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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
PubMed1992

Cytotoxicity of a transferrin-adriamycin conjugate to anthracycline-resistant cells.

Conjugates of adriamycin coupled to transferrin by glutaraldehyde are cytotoxic to human promyelocytic (HL-60) and erythroleukemic (K562) cells. Growth inhibition of adriamycin-sensitive cells, as evaluated by thymidine incorporation and the MTT-assay, was higher for conjugates than for free adriamycin. The cytotoxicity toward adriamycin-resistant K562 and HL-60 cells was 3-fold and more than 10-fold higher, respectively, for the transferrin-adriamycin conjugate than for the free drug. The effect of the conjugate was dependent on its adriamycin content, i.e., on its conjugation number.

Doxorubicin↗
PubMed1992

Transferrin conjugates of adriamycin are cytotoxic without intercalating nuclear DNA.

Studies of the biological chemistry of most anticancer drugs have revealed their cytotoxicity is expressed after the drugs have entered cells. It is thought that anthracycline antitumor drugs exert their cytotoxicity by entering cells, diffusing into nuclei, and inhibiting topoisomerase II and/or intercalating DNA base pairs. In order to deliver anthracyclines to transferrin (TRF) receptors on the plasma membranes of human tumor cells, we have prepared conjugates of adriamycin (ADR) with human TRF. These TRF-ADR conjugates were found to be stable at low pH and to exert more efficient cytotoxicity than free drug. By using spectrofluorometry, we found that the fluorescence of ADR within the conjugate was quenched by native DNA, demonstrating the presence of conformationally available drug to intercalate with nuclear DNA. However, fluorescence was not quenched when conjugate was reacted with viable cells, indicating that ADR did not reach the nucleus. Results of fluorescence microscopy experiments confirmed that free but not conjugated ADR reached the nuclei of viable cells, and TRF-ADR conjugates labeled with fluorescein isothiocyanate were found to initiate lateral diffusion as determined by patch and cap reactions. The involvement of TRF receptors was shown by flow cytometry experiments in which native TRF inhibited binding of fluorescein-labeled TRF-ADR conjugates. These data suggest that TRF-ADR conjugates mediate cytotoxicity by a mechanism other than intercalation with nuclear DNA. This mechanism, revealed by conjugating ADR to a TRF carrier, may not initiate complications such as cardiotoxicity and drug resistance.

Cell Nucleus↗
PubMed1992

Inhibition of transplasma membrane electron transport by transferrin-adriamycin conjugates.

Transplasma membrane electron transport from HeLa cells, measured by reduction of ferricyanide or diferric transferrin in the presence of bathophenanthroline disulfonate, is inhibited by low concentrations of adriamycin and adriamycin conjugated to diferric transferrin. Inhibition with the conjugate is observed at one-tenth the concentration required for adriamycin inhibition. The inhibitory action of the conjugate appears to be at the plasma membrane since (a) the conjugate does not transfer adriamycin to the nucleus, (b) the inhibition is observed within three minutes of addition to cells, and (c) the inhibition is observed with NADH dehydrogenase and oxidase activities of isolated plasma membranes. Cytostatic effects of the compounds on HeLa cells show the same concentration dependence as for enzyme inhibition. The adriamycin-ferric transferrin conjugate provides a more effective tool for inhibition of the plasma membrane electron transport than is given by the free drug.

Animals↗
PubMed1992

The endothelial heparan sulfate-antithrombin III natural anticoagulant pathway in normal and transplanted human kidneys.

This is the first study of the antithrombin III-heparan sulfate natural anticoagulant pathway in human kidneys. Immunocytochemical experiments were done to demonstrate the pathway on normal renal endothelial cells. Enzymatic studies were done to show that the antithrombin III was anchored to endothelium by molecules of heparan sulfate. Displacement studies were done with glycosaminoglycans to show that the antithrombin III was bound to its glycosaminoglycan anchor via a heparinlike binding site, and replacement studies showed that antithrombin III could be returned to the same endothelial cells from which it was displaced. Immunocytochemical studies of biopsies showed that normally functioning renal allografts manifested the endothelial antithrombin III-heparan sulfate anticoagulant pathway. The pathway was compromised or absent from the microcirculation of biopsies from rejecting or rejected renal allografts, and the diminishment of endothelial ATIII was associated with the presence of fibrin deposition. It is concluded that compromise of the antithrombin III-heparan sulfate natural anticoagulant pathway results in compromised renal function in transplanted kidneys.

Adolescent↗
PubMed1992

Tissue factor in normal and transplanted human kidneys.

Tissue factor (TF) plays a central role in the initiation of blood coagulation that frequently is enhanced in renal allografts. The identification and localization of TF was studied immunocytochemically in biopsies from normal and transplanted human kidneys and classified according to its distribution. The clinical status of each allograft was then correlated with the TF classifications. From these correlations, four distributional types of TF were identified. In normal kidneys, TF was localized to glomerular epithelium and basement membranes. Glomerular TF expression did not colocalize with mesangial or endothelial HLA-DR reactivity as determined by double antibody techniques. Tissue factor in donor kidneys also was identified in the renal capsule and in the adventitia of large arteries. These structures were not reactive in long-term transplanted grafts. Some cadaver kidneys prepared for transplantation had depleted glomerular TF, and exhibited TF reactivity within stromal tissues. Long-term allografts with progressive loss of renal function and kidneys with advanced rejection exhibited diminished TF reactivity of glomerular epithelium and basement membranes. This was frequently associated with fibrin deposition within the glomeruli and in the intertubular microcirculation. These findings indicate that the evaluation of TF in transplanted kidneys is related to the prognosis of graft survival.

Adult↗
PubMed1992

Novel immunohistochemical markers of human renal allograft dysfunction--antithrombin III, Thy-1, urokinase, and alpha-smooth muscle actin.

We have studied the expression of alpha-smooth muscle actin (alpha sm-1) by mesangial cells, and the expression of Thy-1 glycoprotein, antithrombin III (ATIII), and urokinase by tubular epithelial cells in normal kidneys and dysfunctional renal allografts. Kidney biopsies were studied immunocytochemically for changes in each of these markers and the findings were classified into two groups and compared with creatinine plasma levels at the time the biopsies were taken. In dysfunctional grafts, mesangial alpha sm-1 and tubular epithelial Thy-1 reactivities were greatly diminished, and urokinase and ATIII were missing from proximal renal tubular epithelial cells. Urokinase, which was absent from normal renal glomeruli, appeared in glomeruli of some dysfunctional allografts. The possible usefulness of these markers in patient evaluations was supported by our finding that the distribution of vinculin, fibronectin, myosin, actin B4, desmin, glomerular HLA-DR, and the tubular expression of CD15 remained unchanged. These data prompt us to suggest that the immunocytochemical localization and evaluation of alpha sm-1, Thy-1, ATIII, and urokinase in kidney allografts may be useful adjuncts in the assessment of function in renal allografts.

Actins↗
PubMed1992

Vascular immunopathology and atheroma development in human allografted organs.

Human atherosclerosis requires decades to develop spontaneously, and its development customarily is not monitored by serial biopsies. Atherosclerosis in allografts develops within months, and biopsy specimens are usually obtained from the grafts. We have used immunocytochemical techniques to study biopsy specimens of cardiac and renal allografts for parameters of vascular changes. The antibodies used in this investigation were specific for T lymphocytes, macrophages, IgM, and complement, and detailed studies were done with the use of antibodies specific for components of the hemostatic, fibrinolytic, and natural anticoagulant pathways. The results indicated a lack of association between the appearance of cellular infiltrates and measurable alterations in vascular endothelium and smooth-muscle cells. Although infiltrating macrophages and lymphocytes were identified in biopsy specimens with vascular change, such changes were also observed in biopsy specimens that were devoid of cellular infiltrates. The most prominent vascular changes in endothelium and smooth-muscle cells were accelerated hemostasis, depressed fibrinolysis, and deranged anticoagulant pathways. A negative association between the presence of IgM and fibrin on endothelial cells was also identified. Whether vascular changes are due to immunologically mediated reactions or to as yet undefined metabolic stresses of the graft-host relationship remains to be determined. This study provides a model for the study of vascular changes in atheroma development as viewed through the window of transplant-induced atherosclerosis.

Arteriosclerosis↗
PubMed1992

Iron-reducing activity of plasma membranes.

Human placental trophoblast plasma membranes were prepared by differential centrifugation and solubilized in nonionic detergent. Transferrin receptors were isolated from the solubilized membranes by affinity chromatography on diferric transferrin-coupled Sepharose 4B. The trophoblast plasma membrane vesicles demonstrated NADH-ferricyanide oxidoreductive activity. However, NADH-Fe(III) oxidoreductive activity was very weak when Fe(III)-ammonium citrate or diferric transferrin was used as electron acceptor in the presence of bathophenanthroline disulfonate as an indicator of the reaction. After solubilization, only NADH-ferricyanide oxidoreduction was recovered. Affinity chromatography-purified transferrin receptors did not exhibit any measurable oxidoreductase activity. Thus, when these receptors are present in plasma membranes they mediate redox reactions, but biochemically isolated receptors do not mediate such reactions. These observation suggest that transferrin receptors in plasma membranes bind diferric transferrin, and, in an undetermined way, facilitate Fe(III) release so that iron reduction can occur.

Cell Fractionation↗
PubMed1991

Inhibition of transplasma membrane electron transport by monoclonal antibodies to the transferrin receptor.

Reduction of iron in diferric transferrin is inhibited by monoclonal antibodies to the transferrin receptor which bind at sites other than the high affinity transferrin binding site. These antibodies include B3/25, GB16 and GB22. Two antibodies which bind at the high affinity site for transferrin, 42/6 and GB18, do not inhibit iron reduction by transplasma membrane electron transport. The results are consistent with the proposal that differric transferrin reduction or stimulation of transmembrane NADH oxidase activity involves a site different from the high affinity diferric transferrin binding site. A synergistic action of antibodies with epitopes at the tight binding site involved in iron uptake and the antibodies which inhibit electron transport, B3/25 and GB16, can explain the increased inhibition of growth observed when both 42/6 and B3/25 are added to proliferating cells.

Animals↗
PubMed1991

Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.

Amiloride↗
PubMed1991

Diferric transferrin reduction by K562 cells. A critical study.

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.

Ferricyanides↗
PubMed1991

Factor VIII procoagulant: a marker of fibrinoid necrosis in normal term human placentae.

Factor VIII procoagulant (FVIIIPC) is a plasma glycoprotein cofactor for activated factor IX that activates factor X in prothrombin convertase and results in fibrin deposition. We have studied 50 normal term placentae for the presence of FVIIIPC, and found it in granular deposits almost exclusively in areas of fibrinoid necrosis. This observation was made in all fibrinoid areas. These deposits were associated with specific factors of the coagulation, anticoagulation and fibrinolytic pathways. These findings suggest coagulation and fibrinolysis are activated in areas of fibrinoid necrosis and indicate FVIIIPC is a marker of these areas.

Biomarkers↗
PubMed1991

Tissue factor in chronic villitis of unestablished etiology.

We have previously reported the presence in normal human placentae of coagulation, macrophages and helper T lymphocytes in inflammatory foci known as villitis of unestablished etiology. In order to investigate the link between coagulation and immunity, we have studied fetal stem vessel endothelium for tissue factor, which is made available by cytokines and activates coagulation via the extrinsic pathway. We found that fetal stem vessel endothelial cells of normal chorionic villi did not react with antibody to tissue factor. Normal placentae contain small numbers of villitis areas and endothelium in these areas was reactive with antibody to tissue factor. Endothelial tissue factor reactivity was more prominent in placentae from secondary recurrent spontaneous aborters and these placentae have greatly increased numbers of villitis areas. The tissue factor availability on fetal stem vessel endothelium may result from immunologically mediated cytokine release. The net effect of these reactions is the presence of lymphocytes, macrophages, coagulation, necrosis and vasculitis in villitis.

Abortion, Habitual↗
PubMed1991

Immunogenetic studies of soluble Fc receptor (CD16) antigens in human seminal plasma.

Two forms of CD16 Fc receptors (FcRIII) have been defined on human leukocytes. Type 1, present on polymorphonuclear neutrophils (PMN), expresses the NA alloantigen system and type 2, present on natural-killer (NK) cells and macrophages, has no known polymorphic variations. We have described the presence of soluble FcRIII antigens in human seminal plasma (SP). These SP antigens retain an affinity for IgG-Fc and are biochemically distinct from leukocytic FcRIII. Their origin within the male reproductive tract and how they relate to FcRIII types 1 and 2 were not known. By using monoclonal antibodies CLB/Gran 11 and Leu 11c that differentially react with FcRIII type 1 and type 2 from different individuals, we studied how SP FcRIII antigens relate to leukocytic FcRIII. CLB/Gran 11 and Leu 11c reactivities with PMN and NK cells from a selected panel of male donors were compared with enzyme-linked immunosorbent assay reactivities of CLB/Gran 11 and Leu 11c with SP samples from these donors. The reactivity pattern with SP samples was shown to be different from findings with donors' PMN, but it corresponded with their NK cells. Our results indicate that SP FcRIII antigens do not manifest the polymorphic variations that are detected by CLB/Gran 11 and Leu 11c on PMN. These findings suggest that SP FcRIII antigens do not originate from PMN.

Adult↗
PubMed1991

Occurrence of other reproductive failures among women with recurrent spontaneous abortion.

We have previously reported an association between recurrent spontaneous abortion and ectopic pregnancy. We now report that not only ectopic pregnancy, but also other reproductive failures including hydatidiform moles, preterm births, and stillbirths are also associated with recurrent spontaneous abortion. The obstetrical histories of 455 women who had experienced two or more consecutive spontaneous abortions were studied for the occurrence of term births, preterm births, stillbirths, spontaneous abortions, ectopic pregnancies, and hydatidiform moles. The observed frequencies of these reproductive outcomes were compared with the expected frequencies of 920 term births, 80 preterm births, five stillbirths, 150 spontaneous abortions, 14.5 ectopic pregnancies and 0.5 molar pregnancies per 1,000 livebirths. The 455 women had experienced 1,968 pregnancies of which 184 were term births, 30 were preterm births, 16 were stillbirths, 1,669 were spontaneous abortions, 63 were ectopic pregnancies, and six were molar pregnancies. The ratio of observed to expected values was term births 0.1, preterm births 1.6, stillbirths 14.0, spontaneous abortion 6.6, ectopic pregnancy 2.6, and molar pregnancy 7.1. The gravid specific proportions of reproductive outcomes were constant suggesting comorbidity or common cause(s). The commonality that links these types of reproductive failure will provide insight into the mechanisms of reproductive wastage.

Abortion, Habitual↗
PubMed1991

HIV proteins in normal human placentae.

Cryostat sections of human normal term placentae were studied for evidence of immunopathology by using antibodies to lymphocytes, macrophages, platelets, and coagulation factors. Areas of so-called chronic villitis of unestablished etiology were identified in all placentae. The same tissues were examined for HIV protein antigens gp120, p17, p24, and gp41. No evidence for gp41 was found. Antigens gp120 and p17 were identified in normal chorionic villi in vimentin-positive fibroblast-like cells and in endothelium, respectively. Antigen p24 was localized to HLA-DR positive cells that morphologically resembled macrophages in areas of villitis. The distribution of gp120 and p17 was similar to that observed for tissue factor. These findings prompted speculation that retroviral proto-oncogenes that are known to encode for certain placental receptors could be involved in the presentation of tissue factor, and that gp120 may be a hitherto unrecognized immunobiological mechanism for the blockade of CD4 on maternal lymphocytes if and when such cells gain entrance to chorionic villi.

Chorionic Villi↗