Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Detection of isolated tumour cells (TCs) in bone marrow (BM) from epithelial cancer patients by immunocytochemical (ICC) analysis seems to predict future relapse, but the reported percentages of positive BMs among patients with localized cancer show large variations and the number of detected TCs is low. This emphasizes the importance of thoroughly testing the methods in use. This study was performed to clarify to what extent positive staining of haematopoietic cells (HCs) interferes with the ICC detection of epithelial cells in BM. BM mononuclear cells (MNCs) from normal donors and stage I-II breast cancer patients were stained with anti-cytokeratin (CK) and isotype control monoclonal antibodies (MAbs) followed by alkaline phosphatase (AP)-based visualization of immunolabelled cells. In the ICC staining of normal donors by the anti-CK MAbs AE1/AE3 or A45-B/B3, rare immunoreactive cells were detected in 7/20 and 8/19 BMs, respectively. Morphological examination recognized all these cells as typical HCs. In the breast cancer patients (n = 257), anti-CK-positive cells were detected in 26.6 per cent, excluding cells with HC morphology. Using the same morphological criteria, isotype control-positive cells were detected in 5.4 per cent of patients. Some of the false-positive events were further analysed and cells with strong reactivity against the AP enzyme alone were detected. Double ICC staining recognized the majority of these AP directly-reactive cells as CD45-negative and human Ig kappa/lambda-positive, in accordance with the phenotype of mature plasma cells. Morphological evaluation and adequate controls are important to ensure the diagnostic specificity of micrometastases in BM. It is recommended that the number of BM MNCs included in negative controls should equal the number of cells in the diagnostic specimens.
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PMID:Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. 982 43

We describe the expression of 18 different cell adhesion molecules, intermediate filaments and Ki-67 antigen in embryonal childhood tumors. 5 microns frozen sections from 15 nephroblastomas, 13 neuroblastomas, six rhabdomyosarcomas, one Ewing sarcoma and one pulmonary blastoma were analyzed by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method using murine monoclonal antibodies. All tumors exhibited high proliferation rates as did, surprisingly, the nephroblastoma specimens despite pre-treatment with chemotherapy. Polysialylated NCAM was demonstrated on all tumor types, but Ewing sarcoma and expression correlated inversely with cell differentiation. In contrast, E-cadherin was present solely on tubulus like cells in nephroblastomas. This cell type showed a coexpression of cytokeratin and vimentin, giving evidence of its intermediate position between the mesenchyme and epithelium. In neuroblastomas, CD44s (hyaluronate receptor) expression was increased with cell differentiation. ICAM-1, VCAM-1 and E-selectin were mostly expressed in regressive areas of pretreated nephroblastoma specimens where a considerable infiltration of leukocytes was noted as well. Since endothelial and leukocyte adhesion molecules were distinctly less expressed in all other tumors investigated, these findings may indicate immunological processes as a consequence of or as supplement to the chemotherapeutical effect on nephroblastoma cells. Integrin receptors were not found on the surface of tumor cells, and therefore, at least those investigated seem to be of secondary importance to the biology of the tumors studied herein. In conclusion, our investigations demonstrate that, besides achieving a secure and prompt differentiation between various embryonal tumors, applying the panel of monoclonal antibodies proposed herein gives interesting insights into the histogenesis, biology and metastatic potential of pediatric malignancies.
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PMID:Cell adhesion molecules and intermediate filaments on embryonal childhood tumors. 984 36

Single micrometastatic tumor cells encased in mesenchymal tissues, such as bone marrow (BM), are regarded as suitable targets for adjuvant immunotherapy since they are easily accessible for both immunoglobulins and immune effector cells. However, the antigen profile of such cells, to which antibody therapy might be targeted, cannot be deduced from the antigen pattern of the primary tumor. To evaluate the antigen profile of disseminated cells found in BM aspirates from 20 breast cancer patients, we applied a quantitative immuno-cytochemical double-marker assay and typed for 4 common tumor-associated cell-surface antigens (c-erbB-2, CO17-1A, MUC-1, LewisY). Individual breast cancer cells were identified by F(ab) fragments of the pan-cytokeratin (CK) monoclonal antibody (MAb) A45-B/B3, directly conjugated with alkaline phosphatase, which identified cancer cells as sensitively as the standard APAAP procedure (r = 0.998; p < 0.0001). CK+ cells co-expressed c-erbB-2, CO17-1A, MUC-1 and LewisY in 87%, 78%, 79% and 79% of patients, respectively; however, the frequency of double-positive cells per sample varied considerably. The mean percentage of double-positive cells per total number of CK+ cells was 41% for c-erbB-2 (range 0-92%), 47% for CO17-1A (range 0-75%), 49% for MUC-1 (range 0-67%) and 32% for LewisY (range 0-59%). In 14 of these patients, we used an antibody cocktail to type CK+ cells for the combined expression of all 4 antigens. The antibody cocktail labeled significantly more CK+ cells than each of the single MAbs alone, resulting in a mean of 71% double-positive tumor cells (34-100%). We conclude that expression of tumor-associated cell-surface antigens on micrometastatic cancer cells in BM is heterogeneous, which may limit the efficacy of monovalent immunotherapeutic strategies directed against only one particular antigen. Thus, defining target antigens expressed by the actual target cells emerges as a crucial first step in selecting appropriate therapeutic targets.
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PMID:Tumor-antigen heterogeneity of disseminated breast cancer cells: implications for immunotherapy of minimal residual disease. 998 23

Histopathological identification of invasive breast carcinoma in its earliest phases is fraught with pitfalls. Preinvasive malignant lesions complicated by radial scar, sclerosing adenosis, and lobular cancerization, among other lesions, may simulate invasive carcinoma. Fibrosis, inflammatory reaction, and other stromal changes around in situ carcinoma may mask microinvasive foci on routine stains. Conventional immunohistochemistry to demonstrate basement membrane or myoepithelial cell layer may not, by itself, be unequivocally diagnostic of invasion. We performed a novel double immunoenzyme labeling technique using an avidin-biotin complex peroxidase-diaminobenzidine system for smooth-muscle actin followed by an alkaline phosphatase anti-alkaline phosphatase-new fuchsin system for cytokeratin antigen on formalin-fixed, paraffin-embedded histology sections to evaluate 32 such problematic cases. The initial histologic impression with hematoxylin and eosin staining alone was as follows-first group: microinvasive carcinoma-10; second group: carcinoma in situ--"stromal invasion cannot be ruled out"--15; third group: frankly infiltrating carcinoma of various grades and morphologic types-6. The last group served as positive control for invasion. One fibroadenoma with fine-needle-aspiration-induced artifact simulating stromal invasion was also included. The double immunoenzyme labeling technique imparted a dark brown color to the myoepithelial cells and a vivid red color to the epithelial cells, making individual or loosely cohesive groups of malignant epithelial cells infiltrating the stroma easily detectable, whereas their in situ counterparts were contained within dark brown myoepithelial boundaries. The TNM 1997 definition of pT1mic, i.e., extension of malignant cells in the stroma with no focus measuring >0.1 cm, was followed to classify microinvasion. In the first group, microinvasion was confirmed in six cases but was not demonstrable in four. In the second group, definite invasion was identified in five cases, ruled out in nine, and in one case the suspicion of early invasion could not be entirely ruled out even after double immunoenzyme labeling. Thus, it was possible to render a definite opinion regarding presence or absence of invasion in 24 of 25 (96%) cases diagnosed as or suspected to be microinvasive. The precise and simultaneous elucidation of topography between malignant cells and myoepithelial cells on a single permanent section makes this technique a useful diagnostic tool in the evaluation of those cases of breast carcinoma that exhibit equivocal invasion.
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PMID:Double immunolabeling with cytokeratin and smooth-muscle actin in confirming early invasive carcinoma of breast. 998 44

In mice and humans, expression of the tumour necrosis factor receptor-1 (TNF-R1) gene in placental trophoblast cells is constitutive whereas expression of the TNF-R2 gene is developmentally programmed. In order to study the individual functions of TNF-R1 and -R2 in this lineage, cell lines were generated from placental explants of homozygous matings of gestation day 10 outbred mice (Swiss-Webster), TNF-R1-deficient (TNF-R1-/-) and TNF-R2-/- transgenic mice as well as the background strain for the TNF-R2-/- mice (WT, C57BL/6x129). All of the cells exhibited trophoblast markers; they contained cytokeratin intermediate filaments, expressed alkaline phosphatase activity and displayed transferrin receptors, but were negative for vimentin filaments and the macrophage marker, F4/80. Analysis of DNA by polymerase chain reaction demonstrated the expected TNF-R genotype in each line. In experiments testing the effects of recombinant mouse TNF-alpha (rmTNF-alpha) on viability and proliferation of the cell lines, rmTNF-alpha modestly but dose-dependently inhibited the growth of WT and TNF-R2-/- cells while having no effect on TNF-R1-/- cells. Actinomycin D-treated WT and, to a lesser extent, TNF-R2-/- cells, were more sensitive to growth inhibition than untreated cells whereas TNF-R1-/- cell responses remained unchanged. These data indicated that rmTNF-alpha inhibits growth of trophoblastic cells through TNF-R1 and that newly synthesized protein(s) provide partial protection against toxicity. In contrast to the receptor species-specific effects on cell growth exerted by rmTNF-alpha, both TNF-R mediated inhibition of alkaline phosphatase activity. Collectively, the observations support the postulate that receptor expression is the key factor which determines the nature and extent of TNF-alpha effects on trophoblast cell growth and function.
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PMID:Trophoblastic cell lines generated from tumour necrosis factor receptor-deficient mice reveal specific functions for the two tumour necrosis factor receptors. 1019 44

We report four cases of Leydig cell tumor of the testis with a microcystic pattern that mimicked yolk sac tumor. The patients ranged in age from 27 to 35 years and, except for one tumor that was discovered incidentally, presented with testicular masses. All tumors were intratesticular, and three were well circumscribed by a rim of fibrous tissue, whereas one showed minor, focal extension into the adjacent testis. The tumors typically had a vaguely lobular architecture subdivided by fibrous bands. Three of the cases had a complex microcystic appearance caused by individually vacuolated cells and coalescent cystic spaces; this pattern accounted for the majority of two tumors. Another case had focal collections of Leydig cells with prominent cytoplasmic vacuoles but lacked the coalescent spaces. The microcyst contents ranged from optically clear to eosinophilic or lightly basophilic, with the latter having the staining qualities of acid mucopolysaccharide. Three tumors had uniform, bland nuclei and low mitotic rates (<1 mitotic figure per 10 high power fields), but one had marked, random nuclear pleomorphism and an average mitotic rate of five mitotic figures per 10 high power fields. By immunohistochemistry, all were diffusely positive for vimentin; two of three were positive for inhibin, and one showed focal positivity for cytokeratin (CAM 5.2). All were negative for alpha-fetoprotein and placentalike alkaline phosphatase and, apart from having microcystic and solid areas, lacked other features typical of yolk sac tumor. Clinical follow-up ranged from 2 months to 2 years with no patient having recurrence or metastasis. The distinction of Leydig cell tumor from yolk sac tumor has important clinical implications because patients with the former usually receive only clinical follow-up, but the latter often requires chemotherapy.
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PMID:Microcystic Leydig cell tumors mimicking yolk sac tumor: a report of four cases. 1032 86

The presence of disseminated carcinoma cells in bone marrow and peripheral blood has prognostic importance in patients with carcinomas. Much evidence indicates that dissemination of tumor cells may depend on activation of a variety of degradative enzymes. A strong positive correlation has been shown between the expression of tumor cell proteases and tumor invasion. Therefore, phenotypic characterization of disseminated carcinoma cells for expression of protease activators might define the invasive potential of the cells. We present an immunocytochemically enhanced staining method that allows phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows simultaneous and unambiguous immunolabeling of intracellular cytokeratin and of u-PAR intracellularly and on the surface of carcinoma cells. This novel approach can be used for detection and phenotyping of carcinoma cells in blood smears for u-PAR or, presumably, for any other heterogeneously expressed antigen on the surface of the detected cells.
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PMID:Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears. 1037 84

Minimal residual disease in patients with operable pancreatic carcinoma is frequently missed by current noninvasive tumour staging. We applied an immunocytochemical cytokeratin assay that allows identification of individual pancreatic carcinoma cells disseminated to bone marrow. Prior to therapy, bone marrow was aspirated from the upper iliac crest of 48 patients with ductal adenocarcinoma of the pancreas at various disease stages and an age-matched control group of 33 noncarcinoma patients. Tumor cells in cytologic bone marrow preparations were detected with monoclonal antibodies (mAbs) CK2, KL1, and A45-B/B3 to epithelial cytokeratins (CK) using the alkaline phosphatase antialkaline phosphatase method. CK-positive cells were found in 14 (48.4%) of 31 cancer patients treated with curative intent and in 10 (58.8%) of 18 patients with extended disease. The overall frequency of these cells was 1 to 83 per 5x10(5) mononuclear cells with no significant differences between patients at different tumor stages and lymph node involvement. After a median follow-up of 22.8 months (range 3-48 months), 6 (40.0%) of 15 patients who underwent complete surgical resection but had tumor cells in bone marrow presented with distant metastasis and 7 (46.7%) had local relapse compared to none of 12 corresponding patients without such cells (p<0.05). Univariate survival analyses revealed that the presence of CK-positive cells was predictive of reduced overall survival. In conclusion, anticytokeratin mAbs are reliable probes for the immunocytochemical detection of single pancreatic cancer cells disseminated to bone marrow. Thus the described technique may help identify patients with pancreatic cancer and at potentially high risk of early metastatic relapse. The results promise to be of important assistance for determining prognosis and the consequences in therapy of early stage pancreatic cancer.
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PMID:Micrometastases in bone marrow: prognostic indicators for pancreatic cancer. 1044 15

Human renal proximal and distal (thick ascending limb and early distal convoluted tubule) epithelial cells have been isolated according to their specific antigen expression. The cells were well characterized by flow cytometry, enzyme cytochemistry and electron microscopy and cultured for up to 3 months. Cultured tubular cells coexpressed cytokeratin and vimentin as intermediate filament proteins. While primary isolated cells, proximal as well as distal, revealed the phenotypic characteristics of their nephron origin, cultured distal cells showed the tendency to dedifferentiate/transdifferentiate. Distal cells lost their characteristic expression of Tamm-Horsfall glycoprotein and started de novo expression of the proximal marker proteins aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV. The expression of these antigens by distal cells could be shown by flow-cytometric analysis and fluorescence microscopy. Enzyme activity assays revealed the activity of aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV, but not of the proximal marker enzyme alkaline phosphatase. This antigenic shift could not be prevented in different culture media, and the original phenotype could not be restored. Cultured cells displayed characteristic hormonal stimulation patterns indicative of their proximal and distal origins, as shown by activation of adenylate cyclase by different peptide hormones. These results indicate that distal tubular cells possibly transdifferentiate to a more proximal phenotype in view of loss of the distal marker enzyme Tamm-Horsfall protein and de novo expression of proximal marker enzymes like dipeptidyl peptidase IV and aminopeptidase M.
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PMID:Transdifferentiation of distal but not proximal tubular epithelial cells from human kidney in culture. 1045 18

Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue-specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non-specific background staining in pathological liver specimens. We compared peroxidase-anti-peroxidase, alkaline phosphatase-anti-alkaline phosphatase (PAP/APAP), labelled-avidin-biotin (LAB/LAB) and digoxigenin-anti-digoxigenin (dig-a-dig/PAP) techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP-technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig-a-dig/PAP protocol. In contrast to the dig-a-dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue.
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PMID:Comparison of different double immunostaining protocols for paraffin embedded liver tissue. 1060 66


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