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Biomedical subjects

R L Cohen

Publications and source records attributed to R L Cohen.

At least 37 records · Page 2Linked to original sources
PubMed1996

Circulating thrombopoietin concentrations in thrombocytopenic patients, including cancer patients following chemotherapy, with or without peripheral blood progenitor cell transplantation.

Thrombopoietin, the ligand for the c-mpl receptor, promotes proliferation and maturation of megakaryocytes. An ELISA using a chimaeric receptor, mpl-IgG, for capture, and rabbit antibody to thrombopoietin for detection was developed for the quantitation of thrombopoietin in human serum or plasma. This ELISA preferentially detects full-length thrombopoietin compared to the bioactive N-terminal half of the molecule which has homology to erythropoietin. Thrombopoietin was not detected (< 0.16 ng/ml) in 88/89 healthy individuals. However, elevated thrombopoietin concentrations of up to 3 ng/ml were detected in 59/63 thrombocytopenic patients, including cancer patients following chemotherapy. In cancer patients receiving chemotherapy with (n = 12) or without (n = 6) peripheral blood progenitor cell transplantation, thrombopoietin concentrations varied inversely with platelet counts throughout the treatment period. In general, patients who received myeloablative chemotherapy on days -7 to -2 and peripheral blood progenitor cell transplantation on day 0 had high thrombopoietin levels (0.6-2.9 ng/ml) around day 5. Low platelet counts (< 20 x 10(9)/l) occurred between days 4 and 9. Patients who received high-dose chemotherapy on day 1 (equivalent to day -7 for transplantation patients) to day 6 without transplantation had high thrombopoietin concentrations (1.4-2.3 ng/ml) around day 13 and low platelet counts occurred between days 7 and 17.

Adult↗
PubMed1996

A multi-center clinical trial of a new chairside test in distinguishing between diseased and healthy periodontal sites. II. Association between site type and test outcome before and after therapy.

The aim of the present study was to evaluate the association between the outcome of a chairside test measuring gingival crevicular fluid (GCF) levels of the enzyme aspartate aminotransferase (AST) and other clinical measures of disease including probing depth, severity of inflammation, and GCF flow before and after therapy. We studied 91 patients with moderate to severe periodontitis. Eight sites with probing depths between 5 mm and 8 mm and obvious signs of inflammation were selected and designated diseased sites. Four sites with probing depth < or = 3 mm with no or minimal signs of inflammation were selected and designated non-diseased sites in patients. Thirty healthy individuals were enrolled and four sites in each were selected and designated healthy controls. Patients were treated with scaling and root planing and control subjects with supragingival prophylaxis. Measurements including GCF volume, gingival inflammation, and probing depth were performed at screening baseline, 1 week later at pretreatment baseline, and at weeks 2 and 4 after treatment. AST content of GCF was measured using a chairside colorometric test. It was concluded that the outcome of the test is an effective objective measure distinguishing between diseased sites and non-diseased sites in patients and control subjects when evaluated both prior to and following application of therapy. Use of this simple chairside test, when combined with other standard diagnostic procedures, provides an objective measurement permitting improved capacity to distinguish between diseased and non-diseased periodontal sites, and to better assess and monitor the outcome of therapy.

Adult↗
PubMed1995

Enhancement of the enzymatic activity of single-chain urokinase plasminogen activator by soluble urokinase receptor.

Single-chain urokinase (scuPA), the unique form of urokinase secreted by cells, is converted to an active two-chain molecule through the cleavage of a single peptide bond by plasmin and other specific proteinases. Although scuPA may express limited enzymatic activity, its contribution to plasminogen activation on cell surfaces remains uncertain. Further, although it is well known that scuPA binds to a specific extracellular urokinase-type plasminogen activator receptor, the effect of this interaction on the enzymatic activity of scuPA has not been described. In the present paper we report that the binding of scuPA to cellular an to recombinant soluble urokinase-type plasminogen activator receptors (suPAR) increases its catalytic activity as measured by the cleavage of a urokinase-specific chromogenic substrate. suPAR increased the Vmax of scuPA 5-fold with little change in its Km. suPAR also stimulated the plasminogen activator activity of scuPA by decreasing its Km for Glu-plasminogen from 1.15 microM to 0.022 microM and by increasing the kcat of this reaction from 0.0015 to 0.022 s-1. Preincubation of scuPA with suPAR also enhances its susceptibility to inhibition by plasminogen activator inhibitor type 1, consistent with exposure of its catalytic site. The activity of scuPA bound to suPAR is not accompanied by cleavage of scuPA, which continues to migrate as a single band in SDS-polyacrylamide gel electrophoresis under reducing conditions. Moreover, suPAR increases the plasminogen activator activity of a plasmin-insensitive variant, scuPA (scuPA-Glu158), as well. Enhancement of scuPA activity by suPAR is both prevented and reversed by its aminoterminal fragment (amino acids 1-135), which competes for receptor binding, suggesting that continued binding to the receptor is required for expression of scuPA's enzymatic activity. Thus, our data suggest that scuPA may undergo a reversible transformation between a latent and an active state. The urokinase receptor may induce or stabilize scuPA in its active conformation, thereby contributing to the initiation of plasminogen activation on cell surfaces.

Peptide Fragments↗
PubMed1995

Immunohistochemical localization of the plasminogen activator inhibitor-1 in breast cancer.

We used an immunohistochemical assay with an antigen-retrieval technique to study plasminogen activator inhibitor type-1 (PAI-1) expression in paraffin-embedded breast tissue samples at different stages of malignant transformation. We detected PAI-1 in 15/20 invasive tumors. In several cases staining was localized to the stromal component. PAI-1-positive fibroblasts could be seen surrounding tumor nodules or at tumor margins. In addition, tumor-infiltrating macrophages (13 cases) and endothelial cells (5 cases) were positive. In 11 specimens PAI-1-positive cancer cells were also detected. In 2 strongly positive cases secreted PAI-1 was visible in the extracellular matrix surrounding the cells. Six of 9 samples of carcinoma in situ (DCIS) were weakly positive. No staining of endothelial cells was visible in DCIS. Only a few positive adenomatous epithelial cells could be seen in 3 of 7 papillomas. All biopsies of normal breast tissue were negative, with the exception of one sample, obtained from a patient with a previous segmental mastectomy for DCIS. PAI-1 production by invasive breast cancers could reflect a general upregulation of the plasminogen activation system in proliferating cancer cells, as suggested by the finding that normal mammary epithelium cultures expressed PAI-1 in all cases examined. In addition, production of PAI-1 by the tumor stroma could protect the tumor itself from excessive proteolysis.

Base Sequence↗
PubMed1995

Co-expression of urokinase, urokinase receptor and PAI-1 is necessary for optimum invasiveness of cultured lung cancer cells.

We investigated the importance of the urokinase (uPA)-plasmin system in fostering invasion of human lung cancer cells through artificial basement membranes composed of Matrigel. Eight cell lines (including 1 small cell and 7 non-small cell lines) were examined. One cell line did not express any components of the urokinase system. Four cell lines had substantial levels of endogenous uPA detectable on their surfaces. Three of these cell lines co-expressed the plasminogen activator inhibitor PAI-1 in addition to uPA. Assays for invasiveness revealed 4 cell lines capable of traversing a Matrigel barrier, including the 3 which co-expressed uPA, PAI-1 and uPA receptor. Surprisingly, the cell line expressing only uPA and uPA receptor displayed no invasive capacity despite levels of secreted uPA more than 20-fold higher than the other cell lines studied. Based on these observations, we hypothesized that both uPA and PAI-1 might be important for invasion by lung tumor cells, at least in vitro. We therefore tested polyclonal antibodies which inhibit uPA and PAI-1 activity for their effects on the highly invasive H292 cell line. After 3 days, invasive capacity was inhibited by antibodies to both uPA and PAI-1 in a dose-dependent manner. The plasmin inhibitor aprotinin reduced H292 cell invasion by 70%. Taken together, our data demonstrate that in cultured human lung cancer cells the uPA-plasmin system is important in promoting invasion into basement membranes and suggest that a critical balance between uPA and PAI-1 is necessary for optimal invasiveness. Our data are consistent with results from recent clinical studies showing that PAI-1 expression in tumor tissue is an adverse prognostic feature.

Carcinoma, Non-Small-Cell Lung↗
PubMed1995

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma.

We have previously shown that, as a consequence of low-dose melphalan (L-phenylalanine mustard (L-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was "mimicked" by the membrane penetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The beta-adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal or L-PAM TuB spleen cells. Since the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8+ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.

Animals↗
PubMed1995

Molecular characterization of plasminogen activators in human gingival crevicular fluid.

Plasminogen activators (PAs), a family of serine proteases, and their inhibitors (PAIs) are important in fibrinolysis, wound healing and tissue remodelling. Previous studies revealed differences in the localization of PA activity between healthy and diseased gingival tissues, suggesting that PAs and PAIs could play a part in periodontal homeostasis and disease. PAs and PAIs are synthesized by most of the cells types making up the periodontium and can be identified in gingival crevicular fluid (GCF). These studies sought to characterize the molecular species of PAs and their inhibitors in GCF collected from clinically healthy sites. PA enzymatic activity in GCF samples demonstrated by fibrin zymography revealed the presence of only tissue-type PA (tPA) activity. No urokinase-type PA (uPA) enzymatic activity was detected. tPA enzymatic activity appeared predominantly as an uncomplexed 70-kDa species, although some samples contained enzyme-inhibitor complexes. Quantitation of total tPA by enzyme immunoassay showed a mean concentration of 1.6 ng/microl. Analysis of GCF samples for uPA by immunoblotting and enzyme immunoassay disclosed the presence of small amounts of uPA (0.2 ng/microl), which were present predominantly in activator-inhibitor complexes. Immunoblotting showed specific PAI-2 immunoreactivity bands in high molecular-weight complexes and low molecular-weight degradation products, but less than nanogram amounts of free PAI-2 molecules. Enzyme immunoassay revealed that PAI-2 was present in an at least a seven times greater amount than PAI-1. These observations support the hypothesis that PA-generated proteolysis and its regulation by endogenous inhibitors has a role in the diverse biochemical mechanisms underlying periodontal physiology and pathology including host-microbial interaction, polymorphonuclear leucocyte infiltration, turnover and migration of epithelial cells, connective tissue degradation and remodelling, fibrinolysis and wound healing.

Adult↗
PubMed1995

Inhibition of lymphocyte activation by catecholamines: evidence for a non-classical mechanism of catecholamine action.

The effects of noradrenaline and other adrenergic agonists on lymphocyte activation were studied. Spleen and thymus cells from BALB/c mice were stimulated by mitogens and lymphocyte activation was monitored by measuring the incorporation of [methyl-3H]thymidine into DNA. Noradrenaline, adrenaline, isoproterenol and dopamine all inhibited the activation of spleen and thymus cells by concanavalin A, a T-cell specific mitogen, and the activation of spleen cells by lipopolysaccharide, a T-independent B-cell mitogen. The various catecholamines were approximately equipotent, having IC50 of approximately 10 microM. alpha-adrenergic agonists (phenylephrine, clonidine) did not inhibit lymphocyte activation. Noradrenaline, adrenaline and isoproterenol also inhibited DNA synthesis in S49 T lymphoma cells. The effects of adrenergic receptor antagonists on lymphocyte function were also studied. The inhibition of lymphocyte activation by catecholamines could not be reversed by antagonists to beta-adrenergic receptors (propranolol), alpha-adrenergic receptors (phentolamine), or dopaminergic receptors (haloperidol). Experiments with human peripheral blood leucocytes revealed that, as with murine cells, the beta-adrenergic antagonists propranolol and nadalol did not affect the catecholamine-mediated inhibition of lymphocyte activation. Although lymphocytes contain beta-adrenergic receptors that are coupled to adenylyl cyclase activity, catecholamines appear to inhibit murine lymphocyte activation by a mechanism that is independent of these or other classical adrenergic receptors.

Adrenergic Antagonists↗
PubMed1995

Thrombin increases expression of urokinase receptor by activation of the thrombin receptor.

PURPOSE: To investigate the effect of thrombin on the urokinase plasminogen activator receptor (u-PAR) in retinal pigment epithelial (RPE) cells. METHODS: The authors analyzed u-PAR mRNA by Northern blot hybridization. Retinal pigment epithelial cell surface u-PAR was assayed by measuring the amount of functional urokinase plasminogen activator (u-PA) bound to cells at saturation. Retinal pigment epithelial cells were derived from fetal retinal tissue and established in primary cell culture. RESULTS: Thrombin increased u-PAR mRNA 4-fold in RPE cells examined by Northern blot hybridization, whereas the amount of thrombin receptor mRNA was unchanged. Thrombin stimulated u-PA binding to RPE cells 2.5- to 5-fold in a time- and dose-dependent manner. Hirudin, a thrombin antagonist, completely blocked the effects of thrombin on u-PAR expression in RPE cells. Phosphatidylinositol phospholipase C treatment of RPE cells resulted in the abolition of thrombin-induced u-PA binding. Recombinant soluble u-PAR competitively inhibited two-chain u-PA binding to the surface of thrombin-treated RPE cells. A thrombin receptor agonist peptide (SFLLRNPNDKYEPF) also induced a 2.5-fold increase in binding of u-PA to the surface of RPE cells. CONCLUSION: Thrombin increases u-PAR expression by RPE cells by a mechanism involving activation of the seven transmembrane thrombin receptor.

Amino Acid Sequence↗
PubMed1994

Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Møller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thøgersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.

3T3 Cells↗
PubMed1994

The urokinase receptor is expressed in invasive breast cancer but not in normal breast tissue.

We have studied expression of the urokinase receptor (u-PAR) in paraffin-embedded breast tissues at various stages of malignant progression. Forty-nine of 59 invasive cancers studied showed varying degrees of reactivity with our polyclonal antibody. The staining pattern was variable from case to case, although strong surface staining of tumor-associated macrophages was evident in most of these sections. In several cases, blood vessels in selected tumor areas were stained, as confirmed by treatment of adjacent sections with an anti-factor VIII antibody. These could represent regions of recent angiogenesis. Staining of tumor cells was observed in 21 of 59 cases and was extensive in 5 cases but confined to a small percentage of cells in the remaining 16 samples. In contrast with the cancer sections, all normal breast tissue (12 cases) was negative, as well as all fibroadenomas (4 cases), papillomas (5 cases), and hyperplasia with atypia (2 cases) studied. Seven carcinomas in situ examined were also negative for u-PAR, with the exception of few macrophages in two cases, suggesting that u-PAR expression may be associated with invasive tumor. The presence of u-PAR in human breast cancer and its absence from nonmalignant breast tissue supports the idea that plasminogen activation plays an important role in the process of cancer invasion. Expression of u-PAR on macrophages, endothelial cells, and cancer cells suggests the existence of complex paracrine interactions between tumor cells and stroma.

Base Sequence↗
PubMed1994

Immunologic detection of the cellular receptor for urokinase plasminogen activator.

The cellular receptor for urokinase plasminogen activator (uPA-R) is a monomeric phosphatidylinositol-linked glycoprotein (gp40-65) that may contribute to the invasive capacity of tumor and inflammatory cells by focusing the activity of urokinase (uPA) in converting plasminogen to plasmin, a serine protease capable of degrading extracellular matrix proteins. The further characterization of uPA-R has been facilitated by our recent development of a monoclonal antibody, anti-Mo3f, specific for uPA-R. This mAb bound to uPA-R expressed by phorbol myristate acetate-stimulated U-937 cells and by NIH-3T3 cells permanently transfected with uPA-R cDNA. In competitive binding assays, anti-Mo3f inhibited the binding of fluorescein-conjugated uPA ligand to uPA-R expressed by U-937 cells and uPA-R transfectants; conversely, preexposure of cells to saturating quantities of exogenous uPA partially blocked the subsequent binding of anti-Mo3f mAb to uPA-R. Anti-Mo3f mAb was employed as the capture reagent in an ELISA for the quantitation of soluble forms of uPA-R (derived from U-937 cells and recombinant uPA-R) which had a sensitivity of approximately 4-12 ng/ml. Anti-Mo3f mAb was also applied as a serologic probe for the detection of uPA-R expressed by human tumor tissues. By immunoperoxidase staining, anti-Mo3f demonstrated positive tumor cell staining in 4 of 16 breast and 7 of 31 prostate carcinomas in formalin-fixed, paraffin-embedded specimens. These data indicate that the anti-Mo3f mAb detects an epitope proximate to or within the ligand binding domain (domain 1) of uPA-R and may be useful as a tool for the serologic detection of uPA-R in soluble form or associated with human tumors.

3T3 Cells↗
PubMed1994

Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals.

OBJECTIVE: To characterize a Kaposi's sarcoma (KS) cell line established from a tumor biopsy from the oral mucosa of an iatrogenically immunosuppressed HIV-negative man. METHODS: Cells were placed in culture and evaluated by a variety of biologic, serologic, karyotypic, and immunologic procedures. Electron microscopic examination was performed. The ability to produce tumors in nude mice was evaluated, and the nature of the cells within the tumor determined. Assays for urokinase plasminogen activator type (uPA), plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR) were conducted. RESULTS: The SLK cell line has an endothelial cell morphology with very little anaplasia. The karyotype indicates diploid phenotype of human origin. Immunohistochemical and electron microscopic examinations confirmed the endothelial nature of this cell line. No viruses were detected. The tumors induced in nude mice showed hypervascularization, with characteristics of KS. The cell line produces uPA and PAI-1, and also expresses uPAR. CONCLUSIONS: The SLK cell line is of endothelial cell origin and the first human cell line to induce KS-like tumors in recipient animals. The expression of urokinase and its receptor suggests a paracrine and autocrine interaction that may be important for the growth of the tumor. The SLK line should be valuable for studies of KS pathogenesis and therapeutic approaches to this malignancy.

Animals↗
PubMed1994

Another 'dangerous opportunity': child and adolescent psychiatry faces health care reform.

Child and adolescent psychiatry has evolved into its present form through a series of crises over the past 75 years. The current challenge posed by health care reform represents the next developmental opportunity for this subspecialty. The author presents a brief summary of these crises, an overview of other forces affecting practice, and a possible model of adaptive approaches to these dilemmas. He concludes that there is every expectation that the field will mature further in response to this newest crisis.

Adolescent↗
PubMed1993

Prevention of metastasis by inhibition of the urokinase receptor.

The plasminogen activator urokinase (u-PA) mediates proteolysis by a variety of human tumor cells. Competitive displacement of u-PA from cellular binding sites results in decreased proteolysis in vitro, suggesting that the cell surface is the preferred site for u-PA-mediated protein degradation. We studied the effect of u-PA receptor blockade on the metastatic capacity of human PC3 prostate carcinoma cells, using transfectants which expressed chloramphenicol acetyl-transferase (CAT). Eight weeks after subcutaneous inoculation of these cells into nude mice, CAT activity was detected in regional lymph nodes, femurs, lungs, and brain, thereby mimicking the organ tropism observed for naturally occurring metastases of prostate cancer. In a second transfection, CAT-expressing PC3 cells received cDNA encoding a mutant u-PA (Ser356-->Ala) which lacks enzymatic activity but which retains full receptor binding affinity. Three mutant u-PA expressors, each with < 5% of wild-type cell-associated u-PA activity, were compared in vivo with independently derived controls. Primary tumor growth was similar in each group of animals and all tumors expressed comparable CAT activity. In contrast, metastasis (as assessed by CAT activity) was markedly inhibited when cell surface u-PA activity was blocked. Levels of CAT activity were reduced by a factor of > 300 in regional lymph nodes, 40-100 in brain tissue, and 10-20 in lung tissue. Metastatic capacity was inhibited similarly when animals were given intermittent intraperitoneal injections of a u-PA/IgG fusion protein capable of displacing u-PA activity from the tumor cell surface. Our results indicate that cell surface u-PA activity is essential to the metastatic process. In addition, the assay system employed in these experiments may be generally useful in testing other therapeutic modalities to limit the spread of primary tumors.

Adenocarcinoma↗
PubMed1993

Neuroimmune modulation: signal transduction and catecholamines.

In recent years, much interest has centered on the commonalities and bi-directional interactions between the nervous system and the immune system. This review focuses on mechanisms through which, catecholamines, a class of neuro-endocrine molecules, modulate immune functions. Catecholamines can be immune suppressive and inhibit lymphocyte activation of both T and B cells as well as the generation of immune-mediated anti-tumor responses. Some of these catecholamine-regulated activities appear to be modulated through the second messenger, cyclic AMP, whereas others appear to be catecholamine-dependent but cyclic AMP independent. Further delineation of the interacting ligand-receptor complexes, populations of responding cells and signal transduction mechanisms leading to the activation of specifically involved genes and gene products, will lead to enhanced understanding of the integratory functions of the nervous system in immune responses, the biology of stress, the role of stress-associated molecular mechanisms in perturbations of physiological homeostasis and the development of a new biological psychiatry with accompanying rational therapeutic modalities.

Animals↗