplasia to advanced carcinoma. VEGF expression and the neovascularisation coefficient reached statistical significance between Barrett's metaplasia and high grade dysplasia (p<0.001), but were not statistically different between high grade dysplasia and microinvasive carcinoma (p=0.421; p=0.146). Comparing microinvasive to advanced carcinoma the difference was significant for both parameters (p<0.001). CONCLUSIONS: Based on a quantitative computer based evaluation program, the present study suggests, that an angiogenic switch might exist and that it is an early event in the metaplasia-dysplasia-carcinoma sequence of Barrett's carcinoma. The neovascularisation phase in Barrett's carcinoma may precede tumour growth.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Lesiones Precancerosas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Anciano , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neovascularización Patológica , Estadísticas no ParamétricasRESUMEN
Vasoactive intestinal peptide (VIP) is the most important peptidergic transmitter in intestinal relaxation. VIPergic nerves are reduced in aganglionosis (AG). Corresponding findings in intestinal neuronal dysplasia (IND) are sparse. It is unknown whether superficial mucosal VIP receptors are reduced in AG, IND, or hypoganglionosis (HYP) compared to concentrations in morphologically normal innervated colon (MNIC). Cryostat sections from 38 colonic biopsies (23 patients with AG, IND, or HYP, 15 with MNIC) were incubated with radioactive iodinated (125)I-VIP. Receptors were analyzed by autoradiography. Radioactive-marked receptors trigger the precipitation of metallic silver as silver grains within a photographic emulsion. Grains were quantified by image analysis, calculating the percent covered cell surface. Statistical analysis was done by Mann-Whitney and Kruskal-Wallis tests (significance #E5/E5#<0.05). VIP receptors covered 4.31% of the cell surface in MNIC. The values were significantly reduced in AG (2.72%; #E5/E5#=0.012) and IND (2.93%; #E5/E5#=0.008). The two HYP biopsies showed the lowest values (1.83%). Aganglionic colon could be distinguished from healthy proximal segments and IND from habitual constipation. In AG and IND, even the superficial mucosal VIPergic innervation seems to be impaired. The reduction of mucosal VIP receptors in developmental faults of the enteric nervous system may thus be an indicator of a sensomotor disturbance.
Asunto(s)
Colon/inervación , Sistema Nervioso Entérico/patología , Enfermedad de Hirschsprung/patología , Enfermedades Intestinales/patología , Mucosa Intestinal/fisiología , Receptores de Péptido Intestinal Vasoactivo/fisiología , Preescolar , Enfermedad de Hirschsprung/fisiopatología , Humanos , Lactante , Enfermedades Intestinales/fisiopatologíaRESUMEN
PURPOSE AND METHODS: To investigate the potential role of an aberrant cellular DNA repair in target cells during malignant transformation we studied cell type-specific mRNA expression of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (O6-AGT) in normal and regenerating rat liver, chronic hepatitis and preneoplastic liver lesions by in situ hybridization and semiautomatic image analysis. RESULTS: O6-AGT mRNA expression was found to be four to five times higher in hepatocytes than in nonparenchymal cells. A 1.9-fold increase in O6-AGT mRNA was observed after partial hepatectomy. Intraperitoneal injection of diethylnitrosamine led to a 1.3-fold and 2.6-fold rise in periportal and perivenous hepatocytes, respectively. Ethylnitrosourea produced an enhancement of mRNA levels up to 1.6-fold in hepatocytes without regional differences. In megalocytic hepatocytes of Long-Evans Cinnamon rats with chronic hepatitis, a 4.4-fold mRNA induction was found. In small preneoplastic lesions induced after chronic diethylnitrosamine-exposure, O6-AGT mRNA expression was identical to that of adjacent normal tissue. Intermediate and large lesions revealed 1.5- to 1.6-fold higher mRNA levels. CONCLUSIONS: These results suggest an induction of O6-AGT mRNA expression in hepatocellular target tissue under conditions of increased carcinogen sensitivity . The O6-AGT expression in early preneoplastic lesions did not differ from normal surrounding liver tissue, thus excluding the possibility that progression of preneoplasia in rat liver is associated with a deficient mRNA expression of this DNA repair protein. On the contrary, enhanced O6-AGT mRNA expression in more advanced foci and early neoplastic nodules may confer a selective advantage upon early malignant hepatocytes with regard to further tumor progression.
Asunto(s)
Regeneración Hepática , Hígado/enzimología , O(6)-Metilguanina-ADN Metiltransferasa/genética , Lesiones Precancerosas/enzimología , Transcripción Genética , Animales , Dimetilnitrosamina , Inducción Enzimática , Hibridación in Situ , Inflamación/enzimología , Ratas , Ratas Long-Evans , Ratas WistarRESUMEN
Amplification of the Her-2/neu gene is accompanied by overexpression of its cell surface receptor product, c-erbB-2 protein. To investigate the degree of intratumoural heterogeneity we applied immunohistochemistry in primary Barrett's adenocarcinoma (BCA) (n = 6) and dysplasia adjacent to the carcinoma (n = 4). In addition, fluorescence in situ hybridisation (FISH) was performed in primary BCA (n = 5) and dysplastic areas (n = 4). For an objective evaluation digital image analysis and laser scanning microscopy were used. Five of six BCA showed a marked intratumoral heterogeneous staining pattern ranging from areas in which the tumour cells were negative or faintly positive to tumour areas with a strong staining of the entire membrane. Among the two dysplastic areas also a heterogeneous staining pattern was observed. FISH analysis revealed marked heterogeneity of intratumoral gene copy number changes in all BCA showing populations with different fractions of cells with polysomy, low level amplification and high level amplification. One dysplasia showed a minor population with Her-2/neu signal clusters. In conclusion, we observed marked intratumoural heterogeneity of c-erbB-2 protein overexpression and Her-2/neu gene copy number in the majority of the primary BCA analyzed. Digital image analysis and laser scanning microscopy were helpful in quantifying the variations in protein expression and DNA copy number in individual tumour cells. The observed heterogeneity could hamper the exact diagnostic determination of the c-erbB-2 status in small biopsies and possibly influence the effectiveness of a potential c-erbB-2 targeting therapy. Figures on http://www.esacp.org/acp/2000/20-1/walch.htm+ ++.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Receptor ErbB-2/biosíntesis , Adenocarcinoma/patología , Esófago de Barrett/patología , Cromosomas Humanos Par 17/ultraestructura , Dosificación de Gen , Genes erbB-2/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación Fluorescente in Situ , Microscopía Confocal , Microscopía por Video , Receptor ErbB-2/genéticaRESUMEN
Neuroendocrine tumours (NET) of the lung are divided in subtypes with different malignant potential. The first is the benign or low-grade malignant tumours, well-differentiated, called typical carcinoids (TC) and the second is the high-grade malignant tumours, poorly differentiated of small (SCLC) or large cell type (LCLC). Between these tumour types lies the well-differentiated carcinoma with a lower grade of malignancy (WDNEC). In clinical routine it is very important with regard to prognosis to distinguish patients with low malignant potential from those with higher ones. In this study 32 cases of SCLC, 13 of WDNEC and 14 of TC with a follow-up time up to 7 years were collected. Sections 4 microm thick from paraffin embedded tissue were Feulgen stained. By means of high resolution image analysis 100 nuclei per case were randomly gathered to extract morphometric, densitometric and textural quantitative features. To investigate the ploidy status of the tumour the corrected DNA distribution was calculated. Stepwise linear discriminant analysis to differentiate the classes and Cox regression analysis for the survival time analysis were applied. Using chromatin textural and morphometric features in two two-class discriminations, 11 of the 14 TC cases and 8 of the 13 WDNEC cases were correctly classified and 11/13 WDNEC cases and 28/32 SCLC cases, respectively. The WDNEC cases are more similar in chromatin structure to TC than to SCLC. For the survival analysis, only chromatin features were selected to differentiate patients with better and worse prognosis independent of staging and tumour type.
Asunto(s)
Neoplasias Pulmonares/patología , Tumores Neuroendocrinos/patología , Adolescente , Adulto , Anciano , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/patología , Núcleo Celular/genética , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Humanos , Neoplasias Pulmonares/genética , Masculino , Microscopía Confocal , Persona de Mediana Edad , Tumores Neuroendocrinos/clasificación , Tumores Neuroendocrinos/genética , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de SupervivenciaRESUMEN
OBJECTIVE: To detect textural nuclear features correlated with nonprogression and progression in esophageal dysplasia. STUDY DESIGN: Asymptomatic adults from Heshun Commune, Linxian County, China were examined with a balloon sampler in 1983 fifty cases of moderate esophageal dysplasia and 68 cases of mild were selected for study. By means of an Axiomat microscope equipped with a TV camera, 100 visually normal intermediate squamous cell nuclei per specimen were randomly measured from routinely Papanicolaou-stained slides. RESULTS: Of 50 esophageal moderate dysplasia cases, 24 and 7 progressed to carcinoma within three and nine years, respectively. The other 19 cases remained stable or regressed to normal and were used as the control group. By means of chromatin features, correct specimen classification rates of 79.2% (19/24), 73.7% (14/19), 85.5% (6/7) and 84.2% (16/19) were achieved, respectively (P < .001). Of 68 cases classified as mild dysplasia, 16, 13 and 12 progressed to carcinoma within three, five and nine years, respectively. The other 27 cases remained stable or regressed to normal and were used as the control group. The correct specimen classification rates were 93.8% (15/16), 88.9% (24/27), 69.2% (9/13), 74.1% (20/27), 83.3% (10/12) and 77.8% (21/27), respectively, using chromatin features of the nuclei (P < .001). CONCLUSION: In this study, nuclear chromatin features measured by high-resolution image analysis could sufficiently well forecast the outcome of precancerous lesions and discriminate precancerous lesions with progression and nonprogression. It also can be employed as surrogate end point biomarkers in clinical chemoprevention trials. Stoichiometric staining and standard preparations should increase the correct classification rates in further studies.
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Enfermedades del Esófago/patología , Procesamiento de Imagen Asistido por Computador , Lesiones Precancerosas/patología , Adulto , Anciano , Núcleo Celular/ultraestructura , Neoplasias Esofágicas/clasificación , Neoplasias Esofágicas/patología , Humanos , Persona de Mediana Edad , Regresión Neoplásica Espontánea , Lesiones Precancerosas/clasificación , Pronóstico , Factores de TiempoRESUMEN
Canine pancreatic neuroendocrine tumors were studied using different image analysis techniques (nuclear image histometry, analysis of argyrophilic proteins of nucleolar organizer regions, determination of the mouse anti-Ki 67 antigen proliferation index, and DNA densitometry) to correlate their biological behavior with objective phenotypic markers. The methods were compared to determine the best method for distinguishing between metastatic and nonmetastatic tumors. Discrimination between the two types of tumor was possible using nuclear image histometry in combination with morphometric analysis of argyrophilic proteins of nucleolar organizer regions. In contrast, the mouse anti-Ki 67 antigen proliferation index, DNA measurement, and immunohistochemical parameters revealed no significant difference between the two types of tumors.
Asunto(s)
Carcinoma de Células de los Islotes Pancreáticos/secundario , Carcinoma de Células de los Islotes Pancreáticos/veterinaria , Enfermedades de los Perros/patología , Tumores Neuroendocrinos/secundario , Tumores Neuroendocrinos/veterinaria , Neoplasias Pancreáticas/secundario , Neoplasias Pancreáticas/veterinaria , Animales , Carcinoma de Células de los Islotes Pancreáticos/patología , División Celular , Diagnóstico Diferencial , Enfermedades de los Perros/metabolismo , Perros , Femenino , Citometría de Imagen/métodos , Inmunohistoquímica , Masculino , Tumores Neuroendocrinos/patología , Región Organizadora del Nucléolo/patología , Neoplasias Pancreáticas/patología , PlataRESUMEN
Since 1983, a long-term clinical trial of esophageal carcinoma chemoprevention has been conducted in a high-risk area in China. From this study, 25 esophageal severe dysplasia patients without therapy were selected for analysis. After 5-year follow-ups, 14 cases progressed to esophageal carcinoma, while the other 11 cases remained stable. Three Papanicolaou's smears were used for each case, including one from the esophageal cytological examination at the beginning, two from the re-examinations three and five years later respectively. About 100 visually normal intermediate cells were randomly collected per slide by high resolution image analysis. More than 100 features (morphologic, densitometric, textural) were extracted. The classifications were made by means of stepwise linear discriminate analysis at the single cell level as on the specimen level using up to ten features. In all three comparisons of patients with progression and with regression at time of diagnosis, three years after diagnosis and five years later, the correct cell classification rates were about 70%. The subsequent specimen classifications by means of the a posteriori probability (APOP) distribution of the cells in each case led to 80% correct classification. All selected features reflected the chromatin structure of nuclei. The result demonstrated that the chromatin structures of esophageal epithelial cells in severely dysplasic patients are different between cases with and without progression. These results suggest the possibility of the application of image analysis in the clinical trials to find the dysplasia patients with higher risk of progression, in order to reduce the number of patients for therapy.
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Cromatina/patología , Neoplasias Esofágicas/patología , Esófago/patología , Adulto , Anciano , Antineoplásicos/farmacología , Biomarcadores de Tumor , Núcleo Celular/patología , Citodiagnóstico , Medicamentos Herbarios Chinos/farmacología , Neoplasias Esofágicas/etiología , Neoplasias Esofágicas/prevención & control , Humanos , Procesamiento de Imagen Asistido por Computador , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Factores de Tiempo , Tretinoina/análogos & derivados , Tretinoina/farmacologíaRESUMEN
B1 bradykinin receptors were visualized by using the B1 bradykinin receptor agonist [3H]des-Arg10-kallidin in receptor autoradiography experiments. Cryosections were prepared from arterial vessels from a healthy control pig, a pig with pre-existing inflammation and an animal with experimental sepsis induced by an infusion of bacterial lipopolysaccharide (LPS). Only diffusely scattered silver grains with no preference for a distinct tissue structure were detected on emulsion-coated coverslips above the cryosections from the healthy control animal. This indicates that under normal circumstances no or only minute amounts of B1 bradykinin receptors are present in these tissues. In contrast, a 3-fold increase in specific B1 bradykinin receptor binding was observed on both the corresponding preparations of the sick piglet and of that with experimentally induced sepsis. A similar enhancement of specific [3H]des-Arg10-kallidin binding occurred in preparations devoid of endothelium. By comparison with the stained cryosection on the slide the silver grains showed a preferential distribution above smooth muscle cells. Taken together our data are consistent with the hypothesis that B1 bradykinin receptors are induced in the muscle layer of large vessels not only after experimentally-induced sepsis but also in pre-existing inflammatory disease.
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Vasos Sanguíneos/metabolismo , Inflamación/metabolismo , Receptores de Bradiquinina/metabolismo , Animales , Aorta Torácica/metabolismo , Autorradiografía , Calidina/análogos & derivados , Calidina/metabolismo , Lipopolisacáridos/toxicidad , Arteria Pulmonar/metabolismo , Receptor de Bradiquinina B1 , Receptores de Bradiquinina/agonistas , Sepsis/etiología , Sepsis/metabolismo , PorcinosRESUMEN
The spectrum of neuroendocrine lung tumours ranges from highly aggressive small cell carcinomas (SCLC) to carcinoid tumours (CD) of low malignant potential. Between these two extremes, the 'well-differentiated neuroendocrine carcinomas' (WDNEC) form a transitional group with uncertain biological behaviour. This study investigated the prognostic value of the proliferation marker MIB-1 (paraffin Ki-67) in 59 neuroendocrine lung tumours (32 SCLC, 13 WDNEC, 14 CD) by immunostaining of routinely processed paraffin sections. Morphometric evaluation was done by semi-automatic image analysis. The results were compared with survival data (mean follow-up: 42 months). The proliferation rates of the tumours as determined by MIB-1 immunoreactivity (MIB-1-PR) were significantly different between the tumour types (SCLC > WDNEC > CD) and showed a strong inverse correlation with survival time. In CD, the percentage of MIB-1-labelled nuclei never exceeded 1.1 per cent; higher values would therefore favour the diagnosis of WDNEC over that of CD. Among WDNEC, MIB-1 was able to differentiate a subgroup with excellent prognosis (MIB-1-PR: 0.3-3.4 per cent) from another subgroup with a death rate of 50 per cent (MIB-1-PR: 7.3-20.3 per cent). Within each tumour type, all patients without distant metastases at diagnosis survived when MIB-1-PR was < or = 9.4 per cent, suggesting a potential threshold for prognosis. Although the status of metastases are complementary prognostic indicators and are best used in combination to characterize the biological behaviour of neuroendocrine lung tumours.
Asunto(s)
Carcinoma Neuroendocrino/patología , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/análisis , Proteínas Nucleares/análisis , Adulto , Anciano , Anticuerpos Monoclonales , Tumor Carcinoide/patología , Carcinoma Neuroendocrino/química , Carcinoma de Células Pequeñas/patología , División Celular , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Humanos , Antígeno Ki-67 , Neoplasias Pulmonares/química , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de SupervivenciaRESUMEN
BACKGROUND: The self-sustained sequence replication (3SR) reaction is an isothermal method for nucleic acid amplification that has several features that make it an attractive alternative to PCR. We have studied the feasibility of the in situ 3SR reaction in cells using a measles virus-infected cell line as a model. EXPERIMENTAL DESIGN: The study was carried out in four steps. First, using RNA extracted from a measles-infected Vero Green monkey kidney cell line, conditions for the in vitro amplification of a segment of the nucleocapsid portion of the RNA viral genome were optimized for 420- and 119-bp 3SR products, and the results were compared. Second, 3SR was performed on intact infected cells in suspension, and the amount of RNA product was compared with infected cells without 3SR. Then, the 3SR reaction was conducted on cytospin preparation slides, followed by in situ hybridization for detection of the amplification product. Finally, 3SR was carried out on sections of formalin-fixed, paraffin-fixed, paraffin-embedded cells, and the degree of amplification as detected by ISH was quantified and compared between infected cells with and without 3SR reaction. RESULTS: Specific amplification of measles was observed in each of these types of preparations with an 8.5-fold rate of amplification in paraffin sections of formalin-fixed cells (a mean of 272.5 +/- 65.3 grains/cell after 3SR amplification in comparison to 31.97 +/- 4.2 grains/cell without amplification). CONCLUSIONS: A significant amount of amplification of RNA is possible with in situ 3SR (IS-3SR) and, in combination with ISH, offers several advantages compared with in situ PCR (IS-PCR), such as ease of use, lack of conditions that lead to cell damage, and a specificity for RNA amplification. This is the first report of specific amplification of RNA within cells using the IS-3SR procedure, a technique that has a wide range of potential applications in pathology and molecular biology.
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Amplificación de Genes , Hibridación in Situ/métodos , Virus del Sarampión/genética , ARN Viral/genética , Animales , Secuencia de Bases , Northern Blotting , Chlorocebus aethiops , Genes Virales , Datos de Secuencia Molecular , Oligonucleótidos/análisis , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Células VeroRESUMEN
The intercellular adhesion molecule-1 (ICAM-1) is important in mediating intercellular contact in inflammation. Therefore, we have analyzed the expression of this molecule on alveolar macrophages (AM) and in serum of patients with sarcoidosis. Bronchoalveolar lavage (BAL) cells from 13 patients and 11 control donors were stained with an anti ICAM-1 monoclonal antibody (mAb) or an isotype control. Alkaline phosphatase was used as a detection system, followed by digital single cell image analysis. Soluble ICAM-1 in serum (ssICAM-1) was determined by enzyme linked immunoassay (Elisa). Immunocytochemistry revealed a strong increase of ICAM-1 expression on AM from sarcoidosis patients (64%) compared to healthy controls (30%). Furthermore, patients exhibited a fourfold higher antigen density. Serum levels of sICAM-1 were more than twofold increased in the patient group (805.4 micrograms/ml) compared to healthy controls (384.8 micrograms/ml). SsICAM-1 showed an inverse correlation with vital capacity (VC) and diffusing capacity (DCO). This significant correlation with impairment of two important lung function parameters suggests that ssICAM-1 might be useful in serological assessment of disease activity in sarcoidosis.
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Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/sangre , Macrófagos Alveolares/química , Sarcoidosis Pulmonar/metabolismo , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Función Respiratoria , Sarcoidosis Pulmonar/sangre , Sarcoidosis Pulmonar/fisiopatología , Solubilidad , Coloración y EtiquetadoRESUMEN
In a group of 20 patients with extrinsic allergic alveolitis (10 with farmer's lung, 9 with bird-breeder's lung, and 1 with humidifier's lung), we observed increased levels of soluble serum interleukin-2 receptor (ssIL-2R) with an average of 75.2 pmol (control group average, 40.6 pmol). Analysis of bronchoalveolar lavage cells by immunocytochemistry with a CD25 antibody revealed only a slight increase to 4.2% IL-2R+ cells among alveolar lymphocytes but a pronounced rise to 21.2% IL-2R+ cells among alveolar macrophages. Furthermore, cytometry revealed a fourfold higher IL-2R expression on a per cell basis for alveolar macrophages as compared with alveolar lymphocytes. A clear-cut positive correlation (p = 0.006) was found for ssIL-2R and IL-2R+ alveolar macrophages, but only a borderline correlation was found for ssIL-2R and IL-2R+ alveolar lymphocytes (p = 0.04). The finding of a concomitant decrease of IL-2R+ alveolar macrophages and of ssIL-2R on allergen avoidance further supports the notion that alveolar macrophages may be the main source of the increased ssIL-2R in patients with extrinsic allergic alveolitis.
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Alveolitis Alérgica Extrínseca/inmunología , Macrófagos Alveolares/química , Receptores de Interleucina-2/análisis , Adulto , Alérgenos/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores de Interleucina-2/inmunologíaRESUMEN
Subtle cellular changes are known to exist in normal host tissue adjacent to tumours. These are called malignancy associated changes (MAC). To get more insight into the degree of expression and local spread of such changes we performed high resolution image cytometry on visually normal intermediate cell nuclei in smears from patients with laryngeal or pharyngeal squamous cancer. The smears were taken from the tumour surface, from a border region of the tumour and from a distant unsuspicious buccal site. In addition buccal smears from healthy control persons were examined. In a pilot study smears from 12 cancer patients and 11 control persons and in a succeeding validation study 63 controls, 18 non-tumour patients and 25 cancer patients were investigated. In both studies the occurrence of MACs was demonstrated quantitatively. In cancer patients normal appearing intermediate cells from the three different sampling sites could be discriminated with 65% in the pilot study and with 53% correct classification in the validation study. In addition the influences of smoking behaviour and sex were investigated in the control group. Only in the latter case there was a significant difference between female and male with a 63% correct cell and 71% correct specimen classification.
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Carcinoma de Células Escamosas/patología , Neoplasias Laríngeas/patología , Neoplasias Faríngeas/patología , Análisis Discriminante , Epitelio/patología , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Mucosa Bucal/patología , Proyectos Piloto , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
Argyrophilic proteins of nucleolar organizer regions (AgNOR proteins) were stained by a modified silver staining technique. Paraffin sections from 137 cases of invasive ductal carcinoma of the breast were investigated. The nuclei and AgNOR dots were quantitatively measured by means of a semiautomatic image analysis system. Follow-up data for about 120 months (mean, 102 +/- 47) and clinical/histologic and DNA distribution parameters were available for all patients. The prognostic significance of AgNOR parameters was tested by Cox regression analysis. Four AgNOR features showed a significant univariate correlation with survival time. In multivariate analysis offering all the available parameters, one AgNOR parameter (coefficient of variation of relative AgNOR area) ranked at the third position beyond the standard deviation of DNA distribution and pTNM/staging. When considering the distant recurrence-free survival of patients, the same AgNOR feature provided significant additional prognostic information. Performing survival analysis for the prognostically defined subgroup of pTNM/stage I patients and offering all DNA and AgNOR features the same AgNOR parameter yielded the highest prognostic validity. These results show that quantitatively measured AgNORs yield prognostic factors in breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Región Organizadora del Nucléolo/patología , Neoplasias de la Mama/ultraestructura , Carcinoma Ductal de Mama/ultraestructura , Núcleo Celular/patología , Interpretación Estadística de Datos , Femenino , Humanos , Metástasis de la Neoplasia , Pronóstico , Plata , Coloración y Etiquetado , Análisis de SupervivenciaRESUMEN
CD14 expression on alveolar macrophages (AM) was studied in patients with sarcoidosis using immunocytochemistry and cytometric analysis. Compared with healthy control donors, patients had elevated percentages of CD14-positive AM (22 percent vs 34 percent), and the antigen density was threefold higher (92 vs 297 channels). Furthermore, soluble serum CD14 (ssCD14) was significantly elevated in patients with sarcoidosis with an average of 5.3 +/- 1.6 mg/L vs 3.2 +/- 0.7 mg/L in healthy control subjects. Follow-up of one patient, whose lung function test results improved during therapy with corticosteroids, revealed a concomitant decrease of CD14 staining on AM and of ssCD14. Statistical analysis revealed a negative correlation between CD14 expression on AM and PO2 at rest (p = 0.0005), and after labor (p = 0.02). Levels of ssCD14 gave a positive correlation to reduction of Dco (p = 0.006) and VC (p = 0.05). These data suggest that CD14 expression is related to severity of disease and that it may be useful for monitoring in sarcoidosis.
Asunto(s)
Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Enfermedades Pulmonares/fisiopatología , Pulmón/fisiopatología , Macrófagos Alveolares/inmunología , Sarcoidosis/fisiopatología , Adulto , Anciano , Antígenos CD/sangre , Antígenos CD/efectos de los fármacos , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/sangre , Antígenos de Diferenciación Mielomonocítica/efectos de los fármacos , Antígenos de Diferenciación Mielomonocítica/genética , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Femenino , Expresión Génica , Glucocorticoides/uso terapéutico , Humanos , Inmunohistoquímica , Receptores de Lipopolisacáridos , Pulmón/inmunología , Enfermedades Pulmonares/inmunología , Masculino , Persona de Mediana Edad , Oxígeno/sangre , Capacidad de Difusión Pulmonar/efectos de los fármacos , Capacidad de Difusión Pulmonar/fisiología , Sarcoidosis/inmunología , Coloración y Etiquetado , Capacidad Vital/efectos de los fármacos , Capacidad Vital/fisiologíaRESUMEN
The relevance of silver-stained NORs for classifications and prognosis was investigated in breast tissue. Paraffin sections from 137 cases of invasive ductal breast carcinomas and 12 cases with non-tumorous ductus epithelium as controls were stained according to a modified technique and analysed. From the cancer cases follow-up data up to 10 years (45 to 165 months) and in addition clinical, histological and several DNA distribution parameters were available. The nuclei and the silver grains were measured by means of a semiautomatic image analysis system. Significant differences in AgNOR features were found between controls and diploid tumors (p < or = 0.001), diploid and aneuploid tumors (p < or = 0.001), Bloom-Richardson-gradings I, II, and III (p < or = 0.001), and between the tumor cells from patients developing metastases within 5 years and those without (p < or = 0.002). The prognostic significance of AgNORs was estimated using Cox regression analysis. Four AgNOR features were correlated significantly with survival time. In a multivariate approach offering all parameters available an AgNOR parameter (CV of relative area AgNORs) ranked at the third position beyond the SD of DNA distribution and pTNM-staging. Considering the metastases-free interval of patients the same AgNOR feature showed an independent prognostic validity.
Asunto(s)
Neoplasias de la Mama/clasificación , Neoplasias de la Mama/ultraestructura , Carcinoma Ductal de Mama/clasificación , Carcinoma Ductal de Mama/ultraestructura , Región Organizadora del Nucléolo/ultraestructura , Adulto , Anciano , Mama/ultraestructura , Neoplasias de la Mama/mortalidad , Carcinoma Ductal de Mama/mortalidad , ADN de Neoplasias/análisis , Femenino , Humanos , Linfocitos/ultraestructura , Masculino , Análisis Multivariante , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Plata , Coloración y EtiquetadoRESUMEN
Highly sensitive and inexpensive methods that are not time consuming are desirable for monitoring the workplace environment for the detection of cytotoxic hazards, particularly cancerous risks. It is possible to detect precancerous and cancerous lesions in samples taken by brushing the nose, but the cytological diagnoses can be affected by an inability to obtain representative smears from the sometimes very small focal lesions, and uncertainties in the subjective interpretation of suspicious cells when these are small in number. In an attempt to improve diagnosis we applied imaging cytometry (ICM) and tested the concept of malignancy-associated changes (MAC) in routinely Papanicolaou-stained smears. Cells of non-goblet type that visually appeared normal were selected from nickel workers with and without dysplastic lesions of the nasal mucosa. A set of nuclear features was measured by ICM and used for discriminant analysis. We were able to differentiate between workers with non-dysplastic normal and suspicious mucosa smears and those with dysplastic lesions. Unexpectedly, it was found possible to distinguish between workers in the roasting/smelting and the electrolysis departments, who were exposed to different carcinogenic nickel compounds. A further surprising finding was the possibility to distinguish smokers and non-smokers among the nickel workers.