Effect of fibrin bandage fibrinogen concentration on blood loss after grade V liver injury in swine.

OBJECTIVE: To determine the effect of fibrinogen concentration of dry fibrin bandages on blood loss after grade V liver injury. METHODS: Twenty-four pigs were used. Grade V liver injuries were induced and treated with dry fibrin bandages containing 0, 4, 8, or 15 mg fibrinogen/cm2. Animals were monitored for 60 minutes. Blood loss, fluid use, hematological data, and hemostasis were assessed. RESULTS: Post-treatment blood losses (mean and 95% confidence interval [CI]) were 1,560 mL (356-6,844), 372 mL (65-2,134), 225 mL (51-992), and 127 mL (22-732) in the 0-, 4-, 8-, and 15-mg groups, respectively. Only the 15-mg group had results significantly lower than the 0-mg group (p < 0.05). Blood loss was negatively related to fibrinogen concentration (p < 0.05). CONCLUSION: Fibrinogen concentration was inversely related to blood loss after grade V liver injury. The 15-mg formulation was the only one that significantly reduced blood loss.

Dry fibrin sealant dressings reduce blood loss, resuscitation volume, and improve survival in hypothermic coagulopathic swine with grade V liver injuries.

OBJECTIVE: The majority of early trauma deaths are caused by uncontrolled hemorrhage, and are frequently complicated by hypothermic and dilutional coagulopathies. Any hemorrhage-control technique that achieves rapid hemostasis despite a coagulopathy should improve the outcome of these patients. We conducted this study to determine whether dry fibrin sealant dressings (DFSD) would stop bleeding from grade V liver injuries in swine that were hypothermic and coagulopathic. METHODS: Nineteen swine weighing 39.7 kg (mean and 95% confidence interval, 36.3-43.1), underwent a 60% isovolemic, hypothermic exchange transfusion with 33 degrees C 6% hetastarch to produce a dilutional and hypothermic coagulopathy. The animals then received a grade V liver injury and one of three treatments: DFSD, conventional liver packing with gauze sponges, or immunoglobulin G (IgG) placebo sealant dressing (blinded control). All animals were resuscitated with lactated Ringer's solution to their preinjury mean arterial pressure. Blood loss after treatment, mean arterial pressure, resuscitation volume, hematologic variables, and core temperature were monitored for 1 hour. RESULTS: At the time of injury, core temperature = 33.3 degrees C (95% confidence interval, 33.2-33.4), hemoglobin concentration = 4.4 g/dL (4.2-4.6), platelet count = 132 x 10(5)/microL, (93-171), prothrombin time = 21.6 seconds (19.6-23.5), activated partial thromboplastin time = 25.2 seconds (range, 22.9-27.5 seconds), and fibrinogen = 83 mg/dL (range, 76-89 mg/dL) across treatments. The posttreatment blood loss in the DFSD group was 669 mL, (range, 353-1,268 mL), which was lower (p < 0.01) than the means of 3,321 mL (range, 1,891-5,831 mL) and 4,399 mL (range, 2,321-8,332 mL) observed in the packing and IgG groups, respectively. The resuscitation volume in DFSD was 2,145 mL (range 1,310-3,514 mL), which was lower (p < 0.05) than the means of 5,222 mL (range 3,381-8,067 mL) and 5,542 mL (range 3,384-9,077 mL) in the packing and IgG groups, respectively. One-hour survival in the DFSD group was 83%, whereas survival in the packing and IgG groups were 0% (p < 0.05). CONCLUSION: In swine with a grade V liver injury complicated by a dilutional and hypothermic coagulopathy, DFSD provided simple, rapid hemorrhage control, decreased fluid requirements, and improved survival.

Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine.

BACKGROUND: We conducted this study to determine whether the dry fibrin sealant dressing (DFSD) would stop bleeding from a grade V liver injury and to evaluate the effects of leaving the absorbable DFSD in survival animals. METHODS: Twenty-four swine (40+/-3.0 kg) received a uniform grade V liver injury and were randomized to one of four 1-hour treatment groups: (1) gauze packing, (2) DFSD, (3) immunoglobulin G placebo dressing, and (4) no treatment. All animals were resuscitated with lactated Ringer's solution. Total blood loss (TBL), mean arterial pressure, resuscitation volume, and laboratory data were monitored for 1 hour after injury. Four swine were treated with the DFSD after grade V injury and allowed to survive for 7 or 14 days. RESULTS: The TBL was 1,104+/-264 mL (mean +/- SEM), 544+/-104 mL, 4,223+/-1,555 mL, and 6,026+/-1,020 mL for groups 1, 2, 3, and 4 respectively. TBL in DFSD animals was less than that in animals treated with gauze packing (p = 0.06). Grade V injuries were uniform among the 1-hour groups, and no evidence of intrahepatic abscess, unusual adhesions, or hepatic vein, vena caval, or pulmonary thromboses were noted in the long-term survival animals. CONCLUSION: In this model of grade V liver injury, blood loss with the DFSD was 51% of that observed with standard gauze packing (not statistically different). Initial survival data revealed no complications attributable to the fibrin dressing. DFSD may provide simple, rapid, and definitive hemorrhage control in life-threatening liver injuries without the need for reoperation.

Soluble E-selectin levels in sepsis and critical illness. Correlation with infection and hemodynamic dysfunction.

E-selectin, an early mediator of leukocyte-endothelial adhesion, is expressed on activated endothelium. Soluble E-selectin is present in the supernatant of cytokine-activated endothelial cells and elevated serum levels are found in a variety of inflammatory conditions. We documented elevated E-selectin serum levels in 119 critically ill medical ICU patients (log transformed mean E-selectin level, measured by ELISA, was 5.28 ng/ml) compared to normal volunteers (1 ng/ml). Forty-three patients with culture-positive sepsis had higher (p < 0.05) E-selectin levels (15.39 ng/ml) than 24 patients with culture-negative sepsis (4.87 ng/ml), 44 with noninfectious SIRS (2.33 ng/ml), and eight without SIRS (1.97 ng/ml). E-selectin levels related strongly to the degree of hemodynamic compromise (p < 0.0001). Further analysis demonstrated microbiological status and hemodynamic status to be independent variables related to E-selectin level. Day 1 E-selectin levels correlated positively with peak organ failure score over the course of ICU hospitalization (r = 0.30, p = 0.001) and were higher (p < 0.05) for nonsurvivor (10.61 ng/ml, n = 26) than survivors (4.35 ng/ml, n = 93). We conclude that soluble E-selectin levels are higher in serum of patients with microbiologically documented sepsis than in other critically ill medical ICU patients. Day 1 E-selectin levels correlate highly with hemodynamic compromise and modestly with subsequent organ dysfunction and survival.

Detection of the chemokine RANTES and endothelial adhesion molecules in nasal polyps.

BACKGROUND: To better understand the mechanisms of eosinophil recruitment into the upper airways, we examined human nasal polyps for the expression of the chemotactic cytokine RANTES and the endothelial adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1). METHODS: Routine histologic examination and immunostaining with antibodies to RANTES, E-selectin, and VCAM-1 were performed on three types of tissues: nasal polyps, sinus mucosa, or turbinates from patients undergoing other elective procedures (S/T), and nasal biopsy specimens from nonallergic volunteers (NA). To further quantify the expression of endothelial adhesion molecules, some tissue samples were homogenized, and the resulting supernatants were assayed with sandwich ELISAs for VCAM-1 and E-selectin. RESULTS: Polyp eosinophil counts ranged from 19/mm2 to 1818/mm2 (763 +/- 120/mm2, mean +/- SEM) and were significantly higher than those found in the control tissues (5 +/- 2 in S/T samples and 20 +/- 9 in NA samples, p < 0.002). Immunochemical staining for RANTES was observed in 11 of 14 polyps; intense staining for RANTES (grade 3) was observed in six of 14 polyps. None of nine S/T samples or five NA samples demonstrated grade 3 staining. Staining with anti-RANTES was largely localized to airway and glandular epithelium. There was no significant correlation between counts of eosinophils or the combined total of eosinophils plus mononuclear cells and the intensity of epithelial RANTES staining in all nasal tissues. Staining for VCAM-1, as well as for E-selectin, was detected in 11 of 14 polyps and eight of 13 control tissues. VCAM-1 detected by ELISA in polyp tissues (6.8 +/- 1.3 micrograms/gm) was higher than that found in six S/T samples (1.2 +/- 0.3 micrograms/gm, p < 0.005) and in two NA samples (1.8 +/- 0.02 micrograms/gm, p = 0.08). E-selectin values in polyps (1.4 +/- 0.3 micrograms/gm) were not statistically different from those detected in six S/T samples (0.5 +/- 0.2 microgram/gm) or two NA samples (1.6 +/- 0.4 microgram/gm). Counts of eosinophils and eosinophils plus mononuclear cells displayed a strong correlation with VCAM-1 ELISA values (p < 0.005 and p < 0.004, respectively) but not with VCAM-1 staining. VCAM-1 staining correlated with EG2-positive eosinophils in nasal polyp tissues (p < 0.01). E-selectin staining did not correlate with either neutrophil or eosinophil counts. CONCLUSIONS: These studies demonstrate that the chemokine RANTES is produced in vivo predominantly to nasal epithelium. Endothelial activation, as indicated by adhesion molecule expression, occurs in human nasal polyp tissues and in control tissues, possibly reflecting the continued antigen exposure of the nasal mucosa. The correlations found in this study suggest that expression of VCAM-1 plays a role in the selective recruitment of eosinophils and mononuclear cells into nasal polyp tissues and that RANTES may be more important in localizing eosinophils to the epithelium.

CD34+ cell expansion and expression of lineage markers during liquid culture of human progenitor cells.

A 96-well-based suspension culture system for human hematopoietic progenitor cells has been developed to monitor the commitment and differentiation of CD34+ cells to specific lineages and the maintenance and expansion of CD34+ cells in vitro. The expression of maturation and lineage markers on the cells in culture was measured by enzyme-linked immunosorbent assay (ELISA). CD34+ cells were isolated from umbilical cord blood and fetal liver (90% purity) and were grown in liquid culture in 96-well plates for 10 days. The cells were then fixed with a glutaraldehyde-paraformaldehyde mixture, attaching the cells firmly to the plastic. An ELISA was performed, using appropriate primary antibodies directed against cell surface markers. The expression of four different lineage markers was measured: CD14 (monocyte), CD15 (neutrophil), platelet glycoprotein (GP) IIb/IIIa (CD41a, megakaryocyte) and glycophorin A (erythroid). The two-growth factor combination of interleukin 3 (IL-3) and stem cell factor (SCF) stimulated expression of CD14, CD15 and GP IIb/IIIa. Lineage-restricted growth factors such as erythropoietin (EPO), in combination with SCF, stimulated expression of glycophorin A. The three-factor combination of IL-3, SCF and EPO stimulated expression of all four lineage markers. Other multiple growth factor combinations all stimulated myeloid and megakaryocyte growth, as measured by ELISA and flow cytometry, but erythroid growth was present only when EPO was included in the growth factor mixture. In serum-free medium or plasma-containing medium, CD14 expression was markedly reduced, whereas glycophorin A expression was greatly elevated in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of lipopolysaccharide and IL-1 but not of TNF-induced activation of human endothelial cells by suramin.

The binding and trans-endothelium migration of inflammatory cells is believed to play a critical role in a variety of inflammatory conditions. This study investigates the ability of the experimental drug suramin to block the activation of HUVEC by endotoxin and by the proinflammatory cytokines IL-1 and TNF. We demonstrate that the inducible expression of several adhesion molecules by LPS and IL-1 beta but not by TNF-alpha is prevented by suramin. In a dose-dependent manner, suramin inhibits the binding of neutrophils and T lymphocytes to LPS and IL-1 beta but not to TNF-alpha-activated HUVEC. The inhibitory effect of the drug on IL-1 beta-induced but not on LPS-induced cell stimulation can be completely reversed by the addition of excess cytokine but not by excess LPS. Because LPS activation of HUVEC is known to depend on serum/plasma-derived soluble CD14, we set out to determine whether suramin inhibition involves interference with the action of the CD14-LPS complex on HUVEC. Indeed, the drug prevents the binding of radioactive LPS in the presence of serum and inhibits LPS-induced cell activation in serum-free medium supplemented with recombinant soluble CD14. The results suggest that suramin interferes with the CD14-dependent activation of HUVEC and that it also may be a useful agent in blocking infectious endotheliopathies in vivo.

Soluble E-selectin is increased in inflammatory synovial fluid.

OBJECTIVE: To investigate the hypothesis that soluble E-selectin (sE-selectin) may be detected in synovial fluid (SF) and play a role in inflammatory arthritis. METHODS: We used a sandwich ELISA to measure sE-selectin in the SF of 58 patients with rheumatoid arthritis (RA), 9 with psoriatic arthritis (PsA), 30 with osteoarthritis (OA), 13 with gout, and 9 with calcium pyrophosphate dihydrate crystal deposition disease (CPPD). RESULTS: SF sE-selectin values in RA (mean 1.49 ng/ml, 0.18-3.90) and PsA (mean 1.36 ng/ml, 0.88-2.31) were significantly higher than those with OA (mean 0.83 ng/ml, 0.00-1.83), gout (mean 1.04 ng/ml, 0.11-3.42), or CPPD (mean 0.80 ng/ml, 0.20-1.47). Elevated SF sE-selectin was associated with elevated serum sE-selectin, erythrocyte sedimentation rate, and SF white blood cell count. CONCLUSION: Our findings suggest that endothelial cell activation and E-selectin may contribute to the development of inflammatory processes.

Serum ELAM-1 is increased in vasculitis, scleroderma, and systemic lupus erythematosus.

OBJECTIVE: To investigate the state of endothelial cell activation in vasculitis, scleroderma, and systemic lupus erythematosus (SLE). METHODS: We used a sandwich ELISA to quantitate a soluble form of endothelial leukocyte adhesion molecule-1 (sELAM) in serum. RESULTS: sELAM was detected in serum from healthy individuals (mean 0.92 ng/ml). Levels were significantly higher in patients with giant cell arteritis (mean 2.04 ng/ml), polyarteritis nodosa (mean 2.08 ng/ml), scleroderma (mean 2.27 ng/ml), and SLE (mean 3.93 ng/ml). Elevated values were present in patients with both active and inactive disease. sELAM levels of > 3 ng/ml identified most patients with recent onset or active disease. CONCLUSION: Our findings may reflect a low degree of endothelial cell activation in healthy persons that is increased in inflammatory diseases involving blood vessels. Elevated serum sELAM levels may reflect ongoing inflammatory processes in these diseases.

Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in the serum of patients with septic shock.

A quantitative sandwich ELISA for E-selectin in the fluid phase (soluble E-selectin, sEs) has been developed that is sensitive to 100 pg/ml. The assay shows no reactivity with either L- or P-selectins. We have used this to determine the fate of E-selectin after cell-surface expression and to test whether levels measured in vivo may represent the state of endothelial activation. E-selectin was first detectable in supernatants of IL-1-stimulated endothelial cells at 24 h, and increased slowly up until 72 h. However, over this time period the total E-selectin detectable in the system (cells plus supernatants) declined dramatically. 125I-surface-labeled endothelial cells cultured for 24 h show an E-selectin of reduced m.w. in the supernatant, indicating that the molecule is shed from the surface. The shed form also appears to be slightly smaller than the intact membrane form as determined from immunoprecipitation and molecular sieving studies. In addition, the cytoplasmic domain of the molecule found in supernatants of activated endothelial cells and in serum is not intact as determined by loss of reactivity with an antipeptide antibody specific for the cytoplasmic domain. We have examined the sera of 71 normal individuals. Without exception, sEs was found in serum in the range of 0.13 to 2.8 ng/ml, suggesting that even in the absence of overt inflammatory processes E-selectin is being synthesized and released into the bloodstream. In addition, bacteremic patients with hypotension, but not those without, showed markedly elevated sEs values. As determined by cell-binding studies, the blood-derived form of E-selectin is biologically active.

Altered adhesion molecule expression and endothelial cell activation accompany the recruitment of human granulocytes to the lung after segmental antigen challenge.

Mounting evidence suggests that inflammatory cells recruited to the lung can contribute to the pathogenesis of asthma. The factors governing the activation and recruitment of circulating cells to the lung remain unknown, but an early step in this process is the interaction of adhesion molecules on circulating cells with those on endothelial cells. We used a segmental antigen challenge model followed 18 h later by bronchoalveolar lavage (BAL) to study granulocyte recruitment to the lung in 14 allergic subjects. Using immunofluorescence and flow cytometry, we determined the expression of the adhesion molecules CD11b, L-selectin (LECAM-1), and VLA-4 on BAL and peripheral blood granulocytes. Total cell count and percentages of recovered eosinophils and basophils were significantly increased in BAL fluids from antigen-challenged segments. Compared with their peripheral blood counterparts, CD11b expression was increased 2- to 3-fold on BAL eosinophils, basophils, and neutrophils (n = 9, P less than 0.05). In contrast, L-selectin expression was significantly decreased on BAL cells (n = 3 to 4, P less than 0.05). Similar phenotypic changes were observed on all three cell types, and on neutrophils recovered from saline-challenged control lung segments. In two subjects, VLA-4 alpha (CD49d) expression on BAL eosinophils was 78 +/- 5% of that seen on peripheral blood eosinophils. Because ELAM-1 (endothelial leukocyte adhesion molecule-1, E-selectin) expression occurs during allergic inflammation and is shed after endothelial activation, we used a sensitive enzyme-linked immunosorbent assay to analyze BAL supernatants for a soluble form of this molecule (sELAM-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Four molecular pathways of T cell adhesion to endothelial cells: roles of LFA-1, VCAM-1, and ELAM-1 and changes in pathway hierarchy under different activation conditions.

T cell adhesion to endothelium is critical to lymphocyte recirculation and influx into sites of inflammation. We have systematically analyzed the role of four receptor/ligand interactions that mediate adhesion of peripheral human CD4+ T cells to cultured human umbilical vein endothelial cells (HUVEC): T cell LFA-1 binding to ICAM-1 and an alternative ligand ("ICAM-X"), T cell VLA-4 binding to VCAM-1, and T cell binding to ELAM-1. Contributions of these four pathways depend on the activation state of both the T cell and HUVEC, and the differentiation state of the T cell. ELAM-1 plays a significant role in mediating adhesion of resting CD4+ T cells to activated HUVEC. LFA-1 adhesion dominates with PMA-activated T cells but the strength and predominant LFA-1 ligand is determined by the activation state of the HUVEC; while ICAM-1 is the dominant ligand on IL-1-induced HUVEC, "ICAM-X" dominates binding to uninduced HUVEC. Adhesion via VLA-4 depends on induction of its ligand VCAM-1 on activated HUVEC; PMA activation of T cells augments VLA-4-mediated adhesion, both in the model of T/HUVEC binding and in a simplified model of T cell adhesion to VCAM-1-transfected L cells. Unlike LFA-1 and VLA-4, ELAM-1-mediated adhesion is not increased by T cell activation. Differential expression of adhesion molecules on CD4+ T cell subsets understood to be naive and memory cells also regulates T/HUVEC adhesion. Naive T cell adhesion to HUVEC is mediated predominantly by LFA-1 with little or no involvement of the VLA-4 and ELAM-1 pathways. In contrast, memory T cells bind better to HUVEC and utilize all four pathways. These studies demonstrate that there are at least four molecular pathways mediating T/HUVEC adhesion and that the dominance/hierarchy of these pathways varies dramatically with the activation state of the interacting cells and the differentiation state of the T cell.

Vascular cells respond differentially to transforming growth factors beta 1 and beta 2 in vitro.

Transforming growth factor beta 1 (TGF-beta 1) and beta 2 (TGF-beta 2) are equipotent in many cell systems studies thus far. Recent data, however, show different effects elicited by these two growth factors in specific biologic systems. This investigation compares the effects of TGF-beta 1 and TGF-beta 2 bovine aortic endothelial cells (BAECs), rat epididymal fat pad microvascular endothelium (RFCs), and bovine aortic smooth muscle cells (BASCs). In two-dimensional cultures, proliferation of BAECs, BASMCs, and RFCs were all inhibited by TGF-beta 1, while in response to TGF-beta 2, BASMCs were fully inhibited, RFCs were modestly inhibited, and BAECs were unaffected. Bovine aortic endothelial cell migration was significantly inhibited by TGF-beta 1, but only slightly inhibited by TGF-beta 2. In contrast, BASMC migration was enhanced by TGF-beta 1 and was not affected by TGF-beta 2. In three-dimensional cultures, RFCs were stimulated to undergo in vitro angiogenesis in response to TGF-beta 1 and TGF-beta 2 at 10-fold higher concentrations. Three distinct receptor assays demonstrated the presence of type I and type II TGF-beta 1 cell-surface-binding proteins on BAECs, BASMCs, and RFCs. Labeled TGF-beta 1 was competed off completely with 100-fold molar excess unlabeled TGF-beta 1, but only partially with equivalent excess unlabeled TGF-beta 2. Furthermore the ratios of type I to type II TGF-beta receptors in these three vascular cell types vary from 1:1 in BAECs to 1.5:1 in RFCs to 3:1 in BASMCs and can be correlated with the differences noted in cellular responses to TGF-beta 1 and TGF-beta 2 in proliferation, migration, and in vitro angiogenic assays. These findings support the hypothesis that there are different responses to the TGF-beta s, depending on the cell type and experimental conditions as well as the TGF-beta concentration and isoform used.

T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1.

The action of human rIL-1 beta on confluent, quiescent monolayers of human umbilical vein endothelial cells (HUVEC) has been studied for the induction of new membrane proteins. Two approaches have been taken. The first is a quantitative two-dimensional gel analysis of [35S]cysteine-labeled membrane proteins of HUVEC with and without cytokine treatment. This analysis indicates that there are a restricted number of new membrane proteins synthesized in the first 6 h of IL-1 treatment, on the order of 19 out of a total of over 600 detectable proteins. Second, we have prepared two mAb (1E7 and 2G7) to different epitopes of a major inducible sialoglycoprotein with molecular mass of 114 kDa and an isoelectric point of 4.6 to 4.8. These antibodies were compared with two additional antibodies, 3B7 and 7A9, which were shown to react with the endothelial leukocyte adhesion molecule-1 (ELAM-1) protein as expressed in COS cells. The 1E7/2G7 protein is distinct from ELAM-1, based upon biochemical comparisons as well as the inability of the 1E7 and 2G7 antibodies to react with ELAM-1-transfected COS cells. The protein defined as 1E7/2G7 is neither expressed constitutively nor in an inducible manner on PBMC, granulocytes, platelets, fibroblasts, or keratinocytes. The 7A9 and 3B7 antibodies are shown to block granulocyte binding to IL-1-activated HUVEC. The 2G7 antibody is effective at inhibiting the binding of T cells but not granulocytes to IL-1-activated endothelium, suggesting this new protein is an adhesion protein that may be active in vivo in T cell-endothelial cell adhesion-related events such as inflammation or lymphocyte recirculation. In addition, T cells were shown to utilize the ELAM-1 protein in binding to cytokine-activated HUVEC. Antibodies directed to both proteins had additive effects on inhibition of T cell adhesion.

Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. Structure-function analysis and detection of specific binding to cultured bone cells by flow cytometry.

The synthetic bovine parathyroid hormone (PTH) analog (Nle8, Nle18, Tyr34) bovine PTH(1-34)amide (bPTH(1-34)amide) was reacted with biotinyl-epsilon aminocaproic acid-N-hydroxysuccinimide under conditions which yielded five isoforms which were fractionated by a combination of reversed phase and ion-exchange chromatography. These reaction products were analyzed by automated Edman degradation in a manner which allowed us to specify the location and number of biotin residues on picomole quantities of hormone. The ability of each of these isoforms to induce a rise in intracellular cAMP in the ROS 17/2.8 cell line allowed us to evaluate the effect on function of biotinylation at different residues. Derivatized PTH molecules which contained a single biotin at either lysine 13, lysine 26, or lysine 27 possessed full biological activity. However, bioactivity was significantly reduced when position 13 plus either lysine 26 or 27 were biotinylated. Biological activity was lost when all 3 lysine residues were biotinylated. Biotinylation of the alpha-NH2 group of alanine at the NH2 terminus also resulted in a total loss of activity. Hence, unlike the effect of altering the alanine at position 1, modification of a single lysine residue at positions 13, 26, and 27 has a less critical effect on biological activity of the molecule. However, biotinylation of all three lysines results in a biologically inert PTH derivative and suggests that changes in isoelectric point, hydrophobicity, or tertiary structure may strongly influence hormone function. A fully bioactive-mixture of isoforms was used to detect receptors on ROS 17/2.8 cells by flow cytometry using fluorescein isothiocyanate-avidin as a fluorescent indicator. Binding to cell surface receptors was saturable and could be inhibited by native bPTH(1-34) but not by transforming growth factor beta, calcitonin or insulin. Moreover, PTH receptors could also be detected on primary cultures of human bone cells and human fibroblasts.

Modulation of TGF-beta type 1 receptor: flow cytometric detection with biotinylated TGF-beta.

Transforming growth factor beta type 1 (TGF-beta 1) was reacted with NHS-biotin to yield a derivative of TGF-beta 1 which was biotinylated on lysine residues. The biotinylated form of TGF-beta 1 was separated from the unreacted material by reverse phase chromatography. In three separate bioassays, the derivatized peptide was as active as the starting material. The use of FITC-avidin in conjunction with flow cytometry demonstrated that the binding of biotinylated TGF-beta 1 to its receptor is saturable, competable, and specific. A 100-fold molar excess of underivatized TGF-beta 1 gave 85% inhibition of binding of the biotinylated peptide to the mink lung cell line CCL-64, while TGF-beta 2 showed no inhibition of binding, nor did insulin, calcitonin, or TGF-alpha. Both CCL-64 cells and human umbilical vein endothelial cells showed a density-dependent down-regulation of receptor expression in culture. Several factors were examined that might mediate this effect. The down-regulation was shown not to be due to the secretion of an active form of TGF-beta 1. The extracellular matrix from high-density cells did not decrease expression of the receptor. Fibronectin, collagen, and gelatin were also unable to signal changes in receptor expression, even though in other systems such matrix components can regulate the responsiveness of cells to TGF-beta 1. Lastly, staining simultaneously for DNA content and TGF-beta 1 receptor expression showed that there was no correlation between cell cycle and receptor levels.