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Biomedical subjects

W P Faulk

Publications and source records attributed to W P Faulk.

At least 19 recordsLinked to original sources
PubMed1995

Vascular endothelial growth factor expression in transplanted human hearts.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen thought to play an important role in coronary collateral vessel formation. We used immunocytochemistry to determine VEGF expression in biopsies (n = 283) of transplanted human hearts (n = 109) with and without microvascular fibrin. Measures of vascular fibrin, alpha 2 plasmin-inhibitor (a2Pl), macrophages, neutrophils, and serum cardiac troponin T titers were used to evaluate myocardial damage. Antibody to T lymphocytes was used to evaluate cellular rejection, and HLA-DR, ICAM-1, and PAL-E antibodies were used to assess endothelial cell activation and phenotypic changes in the microcirculation. No VEGF immunoreactivity was detected in control donor hearts without fibrin, but the proportion of biopsies demonstrating VEGF immunoreactivity increased significantly in allografts with increasing fibrin and a2PI reactivity (P = 0.0001). VEGF immunoreactivity was confined to areas of fibrin deposition and was associated with infiltrates of macrophages and neutrophils (P < 0.0001), but not with T cells (P = 0.10). Biopsies with fibrin/VEGF reactivity were associated with increased capillary endothelial cell HLA-DR, ICAM-1, and PAL-E reactivity. In a subset of patients, serum cardiac troponin-T values were greater in patients with VEGF-positive (n = 21) than VEGF-negative (n = 19) biopsies (P = 0.05). Nested RT-PCR demonstrated that biopsies with and without fibrin/VEGF immunoreactivities expressed VEGF121, VEGF165, and VEGF189 variants, with VEGF165 being the dominate variant. These results indicate that endogenous VEGF is expressed locally following vascular thrombosis and myocardial cell damage, and that VEGF expression may be related to endothelial cell activation and phenotypic changes found in the microcirculation of cardiac allografts.

Adult↗
PubMed1995

Vascular tissue plasminogen activator and the development of coronary artery disease in heart-transplant recipients.

BACKGROUND: An aggressive and potentially fatal form of coronary artery disease may develop after cardiac transplantation. We studied the role of vascular tissue plasminogen activator (t-PA), the primary mediator of fibrinolysis, in the development of this problem. METHODS: We studied 78 consecutive recipients of cardiac allografts over a five-year period, and we collected follow-up data over a mean (+/- SE) of 32.5 +/- 2.0 months. The patients were studied with ventricular function tests, serial endomyocardial biopsies (16.6 +/- 0.5 per patient), and annual coronary angiography. Measurements of t-PA and its inhibitor were performed immunocytochemically on unfixed cryostat sections of endomyocardial-biopsy specimens with the use of monoclonal antibodies to t-PA and its inhibitor. RESULTS: In biopsy specimens obtained during the first three months of follow-up, 38 allografts had a normal distribution of t-PA in arteriolar smooth-muscle cells, whereas in 40 allografts there was depletion of t-PA that persisted in subsequent follow-up. Coronary artery disease developed during follow-up in 31 of 40 allografts (78 percent) with depletion of t-PA, but the disease developed in only 9 of the 38 allografts (24 percent) with normal t-PA levels (P < 0.001). Allografts with depletion of t-PA also had the t-PA inhibitor and were at greater risk for earlier and more severe disease than were allografts with normal arteriolar t-PA levels. Twelve patients whose allografts were depleted of t-PA either received a second transplant or died, whereas only one of the patients whose allografts had persistently normal t-PA levels died (P < 0.001). CONCLUSIONS: These findings reveal an association between the depletion of t-PA from arteriolar smooth-muscle cells and the subsequent development of coronary artery disease and decreased graft survival. Although we cannot be certain about a cause-and-effect relation, our data suggest a possible role for deficient fibrinolysis in the development of coronary artery disease in transplanted human hearts.

Coronary Angiography↗
PubMed1995

Heparan-dependent endothelial antithrombin binding is increased by butyrate.

Heparin biosynthesis involves a critical early step of N-deacetylation which is inhibited by the short chain fatty acid n-butyrate. Such inhibition causes mast cells to produce heparins with high affinity for antithrombin (AT). We have cultured endothelial cells in media supplemented with short chain fatty acids and have found that isobutyric, propionic and valeric acids cause significant increases in endothelial binding of AT measured by flow cytometry, but n-butyric acid was the most effective fatty acid to increase AT binding. Such binding,was heparan sulfate-dependent, for it was decreased significantly by pre-treatment of the cells with heparinase. These findings suggest that inhibition of N-deacetylation in heparan biosynthesis affects sulfation and results in the distribution of negative charges and conformation changes within the heparan domain that binds AT to endothelial plasma membranes. These changes also were associated with up-regulation of the intercellular adhesion molecule-1, which is a marker of endothelial activation.

Anticoagulants↗
PubMed1995

Antithrombin binding by human umbilical vein endothelial cells: effects of exogenous heparin.

Human umbilical vein endothelial cells cultured in growth media that did not contain exogenous heparin were found to grow less well while binding significantly more antithrombin (AT) than comparable cells cultured in growth media that did contain exogenous heparin (90 micrograms/ml). The binding of AT to plasma membranes of cultured endothelial cells was measured immunologically by flow cytometry. This binding was eliminated completely by reacting the cells with heparinase III before incubating them with AT, indicating that the most likely heparinase-sensitive process responsible for AT binding to plasma membranes was heparan sulfate proteoglycan. Increased AT binding also was promoted by addition of heparin-binding molecules (protamine, AT, or ECGF) to growth media, and the effects of other glycosaminoglycans and dextran on AT binding were found to be dependent on their sulfation. Thus, one response of endothelial cells to heparin deficiency is up-regulation of the ability to bind AT to plasma membranes.

Antithrombins↗
PubMed1995

Antiphospholipid antibodies in heart transplant recipients.

Antiphospholipid antibodies (aPA) are found in patients with systemic lupus erythematosus, aPA syndrome, myocardial infarction, and stroke. The presence of aPA may predict recurrent events in certain victims of heart attack and stroke. Blood samples from 105 cardiac transplant recipients (81 men, 24 women) were tested by enzyme-linked immunosorbent assay (ELISA) for the presence of IgG, IgM, and IgA aPA to phosphatidylserine (PS), cardiolipin (CL), and phosphatidylethanolamine (PE). Patients' ages ranged from 17 to 70 years (mean 51 years). Collection times ranged from Day 1 to 9 years post transplant (mean 36 months). All patients received triple immunosuppressive therapy. We report our aPA ELISA results in multiples of the normal median (MoM) of the OD405 values calculated for 252 healthy individuals. A positive MoM is greater than the MoM that encompasses 95% of the controls; for example, above 3 MoM is considered positive for IgG anti-CL, IgA anti-PS, and CL. Above 4 MoM is positive for IgG anti-PS and PE and IgM anti-PS and CL. Thirty-nine patients had IgG anti-PS (range 4.1-14.8 MoM), 63 had IgG anti-CL (3.1-9.4 MoM), 7 had IgM anti-PS (4.1-12.1 MoM), 1 had IgM anti-CL (14 MoM), 47 had IgA anti-PS (3.1-13.1 MoM), and 58 had IgA anti-CL (3.1-11.5 MoM). In our patient population, the incidence of IgG and/or IgA aPA was significantly higher (p < 0.001) than IgM. Few patients showed specificity for either PS, CL, or PE, and many were positive with more than one antibody isotype. Because aPA were evaluated in these patients, we investigated pretransplant serum samples which are available from 79 of the 105 recipients, and found aPA in 52 of 79 (66%) patients before transplantation. Longitudinal studies were done in three patients: two had increasing IgA aPA, beginning on Days 13 and 26 post transplant, whereas the third patient showed an increased aPA on Day 8 but a decrease on Day 23. Studies are in progress to determine whether a correlation exists between the presence of aPA, immunocytochemical (biopsy) findings, and clinical outcome.

Adolescent↗
PubMed1995

Intercellular adhesion molecule-1 (ICAM-1) and HLA-DR antigens are expressed on endovascular cytotrophoblasts in abnormal pregnancies.

PROBLEM: We asked if the lack of normal trophoblastic invasion of spiral arteries in the basal plate of abnormal pregnancies was associated with the expression of HLA-DR antigens and intercellular adhesion molecules (ICAM-1) on endovascular cytotrophoblasts. METHOD: The basal plates of placentae from 15 normal and 55 abnormal pregnancies, including preeclampsia, small-for-gestational age infants, and mothers with history of secondary recurrent spontaneous abortion, were studied immunocytochemically by using monoclonal antibodies to HLA-DR and ICAM-1. Spiral and uteroplacental arteries were identified by using a triple antibody technique with antibodies to cytokeratin, alpha-smooth muscle actin, and von Willebrand factor to detect cytotrophoblasts, arterial smooth muscle cells, and endothelium, respectively. RESULTS: Placentae with normal placentation showed the presence of uteroplacental arteries that contained endovascular cytotrophoblasts that were negative for HLA-DR and ICAM-1 antigens. Placentae from abnormal pregnancies showed the presence of spiral arteries without trophoblastic invasion and uteroplacental arteries that were surrounded by numerous macrophages and T lymphocytes. Endovascular cytotrophoblasts in uteroplacental arteries of placentae from abnormal pregnancies reacted with antibodies to HLA-DR and ICAM-1 antigens. CONCLUSION: Placentae from normal pregnancies show uteroplacental arteries that contain endovascular cytotrophoblasts that do not react with antibodies to ICAM-1 and HLA-DR antigens, and placentae from abnormal pregnancies with uteroplacental arteries that are associated with arteries that do not show physiological changes contain endovascular cytotrophoblasts that react with antibodies to ICAM-1 an HLA-DR antigens. Normal uteroplacental arteries were found to be not surrounded by round cell infiltrates, but uteroplacental arteries associated with arteries that lack physiological changes were surrounded by round cell infiltrates, indicating that round cell infiltrates and endovascular cytotrophoblasts which react with antibodies to ICAM-1 and HLA-DR antigens are associated with abnormal pregnancies. These findings suggest that the cellular infiltrates are associated with endovascular cytotrophoblasts that react with ICAM-1 and HLA-DR antigens.

Abortion, Habitual↗
PubMed1995

Maternal cells in chorionic villi from placentae of normal and abnormal human pregnancies.

PROBLEM: We asked if activated macrophages and CD4 positive T lymphocytes in placental chorionic villi with villitis were of maternal or fetal origin. METHOD: We employed a double antibody immunocytochemical technique on placental sections from three normal and four abnormal pregnancies with small-for-gestational-age infants. All studied placentae were mismatched for the maternal-fetal HLA-DRw 52 antigen. Areas of immunopathology were identified by using a monoclonal antibody to a monomorphic determination on HLA-DR, and the origin of immunological cells in areas of immunopathology was identified by using a monoclonal antibody to a polymorphic determinant on HLA-DRw 52. RESULTS: We used a double antibody technique that employed monoclonal antibodies to HLA-DR and HLA-DRw 52 antigens and placentae that were mismatched for the maternal-fetal HLA-DRw 52 antigen. We found that the vast majority of immunological cells within villi with inflammation were of maternal origin. Quantitative studies showed that between 75 and 100% of the cells in normal as well as in abnormal pregnancies were of maternal origin, and that abnormal pregnancies had a significantly higher percentage of villi with maternal cellular infiltrates. CONCLUSION: Our data show unequivocally that cells in areas of placental immunopathology are predominantly of maternal origin, and that abnormal pregnancies are associated with significantly more villi containing immunological cells of maternal origin.

Adult↗
PubMed1994

Natural anticoagulant and fibrinolytic pathways in renal allograft failure.

This is an immunocytochemical study of the relationship between depletion of natural anticoagulant and fibrinolytic pathways and allograft survival following renal transplantation. Patients (n = 44) were classified in three groups according to the length of time between transplantation and allograft failure: group 1 (n = 14) failed within a month of transplantation; group 2 (n = 14) failed between one month and one year after transplantation; and group 3 (n = 16) failed after one year of transplantation. Control biopsies were from donor kidneys (n = 16) prior to transplantation. There were no statistically significant differences in recipient age, gender, donor kidney type (living-related versus cadaver), histocompatibility, and plasma cholesterol, triglycerides, or creatinine concentrations between groups. However, group 1 allografts had a greater depletion of the vascular heparan sulfate proteoglycan-antithrombin III natural anticoagulant pathway than allografts in group 2 or 3 (P < or = 0.05), and this depletion was associated with significantly greater fibrin deposition in group 1 than in either group 2 or 3 (P < or = 0.05). All three groups demonstrated severe depletion of tissue plasminogen activator from arteriolar smooth muscle cells and depressed fibrinolysis as evidenced by increased fibrin/plasmin ratios. However, no significant differences were found for either endothelial thrombomodulin or T cell, neutrophil, or macrophage infiltration between the groups. These data indicate that differences in graft outcome may be determined more by compromised vascular function than by the presence of cellular infiltrates.

Adult↗
PubMed1994

Antigenic identification of cells in spiral artery trophoblastic invasion: validation of histologic studies by triple-antibody immunocytochemistry.

OBJECTIVE: Trophoblastic invasion of spiral arteries in the placental bed is essential for normal human placentation. Because the absence of these physiologic changes is associated with abnormal pregnancies, we developed a simple immunocytochemical approach that allows simultaneous study of three cell types involved in spiral artery changes (i.e., trophoblastic, endothelial, and smooth muscle cells). STUDY DESIGN: Spiral and uteroplacental arteries in the basal plate of 70 term placentas were studied simultaneously with antibodies to keratin to identify cytotrophoblasts, alpha-smooth muscle actin to identify vascular smooth muscle cells, and von Willebrand factor to identify endothelial cells in 15 normal placentas and 55 placentas from abnormal pregnancies. RESULTS: Normal term placentas showed keratin-positive, alpha-smooth muscle actin-negative and von Willebrand factor-negative uteroplacental arteries. Most (37/55) placentas from abnormal pregnancies had keratin-negative, alpha-smooth muscle actin and von Willebrand factor-positive spiral arteries. Spiral artery atherosclerosis was keratin negative, alpha-smooth muscle actin positive, and von Willebrand factor positive and was identified in 22 abnormal and 0 normal placentas. CONCLUSION: Simultaneous identification of three principal cell types involved in human placentation allowed qualitative and quantitative studies of placentas from normal and abnormal pregnancies that validated previous histologic investigations and provided a new approach to detect spiral arterial changes.

Abortion, Habitual↗
PubMed1994

Increase of lymphocytes expressing Fc-receptor class III (CD16) by exposure to human seminal plasma.

OBJECTIVE: To characterize conditions for the seminal plasma-induced increase of lymphocytes that express immunoglobulin G-Fc receptor class III (Fc gamma RIII). DESIGN: Peripheral blood lymphocytes were incubated in diluted seminal plasma or control media. Cells expressing Fc gamma RIII were quantified by using flow cytometry and fluorochrome-labeled monoclonal antibodies specific for Fc gamma RIII. The influence of incubation time, temperature, and seminal plasma concentration was investigated. Physical properties of the active seminal plasma substance were characterized by studying effects of dialysis and ultracentrifugation. The origin of the active seminal plasma substance was investigated by studying activity of autopsy materials from male accessory glands. Identity of cells that are influenced by seminal plasma activity was investigated by using two-color flow cytometry with monoclonal antibodies specific for Fc gamma RIII and different phenotypic markers of leukocytes. RESULTS: Incubation of lymphocytes in seminal plasma significantly increased percentages of cells expressing Fc gamma RIII. Maximal increases were observed after seminal plasma incubation at 37 degrees C for 90 to 120 minutes and increases were significantly correlated with seminal plasma concentration. Seminal plasma activity was not altered by ultracentrifugation (100,000 x g for 30 minutes) but completely removed by dialysis (12,000 to 14,000 pore size). Fc gamma RIII-positive lymphocytes markedly increased after incubation in prostatic but not in seminal vesicle secretions. Two-color flow cytometry showed that increases of Fc gamma RIII-expressing cells occurred within the subset of CD56-positive natural killer (NK) cells. CONCLUSIONS: Dialyzable compounds of prostatic origin induce significant increases of NK cells expressing Fc gamma RIII. These findings might reflect a novel regulatory mechanism acting on CD56-positive cells within the female reproductive tract after insemination.

Analysis of Variance↗
PubMed1994

Tissue plasminogen activator, plasminogen activator inhibitor-1, and fibrin as indexes of clinical course in cardiac allograft recipients. An immunocytochemical study.

BACKGROUND: Tissue-type plasminogen activator (TPA) is the principal activator of plasminogen. Since hemostasis in the microcirculation of allografts is a well-recognized complication of transplantation, we asked (1) whether the distribution and amount of cellular TPA in biopsies of transplanted human hearts are associated with fibrin deposits in and around the microcirculation, (2) whether such changes involve the physiological inhibitors of TPA and plasmin, and (3) whether the presence of these activators and inhibitors of fibrinolysis in tissue is correlated with clinical outcome. METHODS AND RESULTS: We immunocytochemically quantified the presence of fibrin, plasmin, TPA, and the TPA inhibitor PAI-1 in 938 biopsies from 68 consecutive cardiac allografts over a 54-month period. The localization, distribution, and quantification of TPA in arteriolar smooth muscle cells revealed that 35 of the 68 allografts maintained vascular TPA reactivity consistent with time-zero biopsies of autologous donor hearts: this was designated as the normal TPA group. In contrast, 33 of the 68 allografts significantly lost vascular TPA reactivity compared with time-zero biopsies of autologous donor hearts: this was designated as the depleted TPA group. Analysis of sequential biopsies from both groups during 54 months revealed that the mean cumulative quantitative TPA value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.9 +/- 0.02 (P = .0001), and the mean cumulative quantitative fibrin value for the normal TPA group was 1.0 +/- 0.01, whereas the depleted TPA group value was 1.5 +/- 0.05 (P = .0001). Biopsies of allografts in the depleted TPA group contained endothelial reactivity for TPA-PAI-1 complexes, whereas biopsies from the normal TPA group did not. Plasmin-associated molecules were rarely identified in biopsies of the normal TPA group but were present in the depleted TPA group, and the fibrin-to-plasmin ratio in the normal TPA group always was less than the fibrin-to-plasmin ratio in biopsies from the depleted TPA group. Analysis of demographic and risk factors revealed no significant differences between patients in the normal and depleted TPA groups, but none of the 35 patients in the normal TPA group died or were retransplanted, and 13 of the 33 patients in the depleted TPA group died or required retransplantation (P = .0001). CONCLUSIONS: Time-zero hearts (n = 68) and 34 of 38 stable allografts contained immunocytochemically detectable TPA only in vascular smooth muscle cells. Twenty-nine of 30 patients with normal TPA in their time-zero biopsies who subsequently developed a poor clinical outcome were found to have depleted TPA in biopsies evaluated during their first postoperative month and remained depleted throughout the study. Of 33 patients with depleted TPA, 39% died or required retransplantation. Depleted arteriolar TPA associated significantly with vascular and interstitial deposits of fibrin, plasmin, and endothelial TPA-PAI-1 complexes. These findings indicate that hemostatic and fibrinolytic pathways are activated in falling allografts, and they reveal evidence of depleted TPA before clinical or histopathological signs of failure. Patients with such allografts were found to be at high risk of death independently of other widely used clinical/laboratory parameters of prediction.

Biopsy↗
PubMed1993

Transferrin receptors associate with drug resistance in cancer cells.

Transferrin mediates its growth promoting properties through transferrin receptors, which are increased on cancer cells. We have studied drug-sensitive and drug-resistant cancer cells by using quantitative flow cytomerty with fluorochrome labelled transferrin and antibody to transferrin receptor, and we have found that drug-resistant cells have more transferrin receptors than drug-sensitive cells. This finding was confirmed and extended by using isotopically labelled ligand and by correcting for differences in cell surface areas. We also found that the number of transferrin receptors could be down-regulated by calcium channel blockers, and that such down-regulation diminished drug resistance, suggesting a hitherto unrecognised role for transferrin receptors in drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Adriamycin conjugates of human transferrin bind transferrin receptors and kill K562 and HL60 cells.

Adriamycin (ADR) was coupled to human transferrin (TRF) by using a glutaraldehyde crosslinking method. The TRF-ADR conjugates were separated by column chromatography and the molar ratio of ADR to TRF (i.e., conjugation number) for the studied conjugates was found to be 1.2. Analysis in sodium dodecyl sulfate-polyacrylamide gels demonstrated that TRF-ADR conjugates with this molar ratio had the same mobility as native TRF and contained few aggregates. The ADR remained conjugated to TRF under conditions of decreased pH known to occur in many intracellular compartments, and analysis by spectrofluorometry revealed that the conjugated ADR retained its ability to intercalate DNA. The TRF-ADR conjugates were shown by flow cytometry to preferentially bind tumor cells and cell-bound conjugates were found to be laterally mobile within plasma membranes. The binding of TRF-ADR conjugates was determined to be saturable, and competition experiments done with both radioiodinated and fluorescein-labeled TRF-ADR conjugates demonstrated dose-dependent inhibition of conjugate binding by unlabeled TRF, indicating that TRF-ADR conjugates were bound by TRF receptors. Cytotoxicity studies performed with tritiated thymidine incorporation and tetrazolium reduction assays revealed that TRF-ADR conjugates inhibited the proliferation of both K562 and HL60 cells in culture more effectively than free ADR. Such conjugates could provide a delivery system for ADR that would target the drug and possibly diminish its dose-associated complications.

Cell Survival↗