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The clinical significance of adhesions: focus on intestinal obstruction.

Postoperative adhesions occur after almost every abdominal surgery and are the leading cause of intestinal obstruction, accounting for more than 40% of all cases and 60% to 70% of those involving the small bowel. This contrasts with earlier experience in the Western World and current practice in the Third World, where abdominal operations are infrequent, hernias remain untreated, and strangulated hernia is common. These are among the findings of prospective and retrospective studies on adhesions conducted at the Westminster Medical School, University of London, London, UK, and of other published studies on the clinical consequences of postoperative intra-abdominal adhesions and resultant intestinal obstruction. In an analysis of 210 patients who had undergone at least one previous abdominal operation, 92.9% had postsurgical adhesions. This is not surprising, given the extreme delicacy of the peritoneum and the fact that apposition of two injured surfaces nearly always results in adhesion formation. Problems resulting from postsurgical adhesions create a considerable workload. At Westminster Hospital over 24 years, intestinal obstruction accounted for 0.9% of all admissions, 3.3% of major laparotomies and 28.8% of cases of large or small bowel obstructions. A 1992 British survey reported an annual total of 12,000 to 14,400 cases of adhesive intestinal obstruction. In 1988 in the United States, admissions for adhesiolysis accounted for nearly 950,000 days of inpatient care. Risk factors, such as type of surgery and site of adhesions, as well as timing and recurrence rate of adhesive obstruction, remain unpredictable or poorly understood. The type of surgery most frequently leading to adhesive obstruction includes colonic, and especially rectal surgery, appendicectomy, and gynecological procedures. Laparoscopy does not seem to eliminate the risk of adhesions and adhesive obstruction. Adhesions involving the small intestine occur less frequently than those involving the omentum, but are more likely to become obstructive. Follow-up of over 2,000 laparotomies at the Westminster Hospital demonstrated that 1% of patients developed adhesive obstruction within one year of surgery, and half of these occurred within the first postoperative month. However, obstruction may occur at any time, and some 20% of cases appeared more than 10 years later. Recurrent obstruction following adhesiolysis is common, but actuarial tables still need to be constructed. Adhesive obstruction is clinically challenging, since there is no simple way to differentiate between adhesive and strangulated obstructions. Mortality rates escalate from 3% for simple obstructions to 30% when the bowel becomes necrotic or perforated.

Abdomen↗

The role of soluble adhesion molecules in evaluating endothelial cell activation in preeclampsia.

OBJECTIVE: Adhesion molecules, such as vascular cell adhesion molecule 1, are known to be increased in the serum of patients with preeclampsia, indicating that these molecules are possible markers of endothelial cell activation. We investigated the influence of serum from women with preeclampsia on the expression of adhesion molecules by cultured endothelial cells. STUDY DESIGN: Human umbilical vein endothelial cells were cultured in Ham/Iscove modified Dulbecco's medium containing 20% pooled human serum, l -glutamine (200 mmol/L), penicillin, and streptomycin. We stimulated these cells for 24 hours with sera from patients with preeclampsia and then determined the levels of vascular cell adhesion molecule 1, intercellular cell adhesion molecule 1, E-selectin, and P-selectin in the supernatant and in the maternal serum by means of enzyme-linked immunosorbent assay. These results were compared with those of sera obtained from normotensive pregnant and nonpregnant women. In addition, the expressions of these adhesion molecules on the endothelial surface were determined by immunofluo-rescence microscopy. RESULTS: Only for vascular cell adhesion molecule 1 and E-selectin were elevated plasma levels found in hypertensive patients, whereas intercellular cell adhesion molecule 1 and P-selectin showed similar plasma levels in all the patients. No differences in the levels of the adhesion molecules were found between the supernatants of endothelial cell cultures after stimulation with sera from patients with preeclampsia and those after stimulation with normotensive control sera. In contrast, with immunofluorescence microscopy we could detect higher amounts of vascular cell adhesion molecule 1, intercellular cell adhesion molecule 1, and E-selectin on the endothelial surface after stimulation with sera from women with preeclampsia. CONCLUSION: Although vascular cell adhesion molecule 1 and E-selectin were elevated in maternal serum samples from women with preeclampsia and on the endothelial surface after stimulation with such sera, there were no detectable increases in the levels of both of these adhesion molecules in the supernatant of cultured endothelial cells. We therefore assume that sera from women with preeclampsia may cause endothelial cell activation. Because we could not detect elevated concentrations of any of the investigated adhesion molecules in the supernatant of endothelial cells, we believe that factors other than sera from women with preeclampsia seem to play a major role in the release of soluble forms of adhesion molecules from the endothelial membrane.

Adult↗

Are circulating adhesion molecules specifically changed in cardiac surgical patients?

BACKGROUND: Soluble adhesion molecules are considered to be markers of inflammation, endothelial activation, or damage. This study was designed to assess whether adhesion molecules are specifically altered in patients undergoing cardiac surgical procedures. METHODS: Three groups of 20 patients each were prospectively studied: patients undergoing elective coronary artery bypass grafting; patients scheduled for a Whipple pancreatoduodenectomy; and patients undergoing elective pneumonectomy for lung cancer. Plasma levels of soluble adhesion molecules (endothelial leukocyte adhesion molecule-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and granule membrane protein 140) were measured from arterial blood samples after induction of anesthesia (baseline), at the end of the operation, 2 hours and 5 hours after operation, and on the morning of the first postoperative day. RESULTS: Duration of operation was longest in the group having a Whipple operation (289 +/- 50 minutes) and did not differ between the other two groups. Plasma levels of all measured adhesion molecules at baseline were within normal ranges. After cardiopulmonary bypass, levels of adhesion molecules were significantly increased in the cardiac surgical patients (soluble endothelial leukocyte adhesion molecule-1, from 38 +/- 11 ng/mL at baseline to 68 +/- 12 ng/mL; soluble intercellular adhesion molecule-1, from 241 +/- 50 ng/mL to 498 +/- 78 ng/mL; and granule membrane protein 140, from 69 +/- 12 ng/mL to 150 +/- 25 ng/mL). On the morning of the first postoperative day, all levels had returned to baseline except that of soluble vascular cell adhesion molecule-1, which was still elevated (p < 0.05). In both the other groups, concentrations of adhesion molecules remained almost unchanged. CONCLUSIONS: Cardiac operation was associated with increased plasma levels of soluble adhesion molecules, a finding indicating endothelial activation or dysfunction. In contrast, in patients undergoing complex, long-lasting abdominal or lung operations, soluble adhesion molecules remained unchanged. Activation of proinflammatory cascades, ischemia/reperfusion phenomenon, and microcirculatory dysfunction appear to be the most likely reasons for this difference between groups. Whether modulation of adhesion molecules may influence organ function after cardiopulmonary bypass remains to be elucidated in further studies.

Aged↗

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.

The attachment, spreading, spreading rate, focal contact formation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) were investigated on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR). This approach was used to provide substrates that were adhesive to cells even in the absence of serum proteins and with no prior pretreatment of the surface with proteins of the cell adhesion molecule (CAM) family. This approach was used to dramatically enhance the cell-adhesiveness of substrates that were otherwise cell-nonadhesive and to improve control of cellular interactions with cell-adhesive materials by providing stably bound adhesion ligands. Glycophase glass was examined as a model cell-nonadhesive substrate prior to modification, and polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) were examined as representative materials for biomedical applications. The peptides were surface-coupled by their N-terminal amine to surface hydroxyl moieties using tresyl chloride chemistry. Prior to peptide grafting, the PET and PTFE were surface hydroxylated to yield PET-OH and PTFE-OH. The PET-OH was less cell-adhesive and the PTFE-OH was much more cell-adhesive than the native polymers. Radioiodination of a C-terminal tyrosine residue was used to quantify the amount of peptide coupled to the surface, and these amounts were 12.1 pmol/cm2 on glycophase glass, 139 fmol/cm2 on PET-OH, and 31 fmol/cm2 on PTFE-OH. Although the glycophase glass did not support adhesion or spreading even in the presence of serum, the RGD- and YIGSR-grafted glycophase glass did support adhesion and spreading, even when the only serum protein that was included was albumin. Although PET and PTFE-OH supported adhesion when incubated in serum-supplemented medium, neither of these materials supported adhesion with only albumin present, indicating that cell adhesion is mediated by adsorbed CAM proteins. When these materials were peptide-grafted, however, extensive adhesion and spreading did occur even when only albumin was present. Since the peptide grafting is quite easily controlled and is temporally stable, while protein adsorption is quite difficult to precisely control and is temporally dynamic, peptide grafting may be advantageous over other approaches employed to improve long-term cell adhesion to biomaterials.

Biocompatible Materials↗

Adhesion molecules in autoimmune disease.

Leukocyte activation, circulation, and localization to inflammatory sites are dependent on adherence to molecules on other cells or to extracellular matrix ligands. Adhesion molecule expression and interactions are probably involved in initiation and propagation of autoimmune diseases. Adhesion molecules pertinent to the development of autoimmunity are the subject of this review. Material in this review was generated by a manual and a computerized search of medical literature pertaining to adhesion molecules and specific autoimmune diseases. Topics covered include adhesion molecule classification, regulation of adhesion, and characterization of adhesion receptors in specific autoimmune diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus, Sjögren's syndrome, autoimmune thyroid disease, multiple sclerosis, and diabetes mellitus. Adhesion molecules are classified into selectin, integrin, and immunoglobulin supergene family groups. Increased adhesion molecule expression and avidity changes occurring with cellular activation are the principal methods regulating leukocyte adhesion. Tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN-gamma), and interleukin-1 (IL-1) stimulate adhesion receptor expression on lymphoid and nonlymphoid tissues. Although differences between specific autoimmune diseases exist, key interactions facilitating the development of autoimmune inflammation appear to include L-selectin/P-selectin/E-selectin, lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1), very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1), and alpha 4B7/MadCAM or VCAM-1 adhesion. Administration of anti-adhesion molecule antibodies in experimental animal models of autoimmunity and in a preliminary trial with RA patients has been successful in preventing or reducing autoimmune disease severity. A vast array of adhesive interactions occurs between immunocompetent cells, endothelium, extracellular matrix, and target tissues during the evolution of an autoimmune disease. Further characterization of leukocyte migration patterns and adherence should clarify pathogenic processes in specific autoimmune diseases and identify potential therapeutic targets for their treatment.

Arthritis, Rheumatoid↗

Effect of adhesive retention of maxillofacial prostheses. Part 2: Time and reapplication effects.

STATEMENT OF PROBLEM: The success of most non-implant-retained extraoral prostheses depends on retention derived from skin adhesives. Part 1 of this study found that Skin-Prep Protective Dressing improved the retentive properties of adhesives and that Secure(2) Medical Adhesive was stronger than Epithane-3. Part 2 investigates the application of a second layer of adhesive to the prosthesis, which was earlier noted to improve retention at later time periods. PURPOSE: This study measured the force needed to remove silicone elastomer strips with Secure(2) Medical Adhesive from the skin of human subjects. Testing was performed before and after the removal of the strips and reapplication of the adhesive. MATERIAL AND METHODS: Secure(2) Medical Adhesive was painted on silicone rubber strips and placed in a nonsequential random order of the 3 variables to 3 sites on the ventral forearms of 21 human subjects and tested over an 8-hour period. The bond strength was measured at 0, 4, and 8 hours. After a reapplication of adhesive over the existing adhesive, additional bond strength measurements were made at 4 and 8 hours. Testing was at 10 cm/min in an Instron. All subjects had Skin-Prep coating applied before adhesive application. RESULTS: Bond strengths for both single applications and reapplications of the adhesive were greater at 0 hours and became significantly weaker after the 4- and 8-hour periods. The second application of the adhesive produced the strongest bonds when measured at 4 hours (110 N/m). Bonding was significantly higher at 8 hours if a second application of adhesive was applied at 0 or 4 hours. CONCLUSION: The results of this study indicate that the bond strength of silicone elastomer to skin decreased over an 8-hour interval. After removal of the silicone rubber strip and reapplication of Secure(2) Medical Adhesive over the existing adhesive, bond strengths increased.

Adhesiveness↗

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response.

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.

Animals↗

Role of the cytoskeleton in adhesion stabilization of human colorectal carcinoma cells to extracellular matrix components under dynamic conditions of laminar flow.

Adhesion stabilization of malignant cells in the microcirculation is necessary for successful metastasis formation. The adhesion of colon carcinoma cells to microcirculation extracellular matrix (ECM) components is mediated, in part, by integrins that can be intracellularly linked to cytoskeletal proteins. Thus the functional status of at least certain integrins can be regulated by complex interactions with cytosolic, cytoskeletal and membrane-bound proteins. Wall shear stress caused by fluid flow also influences cellular functions, such as cell morphology, cytoskeletal arrangements and cell signaling. Using a parallel plate laminar flow chamber dynamic adhesion of human HT-29 colon carcinoma cells to collagen was investigated and compared with cell adhesion under static conditions. Cells were pretreated with cytochalasin D, nocodazole, colchicine or acrylamide to disrupt actin filaments, microtubules or intermediate filaments. Disruption of actin filaments completely inhibited all types of adhesive interactions. In contrast, impairment of tubulin polymerization or disruption of intermediate filaments resulted in different effects on static and dynamic adhesion. Treatment with acrylamide did not interfere with dynamic cell adhesion, whereas under static conditions it partially reduced adhesion rates. Under dynamic conditions increased initial adhesive interactions between HT-29 cells and collagen were found after disruption of microtubules, and the adherent cells demonstrated extensive crawling on collagen surfaces. In contrast, under static adhesion disrupting microtubules did not affect cell adhesion rates. Cytochalasin D and acrylamide were found to inhibit Tyr-phosphorylation of FAK and paxillin, whereas microtubule disrupting agents at low but not high concentrations increased phosphorylation of these focal adhesion proteins. Our results revealed that cytoskeletal components appear to be involved in adhesion stabilization of HT-29 cells to ECM components, and hydrodynamic shear forces modulate this involvement. Tyr-phosphorylation of focal adhesion proteins, such as paxillin and FAK, appears to be a part of this cytoskeleton-mediated process.

Acrylamide↗

IL-4, but not tumor necrosis factor-alpha, increases endothelial cell adhesiveness for lymphocytes by activating a cAMP-dependent pathway.

IL-4 and TNF-alpha increase endothelial cell adhesiveness for PBL by promoting the expression of adhesion molecules. We investigated the intracellular cAMP involvement in the increased endothelial cell adhesivity induced by IL-4 or TNF-alpha. We showed that both IL-4 and TNF-alpha increased intracellular cAMP in endothelial cells (EC). Furthermore, dibutyryl-cAMP and forskolin (which increased intracellular cAMP) increased basic EC adhesivity for PBL. The co-stimulation of EC with cAMP elevating agents and TNF-alpha, but not IL-4, resulted in an additive increase in EC adhesiveness. 2',5' dideoxyadenosine, an inhibitor of adenylate cyclase, decreased PBL adhesion to IL-4- but not TNF-alpha-treated EC. Similarly, HA1004, a protein kinase A inhibitor, totally reversed the IL-4 but not TNF-alpha effect on EC adhesiveness, whereas H7, a protein kinase C inhibitor, did not antagonise cytokine-enhanced EC adhesivity. These results indicate that IL-4, but not TNF-alpha, uses a cAMP-dependent pathway to increase PBL adhesion. Furthermore, we showed that cAMP elevation in EC did not induce vascular cell adhesion molecule 1, the only identified adhesion molecule induced by IL-4, indicating that a rise in cAMP in EC promotes an as yet unidentified adhesion pathway. Our results show that IL-4 increases EC adhesiveness for PBL through activation of protein kinase A by promoting an unidentified adhesion pathway.

Cell Adhesion↗

Fluid and pharmacological agents for adhesion prevention after gynaecological surgery.

BACKGROUND: Pelvic surgery is associated with high rates of both de novo adhesion formation and adhesion reformation. Since subsequent fertility is reduced with increasing severity of periadnexal adhesions, pelvic adhesions will remain a clinical problem in infertility patients. Steroids, antihistamines and heparin were amongst the first substances to be advocated for adhesion prevention. More recently icodextrin 4%, hyaluronic acid agents and SprayGel have been used. This review aims to evaluate the role of fluid and pharmacological agents in the prevention of adhesions in fertility-conserving gynaecological surgery. OBJECTIVES: To investigate fluid and pharmacological agents for adhesion prevention when used as adjuvants during pelvic surgery. SEARCH STRATEGY: This review has drawn on the search strategy developed for the Menstrual Disorders and Subfertility Group. The following databases were searched: the Cochrane Menstrual Disorders and Subfertility Group Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE. SELECTION CRITERIA: Randomised controlled trials investigating the use of fluid and pharmacological agents to prevent adhesion formation after fertility-conserving gynaecological surgery. DATA COLLECTION AND ANALYSIS: Data extraction and analysis was performed independently by two authors (Metwally M and Watson A). Two-by-two tables were generated for dichotomous outcomes and expressed as odds ratios (OR) with 95% confidence intervals (CI). For continuous outcomes a standardised mean difference was used. MAIN RESULTS: There is no evidence of benefit from the use of steroids, dextran or other pharmacological agents in any of the outcomes. The use of hyaluronic acid agents may decrease adhesion formation (OR 0.31, 95% CI 0.19 to 0.51) and prevent the deterioration of pre-existing adhesions (OR 0.28 (95% CI 0.12 to 0.66). There is insufficient evidence for the use of icodextrin 4% or SprayGel as adhesion-preventing agents. None of the studied agents has been shown to improve the pregnancy rate when used as an adjunct during pelvic surgery. AUTHORS' CONCLUSIONS: The current evidence for the use of fluid and pharmacological agents for the prevention of adhesions is limited. There is no evidence on any benefit for improving pregnancy outcomes when pharmacological and fluid agents are used as an adjunct during pelvic surgery. There is insufficient evidence for the use of the following agents: steroids, icodextrin 4%, SprayGel and dextran in improving adhesions following surgery. There is some evidence that hyaluronic acid agents may decrease the proportion of adhesions and prevent the deterioration of pre existing adhesions. However, due to the limited number of studies available, this evidence should be interpreted with caution and further studies are needed.

Anticoagulants↗

Adhesion of T lymphocytes to human endothelial cells is regulated by the LFA-1 membrane molecule.

Human T lymphocyte adhesion to human endothelial cells is the initial event in T cell migration to areas of extravascular inflammation. The molecular basis for T cell-endothelial cell adhesion was investigated using two different cell-cell adhesion assays: a) a fluorescein cell-cell adhesion assay using nonadherent endothelial cells and fluorescein-labeled T lymphocytes, and b) a radionuclide cell-cell adhesion assay using adherent endothelial cells and 51Cr-labelled T cells. Both assay systems demonstrated comparable quantitative assessment of cell-cell adhesions. The assays were performed at 22 degrees C and adhesions were maximal at 30 min. The results of these adhesion assays confirmed previous reports that T cells adhere to endothelial cells. In addition, we have shown that T cells adhere only marginally to foreskin fibroblasts or bone marrow derived fibroblasts. T cell-endothelial cell adhesions were significantly stronger than either monocytes or B lymphoblastoid cells adhesion to endothelial cells. To demonstrate the molecular mechanisms involved in regulating T cell-endothelial cell adhesions, a panel of function-associated monoclonal antibodies (MAb) were tested for their ability to inhibit T cell adhesion. MAb reactive with the leukocyte surface glycoprotein LFA-1 significantly inhibited T cell-endothelial cell adhesions in both assay systems. In contrast, MAb directed at other surface antigens did not inhibit T cell adhesion. The involvement of the LFA-1 glycoprotein in T lymphocyte adhesion to endothelial cells suggest that the LFA-1 molecule may be important in the regulation of leukocyte interactions.

Antibodies, Monoclonal↗

The effect of different adhesives on vinyl polysiloxane bond strength to two tray materials.

STATEMENT OF PROBLEM: Although stock trays often provide mechanical retention for elastomeric impression materials, manufacturers typically recommend the use of an adhesive, whether a stock or custom tray is used. Universal adhesives for vinyl polysiloxane (VPS) are used in practice, but comparative bond strengths have not been reported. PURPOSE: The aim of this study was to evaluate the bond of 3 VPS materials with a methylmethacrylate autopolymerizing and a light-polymerizing tray material, using the adhesive recommended by the manufacturer of the impression material, and 2 universal adhesives (paint-on and spray-on). MATERIAL AND METHODS: Ten specimens per group, for a total of 180 flat 15 x 15 x 20-mm specimens, were made from the 2 tray materials and finished to 320 grit with silicon carbide paper. Four paint-on adhesives (Coltene, Caulk, Kerr VPS, or universal VPS) and 1 spray-on adhesive (Sili Spray) were applied and dried following manufacturers' instructions or for 10 minutes. Three impression materials, Affinis, Aquasil, and Take I, were automixed and injected into a perforated cylinder positioned in a universal testing machine. Tray specimens were positioned against the open cylinder end in contact with the VPS material. Tensile tests (cross-head speed 5 mm/min) were conducted until adhesive separation failure. Mean values and standard errors of the adhesive strength were recorded in MPa for each material combination. Data were first analyzed with Welch analysis of variance (ANOVA) to determine overall significance, and then with a 3-way ANOVA and the Tukey HSD test to make pairwise comparisons, with alpha=.05 for all testing. RESULTS: For all impression materials tested, the universal spray-on adhesive consistently demonstrated significantly lower bond strengths than all other adhesives (P<.05). Equivalent or significantly (P<.05) higher bond strength values were found for the universal paint-on adhesive for the 3 impression materials tested. CONCLUSION: The use of GC paint-on universal adhesive provided significantly higher adhesive values than those obtained with the adhesives supplied by the manufacturers of the impression materials tested, with the exception of the Kerr impression and adhesive material combination where no significant differences were found.

Adhesives↗

Thrombin causes increased monocytic-cell adhesion to endothelial cells through a protein kinase C-dependent pathway.

The coagulation protein thrombin has been shown to stimulate multiple endothelial-cell (EC) functions, including production of platelet-derived growth factor and of platelet-activating factor (PAF), and neutrophil adhesion. We have found that thrombin causes increased binding of monocytic cells (U937 cells and normal human monocytes) to cultured EC of various species. Maximum adhesion of monocytes to pig aortic EC occurred 6 h after thrombin treatment and remained elevated through 24 h. Stimulation of adherence by bovine alpha-thrombin was half-maximal at 15 units/ml, and reached a plateau at 50 units/ml. Catalytically inactive thrombin (phenylmethanesulphonyl fluoride-treated) had no effect on monocyte adhesion to EC. Heparin, but not the endotoxin antagonist polymyxin B, suppressed the stimulation of adhesion by thrombin without altering basal adhesion. Two lines of evidence suggested that protein kinase C (PKC) was involved in the intracellular signalling to increase monocyte adhesion to EC. First the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated monocytic-cell adhesion to EC at a dose consistent with stimulation of PKC (half-maximal response at 1-3 nM) and with a time course similar to that for thrombin stimulation (maximal by 4 h). Diacylglycerol, a physiological activator of PKC, also stimulated U937-cell adhesion to EC. Secondly, H7, a PKC inhibitor, completely blocked stimulation of monocyte adhesion to EC by thrombin or PMA. The structural analogue of H7, HA1004, which preferentially inhibits cyclic-AMP- and cyclic-GMP-dependent protein kinases, had no effect on stimulated monocyte adhesion. The PKC inhibitor also blocked the stimulation of monocyte adhesion to EC by interleukin-1 and endotoxin, but did not alter the basal level of monocyte binding to unstimulated EC. Thrombin stimulation of monocyte adhesion differed from the reported stimulation of neutrophil adhesion by thrombin in that the latter process reached a maximum in minutes rather than hours. In addition, neither PAF itself nor agents known to stimulate PAF production by EC, such as arachidonate and the Ca2+ ionophore A23187, had any effect on monocyte adhesion. These results demonstrate a PKC-dependent cytokine-like action of the coagulation protein thrombin in modulating monocytic-cell adhesion to EC, a phenomenon of potential importance in many pathological and physiological processes.

Animals↗

Effects of in vivo administration of G-CSF on neutrophil and eosinophil adhesion.

The effect in vivo of G-CSF on neutrophil and eosinophil adhesion was studied after subcutaneous administration to six healthy individuals of human recombinant glycosylated G-CSF (lenograstim) (3 micrograms/kg) for 6 consecutive days. Basal adhesion and adhesion to E-selectin. VCAM-1 and ICAM-1 of neutrophil and eosinophil granulocytes were measured selectively. During G-CSF administration neutrophil basal adhesion increased from 7.4 +/- 3.9% (mean +/- SD) to 55.8 +/- 12.9% and 23.2 +/- 4.4%, 4 and 7 d, respectively, after start of the administration. At the same time points eosinophil basal adhesion increased from 7.1 +/- 2.4% to 37.7 +/- 6.1% and 13.1 +/- 5.3%, respectively. When adhesion was measured in the presence of Mn2+, which increases the functional activity of integrins, an even higher increase of neutrophil and eosinophil basal adhesion was noted 4 and 7 d, respectively, after start of G-CSF administration. In parallel with the enhanced basal adhesion neutrophil adhesion to E-selectin and ICAM-1 and eosinophil adhesion to E-selectin. VCAM-1 and ICAM-1 were significantly (P < 0.05) increased after 4 d of G-CSF administration as was neutrophil cell surface expression of CD11b and CD18. In vitro G-CSF induced minimal changes of granulocyte basal adhesion and inhibition of the adhesion to E-selectin. 10 ng/ml TNF alpha significantly increased neutrophil and eosinophil basal adhesion and adhesion to VCAM-1 and ICAM-1. In summary, administration of G-CSF to healthy subjects induced enhanced adhesion of neutrophil and eosinophil granulocytes, probably mediated by an increase of the functional capacity of beta 1- and beta 2-integrins. The induction of increased levels of TNF alpha might be one mechanism behind the in vivo effect of G-CSF administration.

Cell Adhesion↗

Beta 1-integrin-mediated adhesion of melanoma cells to the propolypeptide of von Willebrand factor.

Cell-adhesion activity of the bovine propolypeptide of von Willebrand factor (pp-vWF) was assessed by means of an in vitro assay with several cell lines of both normal and tumor-cell origin. pp-vWF promoted adhesion and spreading of B16 mouse melanoma cells and G-361 human melanoma cells. However, it could not induce adhesion of any other cell lines tested including endothelial cells, normal fibroblasts, and tumor cells of sarcoma, carcinoma, neuroblastoma and leukemia origin. A monospecific polyclonal antibody against pp-vWF, but not against fibronectin, laminin, and von Willebrand factor (vWF), completely blocked the pp-vWF-mediated adhesion, indicating that the cell adhesion was due to the pp-vWF molecule and not due to possible contamination of these three well-known adhesive proteins. The cell-adhesion activity was also observed with human pp-vWF and, furthermore, the adhesion to both bovine and human pp-vWF was not affected by a peptide containing the Arg-Gly-Asp sequence while the peptide abolished the cell adhesion to vWF. The adhesion was completely dependent on Mg2+ and inhibited by Ca2+. Inhibition by an anti-(beta 1 integrin) mAb (4B4) indicates that the receptor for this protein belongs to the beta 1-integrin family. A monoclonal antibody (TC4) among several antibodies directed against bovine pp-vWF inhibited the B16 adhesion to immobilized pp-vWF. The epitope for this monoclonal antibody lies in a central 8-kDa portion of pp-vWF, suggesting that this region is important for the cell-adhesion activity. This idea was supported by the finding that purified 8-kDa fragment promoted adhesion of B16 cells in a concentration-dependent manner. As pp-vWF shows unique cell-type specificity in its adhesion activity, which is completely different from that of fibronectin, laminin, vWF and collagen, it may be a novel type of adhesive glycoprotein that utilizes a beta 1-integrin receptor.

Amino Acid Sequence↗

Antagonism of selectin-dependent adhesion of human eosinophils and neutrophils by glycomimetics and oligosaccharide compounds.

Early in inflammation, adhesion occurs between leukocytes and endothelium when selectins bind to sialyl Lewis X (sLex) and related oligosaccharides. We tested novel compounds that mimic sLex for their ability to inhibit selectin-mediated adhesion of human eosinophils and neutrophils in vitro. Neutrophils and eosinophils were isolated by density gradient centrifugation, and eosinophils were further purified by immunomagnetic negative selection. Adhesion to unstimulated or interleukin-1beta-stimulated (5 ng/ml, 4-6 h) umbilical vein endothelial monolayers was tested under static or rotating conditions, where adhesion is primarily E- or L-selectin dependent, respectively. P-selectin-dependent adhesion was tested on immobilized platelets treated with or without phorbol myristate acetate (10(-7) M, 10 min). Stimulus-induced adhesion was always at least 4-fold higher than without stimulus, and selectin dependence was confirmed with specific blocking monoclonal antibodies. E-selectin-dependent adhesion of eosinophils and neutrophils was inhibited by compound GM2296 (the concentration producing 50% inhibition of adhesion [IC50] approximately 0.5-1 mM). E-selectin-dependent adhesion of neutrophils, but not eosinophils, was also inhibited by another compound, sLex with a lipid tail (30 +/- 6% inhibition at 3 mM), whereas compound GM1292 slightly inhibited adhesion of both (23 +/- 5 and 20 +/- 6% inhibition, respectively, at 1 mM). L-selectin-dependent adhesion was more effectively inhibited by GM2296 (IC50 approximately 0.2-0.5 mM), although P-selectin-dependent adhesion was also inhibited (IC50 approximately 1 mM). Inhibition was reversible without affecting viability, and no effect was seen with these compounds in assays testing neutrophil adhesion to immobilized intercellular adhesion molecule-1. Thus, compound GM2296, a carbon-fucosylated derivative of glycyrrhetinic acid, inhibits E-, L-, and P-selectin-dependent eosinophil and neutrophil adhesion. The ability of these and perhaps other related glycomimetic compounds to interfere with the function of more than one type of selectin makes them desirable candidates as anti-inflammatory agents.

Antibodies, Monoclonal↗

Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V.

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.

Animals↗