Biomedical subjects

R C Landis

Publications and source records attributed to R C Landis.

14 recordsLinked to original sources

PubMed2000

Cloning of porcine intercellular adhesion molecule-1 and characterization of its induction on endothelial cells by cytokines.

BACKGROUND: The transplantation of pig organs into humans requires a detailed knowledge of similarities and differences between the two species in the molecular physiology of host defense mechanisms. We therefore set out to identify porcine intercellular adhesion molecule (ICAM)-1 and to characterize its expression by endothelial cells. METHODS: Porcine ICAM-1 cDNA was isolated from an endothelial cell cDNA library. An anti-pig ICAM-1 monoclonal antibody was generated and used to investigate the regulation by cytokines of ICAM-1 expression by porcine aortic endothelial cells (PAEC), using flow cytometry. RESULTS: We found that porcine ICAM-1 was similar in primary structure to human ICAM-1, with five Ig-like domains. COS-7 cells transfected with porcine ICAM-1 supported beta2 but not alpha4 integrin-dependent adhesion of human T lymphoblasts. There was a low-level surface expression of ICAM-1 on unstimulated PAEC and increased expression after stimulation with tumor necrosis factor (TNF)-alpha. However expression of ICAM-1 seemed to be significantly lower than that of vascular cell adhesion molecule-1, both on unstimulated and TNF-alpha-activated PAEC. Recombinant porcine interferon-gamma weakly stimulated ICAM-1 expression when incubated alone with PAEC but had an inhibitory effect on the increase in ICAM-1 due to TNF-alpha, both at 8 and 24 hr. CONCLUSIONS: Our observations confirm the existence of ICAM-1 in the pig and provide novel insights into how porcine and human endothelial cells differ in terms of adhesion molecule expression and cytokine responsiveness. Such differences are potentially important in interpreting models of inflammation in the pig and also in understanding the process of rejection of porcine xenografts.

Amino Acid Sequence↗

PubMed2000

TNF-alpha, IL-4, and IFN-gamma regulate differential expression of P- and E-selectin expression by porcine aortic endothelial cells.

P- and E-selectin are surface glycoproteins that mediate leukocyte rolling on the surface of endothelium in inflammation. We have cloned porcine P-selectin cDNA and generated a mAb, 12C5, with which to examine P-selectin expression by porcine aortic endothelial cells (PAEC) in comparison with that of E-selectin. Basal expression by PAEC of P-selectin was greater than that of E-selectin, whereas E-selectin expression was more prominently enhanced than that of P-selectin by stimulation with TNF-alpha or IL-1alpha. Both human or porcine IL-4 led to an increase in P-selectin expression, with kinetics that were delayed compared with those seen following stimulation with TNF-alpha or IL-1alpha, but IL-4 did not stimulate expression of E-selectin. When cells were stimulated with TNF-alpha in the presence of IL-4, we observed enhanced P-selectin expression with a parallel reduction in E-selectin expression. Finally, the increase in P-selectin expression due to human IL-4 was reduced in the presence of porcine but not human IFN-gamma. These observations show that E-selectin and P-selectin expression are differentially regulated in PAEC, and that IL-4 leads to a shift in the relative surface density of the two molecules toward P-selectin. The ability of porcine IFN-gamma to inhibit IL-4-induced P-selectin expression suggests that the balance between Th1 and Th2 cytokine production may determine the relative densities of the two selectins in chronic immune-mediated inflammation. Because the increased expression of P-selectin induced by human IL-4 was not inhibited by human IFN-gamma, this balance may be shifted toward P-selectin expression in porcine xenografts infiltrated by human lymphocytes.

Amino Acid Sequence↗

PubMed2000

Noninflammatory phagocytosis of monosodium urate monohydrate crystals by mouse macrophages. Implications for the control of joint inflammation in gout.

OBJECTIVE: We have hypothesized that the process of monocyte to macrophage differentiation may alter the inflammatory response of mononuclear phagocytes to the uptake of monosodium urate monohydrate (MSU) crystals. METHODS: Eight mouse monocyte/macrophage cell lines were arranged in increasing order of differentiation, as judged by expression of the macrophage markers F4/80 and BM 8 and by phagocytic capacity. Secretion of tumor necrosis factor alpha (TNFalpha) in response to MSU was measured by enzyme-linked immunosorbent assay. RESULTS: The panel of monocyte/macrophage cell lines revealed a close linkage between the state of differentiation and the capacity of the cells to ingest MSU crystals. TNFalpha production, however, was not linked to phagocytic ability. Peak TNFalpha levels were synthesized by cells at an intermediate state of differentiation (3.2-14.1 ng/ml), whereas mature macrophages, which efficiently phagocytosed crystals, did not secrete TNFalpha. Mature cell lines produced TNFalpha when stimulated with zymosan (5.9-6.2 ng/ml), but this was abolished by coincubation with MSU crystals. Suppression of the zymosan response was not due to apoptosis or steric hindrance by MSU crystals. Culture supernatants from mature macrophages did not stimulate endothelial cell activation, in contrast to MSU-treated cells at an earlier stage of differentiation, which stimulated intercellular adhesion molecule 1 expression on sEND endothelioma cells through the release of TNFalpha (inhibited 80.6% by anti-TNFa). CONCLUSION: We demonstrated that phagocytosis and TNFalpha production are distinct events in the response of mononuclear phagocytes to urate crystals, and these events can be distinguished at the level of macrophage differentiation. The noninflammatory removal of urate crystals by mature macrophages defines a new pathway that may be important in controlling the development of acute gout in patients with hyperuricemia.

Animals↗

PubMed2000

Leukocyte integrin expression in patients undergoing cardiopulmonary bypass.

BACKGROUND: The recruitment of leukocytes to vascular endothelium is controlled by adhesion events mediated through the beta2 integrins, whereas the response of extravasated leukocytes within the tissues is controlled through the beta1 integrins. Although cardiopulmonary bypass (CPB) has been shown to be associated with a systemic inflammatory response and elevated levels of beta2 integrins on leukocytes, its effect on the beta1 integrins is not known. This study investigated the effect of the protease inhibitor aprotinin on the expression of the beta1 and beta2 integrins on circulating leukocytes in patients undergoing CPB. METHODS: Patients undergoing primary elective coronary artery bypass grafting were randomized into full-dose aprotinin or placebo groups. Blood samples were obtained at nine time points preoperatively, intraoperatively, and up to 6 days postoperatively. The surface expression of the beta1 integrins VLA-1, -3, -4, -5, and -6 and of the beta2 integrins CD11a/CD18, CD11b/CD18, and CD11c/CD18 was measured by flow cytometry on gated neutrophil and monocyte subpopulations in whole blood. RESULTS: Expression of the beta1 integrins was not significantly altered during the study period and, therefore, aprotinin had no effect on the expression of these molecules. Of the beta2 integrins, CD11b/CD18 expression was significantly increased on neutrophils at 15 minutes after onset of CPB in the placebo group (p < 0.01) but not in the aprotinin group. CONCLUSIONS: This study showed that expression of the beta1 integrins on neutrophils and monocytes did not alter during the first 6 days after CPB. Expression of the beta2 integrin CD11b/CD18 increased significantly on neutrophils during CPB in control patients but not in patients treated with full-dose aprotinin.

Aprotinin↗

PubMed2000

An anti-inflammatory property of aprotinin detected at the level of leukocyte extravasation.

BACKGROUND: Aprotinin is a serine protease inhibitor used extensively in cardiac operations to reduce postoperative bleeding. It has also been used in trials aimed at reducing the systemic inflammatory response to cardiopulmonary bypass. It remains unclear whether the anti-inflammatory action of aprotinin is related to its general ability to suppress leukocyte activation or whether aprotinin can exercise effects during the leukocyte-endothelial cell adhesion cascade. METHODS: We used intravital microscopy to study the 3 main stages of the adhesion cascade (leukocyte rolling, firm adhesion, and extravasation) within the mesenteric microcirculation of rats. This in vivo technique allows leukocyte recruitment to be viewed directly through the transparent mesentery of anesthetized animals. RESULTS: Aprotinin, given by continuous infusion at a clinically relevant dose, exerted no effect on the rolling or firm adhesion responses toward local chemoattractant N -formyl-methyl-leucyl-phenylalanine but significantly inhibited extravasation of leukocytes (73% at 40 minutes, P =.04) into surrounding tissues. In parallel in vitro experiments, aprotinin (used at 200, 800, and 1600 kIU/mL) dose dependently inhibited neutrophil transmigration through cultured endothelial cells in response to 3 different chemoattractants: N -formyl-methyl-leucyl-phenylalanine (P <.001 at 800 and 1600 kIU/mL), interleukin 8 (P <.05 at 200 kIU/mL and P <.001 at 800 and 1600 kIU/mL), and platelet-activating factor (P <.05 at 1600 kIU/mL). CONCLUSIONS: Our studies have therefore revealed a novel anti-inflammatory mechanism of aprotinin operating at the level of leukocyte extravasation. These findings may be relevant in the prevention of systemic inflammation after cardiopulmonary bypass through the use of protease inhibitors.

Analysis of Variance↗

PubMed2000

The antithrombotic effect of aprotinin: actions mediated via the proteaseactivated receptor 1.

BACKGROUND: Despite aprotinin being in widespread clinical use to prevent bleeding during cardiac surgery, there remains concern that such a powerful hemostatic agent may also be prothrombotic, particularly in relation to coronary vein graft occlusion. The major thrombin receptor on platelets, protease-activated receptor 1 (PAR1) requires proteolytic cleavage to transmit activating signals. Here we have investigated the effect of aprotinin on thrombin-induced PAR1 activation of platelets. METHODS AND RESULTS: Proteolysis-dependent and -independent responses of washed platelets were studied in vitro. Platelet aggregation induced by trypsin was dependent on PAR1 (inhibited by the PAR1-specific antagonist peptide, FLLRN) and was completely blocked by aprotinin at doses more than 100 KIU/mL. Aggregation in response to thrombin, 1 nmol/L, was predominantly mediated through PAR1 and was inhibited 42.6% to 86.6% (P <.05-.001) by pharmacologic doses of aprotinin (50-160 KIU/mL). Aprotinin did not inhibit the nonproteolytic agonists collagen, epinephrine, adenosine diphosphate, or phorbol 12-myristate 13-acetate. Furthermore, blockade of the thrombin response by aprotinin did not prevent subsequent platelet aggregation through collagen or epinephrine. Experiments with intraplatelet Ca(2+) fluxes, which provided an earlier measure of platelet activation, placed the effect of aprotinin proximal to the PAR1 activation event. Since aprotinin did not inhibit platelet responses to the nonproteolytic PAR1 agonist peptide, SFLLRN, this implied that aprotinin acted by preventing PAR1 receptor cleavage by thrombin. CONCLUSIONS: Aprotinin inhibits thrombin-induced platelet activation by preventing proteolysis of the PAR1 receptor. These findings argue against aprotinin being prothrombotic and suggest instead that aprotinin may have significant antithrombotic effects.

Analysis of Variance↗

PubMed2000

Inhibition of neutrophil L-selectin shedding: a potential anti-inflammatory effect of aprotinin.

The cardiopulmonary bypass (CPB)-related inflammatory response involves leucocyte activation and increased leucocyte-endothelial cell interaction. L-selectin is an adhesion molecule expressed on the surface of leucocytes which participates in the initial rolling step of the leucocyte-endothelial cell adhesion cascade. L-selectin is proteolytically cleaved off the surface of leucocytes when they become activated, an event that is regarded as a marker of leucocyte activation. Aprotinin is a protease inhibitor that has been used in cardiac surgery as a haemostatic agent and also exhibits certain anti-inflammatory properties. In this study, peripheral venous blood from volunteers was pre-incubated with aprotinin at 200, 800 and 1600 kallikrein inhibiting units (kiu)/ml and stimulated with the chemoattractants N-formyl-methyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF). Surface expression of L-selectin on neutrophils was measured using a monoclonal antibody and flow cytometry. The results demonstrate that aprotinin inhibits shedding of L-selectin in a dose-dependent fashion (p=0.0278 and 0.0005, respectively, at 800 and 1600 kiu/ml for fMLP-stimulated shedding; p=0.0017 and 0.0010, respectively, at 200 and 800 kiu/ml for PAF-stimulated shedding). This effect may be of significance with respect to the anti-inflammatory action of aprotinin in patients undergoing CPB.

Aprotinin↗

PubMed1994

Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3.

To analyze the binding requirements of LFA-1 for its two most homologous ligands, ICAM-1 and ICAM-3, we compared the effects of various LFA-1 activation regimes and a panel of anti-LFA-1 mAbs in T cell binding assays to ICAM-1 or ICAM-3 coated on plastic. These studies demonstrated that T cell binding to ICAM-3 was inducible both from the exterior of the cell by Mn2+ and from the interior by an agonist of the "inside-out" signaling pathway. T cells bound both ICAM ligands with comparable avidity. A screen of 29 anti-LFA-1 mAbs led to the identification of two mAbs specific for the alpha subunit of LFA-1 which selectively blocked adhesion of T cells to ICAM-3 but not ICAM-1. These two mAbs, YTH81.5 and 122.2A5, exhibited identical blocking properties in a more defined adhesion assay using LFA-1 transfected COS cells binding to immobilized ligand. Blocking was not due to a steric interference between anti-LFA-1 mAbs and N-linked carbohydrate residues present on ICAM-3 but not ICAM-1. The epitopes of mAbs YTH81.5 and 122.2A5 were shown to map to the I domain of the LFA-1 alpha subunit. A third I domain mAb, MEM-83, has been previously reported to uniquely activate LFA-1 to bind ICAM-1 (Landis, R. C., R. I. Bennett, and N. Hogg. 1993. J. Cell Biol. 120:1519-1527). We now show that mAb MEM-83 is not able to stimulate binding of T cells to ICAM-3 over a wide concentration range. Failure to induce ICAM-3 binding by mAb MEM-83 was not due to a blockade of the ICAM-3 binding site on LFA-1. This study has demonstrated that two sets of functionally distinct mAbs recognizing epitopes in the I domain of LFA-1 are able to exert differential effects on the binding of LFA-1 to its ligands ICAM-1, and ICAM-3. These results suggest for the first time that LFA-1 is capable of binding these two highly homologous ligands in a selective manner and that the I domain plays a role in this process.

Antibodies, Monoclonal↗

PubMed1993

Adhesion molecules in cell interactions.

During a successful immune response, several families of adhesion molecules participate in a cascade of binding events that lead to the binding of leukocytes, both to each other and to cell types such as the endothelium and epithelium. A central theme emerging from recent studies is that the function of an adhesion receptor cannot be inferred from its expression alone; rather, adhesion receptors are 'selected' to perform distinct effector functions based on their cell-background and factors present in the local microenvironment. Thus, adhesion receptors expressed on different cell-types may find themselves in different states of 'activation-readiness' and may be further selected by prevailing conditions in the microenvironment to bind tissue-specific ligands and mediate leukocyte effector functions such as homing or transendothelial migration.

Animals↗

PubMed1993

A novel LFA-1 activation epitope maps to the I domain.

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA-1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM-1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA-1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.

Antibodies, Monoclonal↗

PubMed1993

Control of leukocyte integrin activation.

The integrin receptors on leukocytes are transiently activated by "triggering" molecules that may be other leukocyte membrane structures such as the T-cell receptor complex or small molecules such as PAF, which bind to their own specific receptors. This "inside out" signaling is essential for high affinity integrin/ligand pairing. In the example of LFA-1/ICAM-1, binding is positively supported by Mg2+ but negatively supported by Ca2+. How specific divalent cations affect receptor activation and subsequent ligand binding has still to be fully understood. However, the fact that activation can be mimicked from outside the cell via special anti-LFA-1 monoclonal antibodies such as MEM-83 suggests that activated integrins undergo conformational changes. Further alteration occurs as a result of the interaction of integrin with ligand, and the resulting novel epitopes are named "ligand-induced binding sites." For a brief period of time the integrin/ligand complex is able to transmit signals from "outside in." The transient activation of leukocyte integrins determines that cell-cell adhesion will be short lived and serves the purpose of permitting recycling of effector cells with their targets.

Animals↗

PubMed1991

Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.

Antibodies, Monoclonal↗

PubMed1991

Detection of group A streptococci by aerobic culture and a new simplified immunoassay in three pediatric practices and a hospital laboratory.

Duplicate throat swabs for detection of group A streptococci were collected in three pediatric offices from 1,035 patients with symptoms of pharyngitis. In the collecting office and in the hospital laboratory, one swab from each patient was first inoculated to sheep blood agar (incubated at 35 degrees C aerobically for 2 days) and then tested for group A streptococcal antigen by using the SMART enzyme immunoassay technique (New Horizons Diagnostics Corp) incubated for up to 24 hours. Group A streptococci were recovered in culture (from one or both swabs) and serologically identified from 444 (42.9%) of the patients. Pediatric offices numbers 1, 2, and 3 detected 84.4%, 84.6%, and 82.2%, respectively, of their patients who had positive cultures (in the office and/or laboratory) by using their own culture system and 82.6%, 71.1%, and 84.9%, respectively, of these same patients by using the SMART technique. In the laboratory, SMART test sensitivity and specificity were 71.4% and 98.7%, respectively, after 5 minutes of test incubation. However, SMART test sensitivity improved to 86.5% after overnight incubation of the immunoassay and to 91.3% if the data from one defective lot of seven SMART production lots studied were excluded. SMART test results which are negative after 5 minutes of incubation should therefore be confirmed both by reincubation of the antigen test up to 24 hours and by culture.

Adolescent↗

PubMed1987

Performance of an enzyme immunoassay test and anaerobic culture for detection of group A streptococci in a pediatric practice versus a hospital laboratory.

The ability of pediatricians and hospital laboratory personnel to detect group A streptococci in patients with suspected streptococcal pharyngitis was evaluated using the TestPack Strep A and anaerobic culture. Duplicate throat specimens (for similar processing by both the pediatricians and laboratory technologists) were simultaneously collected on rayon-tipped swabs from patients with symptoms of pharyngitis. Each swab was first inoculated to a 5% sheep blood agar plate, then tested for group A streptococcus antigen using the TestPack Strep A according to the manufacturer's instructions. Cultures were incubated anaerobically at 35 degrees C for 2 nights unless positive after 1 night. Group A streptococci were identified using specific antisera. Pediatric office or laboratory cultures from 112 (31.3%) of the 358 patients contained group A streptococci. Of the patients with positive cultures, 96 (85.7%) and 107 (95.5%) were detected by the pediatricians and laboratory, respectively. Respective findings with the TestPack Strep A by the pediatricians and laboratory were sensitivity 68.8% and 74.8%, specificity 94.3% and 95.6%, predictive value of a positive result 81.5% and 87.9%, and predictive value of a negative result 89.2% and 89.9%. Anaerobic culture was significantly more sensitive than the TestPack Strep A for detection of group A streptococci by both the pediatricians (P less than 0.005) and laboratory personnel (P less than 0.05).

Adolescent↗