Tissue factor induction in human monocytes. Two distinct mechanisms displayed by different alloantigen-responsive T cell clones.

One component of the cellular immune response to antigens is the expression of procoagulant activity (PCA) by monocytes and macrophages. Induction of human monocyte PCA in response to alloantigenic stimulation requires the collaboration of HLA-DR-responsive T cells. In mixed lymphocyte cultures (MLCs), the induction of monocyte tissue factor appears to be mediated exclusively by a T cell-derived lymphokine. We have used a soft agar cloning method to generate alloantigen-responsive T cell clones from MLCs between irradiated Daudi lymphoblastoid cells and human peripheral blood mononuclear cells. Developing clones were screened for the ability to induce PCA in fresh autologous monocytes in response to Daudi stimulator cells. PCA induction was observed with some, but not all, proliferating T cell clones and two modes of induction were apparent. Some T cell clones mediated PCA induction exclusively by lymphokine production, whereas other clones delivered induction signals by direct cellular collaboration with the monocyte effector cells. These two inductive pathways were represented in distinct, non-inclusive functional subsets of T cell clones. Constitutive production of soluble inducer signals was not observed in T inducer clones. The magnitude of the monocyte PCA response increased in response to an increase in the allogeneic stimulator/T clone responder ratio, and third-party allogeneic cells were unable to elicit the PCA-inducing lymphokine signals from T inducer clones. Both modes of induction were shown to generate tissue factor protein activity in monocytes. Collectively, these results suggest that PCA induction can be initiated in response to alloantigens through collaboration with certain OKT3+, OKT4+, OKT8-, OKM1- T inducer clones, and that induction can be mediated by at least two different functional subsets of human T cells. Stimulation with the appropriate alloantigen may elicit both lymphokine and T cell-contact pathways of induction of tissue factor in human monocytes.     

An argument for dental hygiene to develop as a discipline

Abstract:  The practice of dental hygiene was developed to provide oral health education and preventive oral health care, originally for children. It has grown to provide oral health services valued by a broad spectrum of society, but has not attained the desired respect and status accorded to other professional groups. Objective:  Professional disciplines link actions of practitioners with the science that is the foundation of practice. The purpose of this paper is to examine whether dental hygiene practice could benefit from pursuit of development as a discipline. Methods:  Literature on professionalization and disciplines, related to dental hygiene in general and the North American context specifically, was retrieved from databases and grey sources, such as organizational reports. Dental hygiene's current characteristics relative to a discipline were examined. Results:  Dental hygiene has developed some characteristics of a discipline, such as identifying a metaparadigm that includes concepts of the client, the environment, health/oral health and dental hygiene actions, with a perspective that includes a focus on disease prevention and oral health promotion. However, research production by dental hygienists has been limited, and often not situated within theoretical or conceptual frameworks. Conclusion:  Dental hygiene draws its knowledge for practice from a variety of sources. Dental hygiene could strengthen its value to society by prioritizing development of highly skilled researchers to study interventions leading to improved oral outcomes, and transferring that knowledge to practitioners, strengthening links between practice and science. Intentional pursuit of knowledge for practice would lead to dental hygiene's eventual emergence as a professional discipline.     

Plasma Lipoprotein Induction and Suppression of the Generation of Cellular Procoagulant Activity in Vitro: REQUIREMENTS FOR CELLULAR COLLABORATION

Isolated human plasma very low density, intermediate density, and high density lipo-proteins at physiologic concentrations have been demonstrated in the preceding report to induce significant increases in the procoagulant activity of human peripheral blood mononuclear cells in vitro, whereas low density lipoprotein did not. The monocyte was identified in this study by cellular fractionation and by direct cytologic assays as the source of this inducible activity, thus identifying the procoagulant activity as a monokine. The generation of these lipoprotein-induced procoagulant monokines was entirely dependent upon the presence of lymphocytes. Isolated lymphocytes that had been exposed to the stimulatory lipoproteins could induce monocytes to produce the procoagulant activity, whereas neither the culture medium from lipoprotein-stimulated lymphocytes, homogenates of lymphocytes, nor other cells such as platelets could substitute for this requirement. The interaction of the stimulatory lipoproteins with lymphocytes was rapid, reaching completion within 30 min, and was equally effective at either 4° or 37°C. Low density lipoprotein did not stimulate lymphocytes to induce monocyte procoagulant activity, but did actively suppress the production of the procoagulant monokines induced by each of the stimulatory lipoproteins, as well as bacterial lipopolysaccharide. The monocyte was identified as the cell sensitive to low density lipoprotein suppression, and no suppression of lymphocyte triggering was observed. These observations on the interaction of plasma lipoproteins with lymphocytes and monocytes in vitro introduce two new regulatory events by which plasma lipoproteins influence the function of cells, and define a regulatory network by which certain lipoprotein classes trigger lymphocytes, which can in turn induce monocytes to express procoagulant activity. Only this latter phase is subject to lipoprotein suppression by physiologic concentrations of low density lipoprotein.     

Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees.

Knowledge of the pathogenetic mechanisms responsible for the activation of the coagulation system associated with endotoxemia is important for the development of improved modalities for prevention and treatment. We analyzed the appearance in plasma of TNF, IL-6, and indices of coagulation and fibrinolytic system activation in normal chimpanzees after intravenous infusion of endotoxin. Endotoxin infusion elicited reproducible and dose-dependent elevations in serum TNF and IL-6, as well as marked increases in thrombin generation in vivo as measured by immunoassays for prothrombin activation fragment F1 + 2, thrombin-antithrombin III complexes, and fibrinopeptide A. Activation of the fibrinolytic mechanism was monitored with assays for plasminogen activator activity and plasmin-alpha 2-antiplasmin complexes. To potentially intervene in the molecular pathways elicited by endotoxin, pentoxifylline, an agent that interrupts "immediate early" gene activation by monocytes, or a potent monoclonal antibody that neutralizes tissue factor-mediated initiation of coagulation, were infused shortly before endotoxin. Pentoxifylline markedly inhibited increases in the levels of TNF and IL-6, as well as the effects on coagulation and fibrinolysis. In contrast, the monoclonal antibody to tissue factor completely abrogated the augmentation in thrombin generation, but had no effect on cytokine levels or fibrinolysis. We conclude that the endotoxin-induced activation of coagulation appears to be mediated by the tissue factor-dependent pathway, the fibrinolytic response triggered by endotoxin is not dependent on the generation of thrombin, and that the release of cytokines may be important in mediating the activation of both the coagulation and the fibrinolytic mechanisms in vivo.     

Combined Functional and Immunochemical Analysis of Normal and Abnormal Human Factor X

Human Factor X was isolated from Cohn fraction III and characterized by polyacrylamide gel electrophoresis, amino acid composition, and isoelectric focusing. Two molecular forms with biological activity were observed at isoelectric points of 4.8 and 5.0. Antisera generated to Factor X was monospecific and used to establish an equilibrium competitive inhibition radioimmunoassay. This assay was specific for human Factor X and did not cross-react with human prothrombin or bovine Factor X within the sensitivity range of 6-300 ng Factor X antigen/ml. The mean concentration of Factor X based on the antigen was 11.9 mug/ml, whereas concentration values based on coagulant activity was 7.8 mug/ml. This 30% difference in measurement appears to result from the presence of a subpopulation of Factor X molecules devoid of coagulant activity. The radioimmunoassay was used to qualitatively and quantitatively compare purified Factor X to plasmic Factor X obtained from normal, warfarintreated, acquired Factor X-deficient, and congenitaldeficient patients. In all but one case, the Factor X present in these plasmas was immunochemically identical to the purified Factor X and permitted precise quantitation of these abnormal Factor X molecules. Factor X procoagulant activity was analyzed relative to Factor X antigen and the specific activities were used to characterize normal and abnormal Factor X molecules. Reduced Factor X activity in plasmas from warfarin-treated and acquired Factor X-deficient patients was attributed to both decreases in Factor X antigen and decreased function of the Factor X molecules. Congenitally deficient patients, in general, showed a reduction in Factor X antigen in parallel with Factor X procoagulant activities resulting from comparable decreases in specific biological activity of the molecules.     

Identification of a lymphocyte surface receptor for low density lipoprotein inhibitor, an immunoregulatory species of normal human serum low density lipoprotein.

The present study demonstrates the existence on human peripheral blood lymphocytes of a saturable cell surface receptor for low density lipoprotein inhibitor (LDL-In), a subset of normal human serum low density lipoprotein (LDL) that has been previously demonstrated to suppress selected lymphocyte functions in vivo and in vitro. The binding of radioiodinated LDL-In of demonstrable biological activity occurs rapidly and is quantitatively augmented by prior cultivation of the lymphocytes in lipoprotein-depleted serum, suggesting regulation of receptor density by lipoproteins in vivo. Binding is temperature dependent, facilitated by calcium ions, saturable at 4 degrees C within 40-60 min, and blocked by prior exposure to unlabeled LDL-In. The lymphocyte receptor is trypsin sensitive and regenerates in vitro with a t1/2 of 3.6 h. LDL-In receptors are calculated to have a maximum density of 4,860 +/- 460 per cell if uniformly distributed on all lymphocyte subsets. These receptors have an estimated average association constant of 1.47 X 10(7) liters/mol. When considered in context of the estimated concentration of LDL-In in blood, the receptors should be partially occupied in vivo by endogenous plasma LDL-In. Prior site occupancy inhibition experiments designed to analyze the specificity of LDL-In binding demonstrate that (a) LDL-In is 13.7-fold more effective than whole LDL in blocking the subsequent binding of 125I-LDL-In to cells; and that (b) LDL is 11-fold more effective than LDL-In in blocking the binding of 125I-LKL. This is consistent with the degree of contamination of each lipoprotein with the other lipoprotein. An independent identity of the LDL-In receptor is also supported by observations that in contrast to the previously described LDL receptor, synthesis and expression of the LDL-In receptor on lymphocytes are not suppressed by cultivation of the cells in the presence of 25-hydroxycholesterol and cholesterol. These findings suggest the existence of a previously undescribed and discrete receptor on lymphocytes for LDL-In, and that the modulation of lymphocyte function by LDL-In may be mediated by a specific cell surface receptor pathway.     

Carbohydrate of the Factor VIII/von Willebrand Factor in von Willebrand''s Disease

We have examined the plasma Factor VIII/von Willebrand factor (FVIII/vWF) molecule from 16 patients with von Willebrand's disease, and have found no evidence of a significant decrease of carbohydrate content in 15 of these patients. FVIII/vWF was isolated by preparative counter immunoelectrophoresis directly from plasma using antibody to Factor VIII-related antigen, reduced in sodium dodecyl sulfate in the presence of urea, and electrophoresed in 5% polyacrylamide gels to separate the FVIII/vWF subunit from other proteins. Duplicate gels were stained by either the periodic acid-Schiff (PAS) reaction or by Coomassie Brilliant Blue G250. The ratio of Coomassie: PAS was determined by spectrophotometric scanning of the gels. Transferrin was used as an internal reference standard. The ratio for 23 normal individuals was 2.4+/-0.38 and the observed range was 1.8-3.8. 15 patients with von Willebrand's disease fell within this range. One patient independently reported as having decreased FVIII/vWF carbohydrate was also studied by this technique. A ratio of 6.8 was found, indicative of decreased, though not absent, carbohydrate. Cold insoluble globulin did not represent a significant contaminant in these analyses. 11 of the von Willebrand's disease patients with normal FVIII/vWF carbohydrate had abnormal crossed immunoelectrophoretic patterns characterized by absence of the less anodic forms of Factor VIII-related antigen. Four patients had normal patterns. These studies indicate that an absence or decrease of PAS reactive FVIII/vWF carbohydrate is not a consistent abnormality in von Willebrand's disease.