B A Chabner, C J Allegra, G A Curt, N J Clendeninn, J Baram, S Koizumi, J C Drake, J Jolivet
Expression of the transferrin receptor on target cell lines has recently been implicated as a determinant of susceptibility to cytolysis by natural killer (NK) lymphocytes. We have examined this proposed relationship in several ways. First, K562 (a cell line highly vulnerable to NK lysis) cells were grown for 24 h in the iron chelator desferrioxamine. Under these conditions, the cells doubled their surface transferrin receptor expression as determined both by radioligand binding and surface binding of the OK-T9 monoclonal anti-transferrin receptor antibody. In contrast, cells grown for the same period of time in hemin halved their receptor expression. This fourfold change in transferrin receptor expression between the desferrioxamine-treated and hemin-treated cells produced no change in susceptibility to NK cytolysis. Second, HeLa (a cell line which in its native state is very resistant to NK cytolysis) cells were compared with K562 cells with respect to surface transferrin receptor expression. The difference in NK susceptibility of the two cell lines was not reflected in differences in transferrin receptor expression: the K562 cells expressed approximately 1.5 X 10(5) receptors per cell while HeLa cells expressed 2.0 X 10(5) receptors/cell. Third, infection of HeLa cells by measles virus greatly increased their susceptibility to NK lysis but produced no change in surface transferrin receptor expression. Furthermore, when measles-infected HeLa cells were grown for 6 d in medium supplemented with iron-saturated human transferrin they underwent a 50% reduction in receptor expression but no change in NK susceptibility. Finally, possible alterations in the surface expression of NK target antigens on modified cells were further assayed by their ability to serve as cold-target inhibitors of cytolysis of NK-sensitive target cells. We examined two groups of cells in which transferrin receptor expression was reduced. These were the transferrin-treated, measles-infected HeLa cells with the 50% receptor reduction, and K562 cells grown in medium containing hemin and iron salts where the reduction was five- to sixfold relative to control. In neither case was there a change in the apparent expression of NK target antigen(s). We conclude that there is a discordance between transferrin receptor expression and susceptibility to NK cytolysis in the model systems examined. Therefore, it is unlikely that the transferrin receptor per se is the target recognition structure for human NK cells, although a role in concert with other, as yet undefined molecules, cannot be excluded.
K R Bridges, B R Smith
The metabolic and systemic effects of dichloroacetate (DCA) in the treatment of hypoxic lactic acidosis were evaluated in the dog and compared with the infusion of equal quantities of volume and sodium. Hypoxic lactic acidosis was induced by ventilating dogs with an hypoxic gas mixture of 8% oxygen and 92% nitrogen, resulting in arterial PO2 of less than 30 mmHg, pH below 7.20, bicarbonate less than 15 mM, and lactate greater than 7 mM. After, the development of hypoxic lactic acidosis dogs were treated for 60 min with either DCA as sodium salt or NaCl at equal infusions of volume and sodium. Dogs treated with DCA showed a significant increase of arterial blood pH and bicarbonate, and steady levels of lactate, whereas NaCl resulted in further declines of blood pH and bicarbonate, and rising blood lactate levels. Overall lactate production decreased during therapy with either regimen, but hepatic lactate extraction increased significantly with DCA, while it remained unchanged with NaCl. Tissue lactate levels in liver and skeletal muscle decreased significantly with DCA treatment but were unchanged with NaCl. Additionally, an increase in muscle intracellular pH was observed only in DCA treated dogs. A possible mechanism for the observed actions of DCA might be related to a significant increase in oxygen delivery to tissues. Such an effect was found with DCA administration, but was not observed with NaCl therapy. In conclusion, DCA therapy in hypoxic lactic acidosis has beneficial systemic effects compared with therapy with NaCl. DCA administration is accompanied by increases of blood pH and bicarbonate, a decrease in lactate production, and enhanced liver lactate extraction, and a lowering of tissue lactate levels.
H Graf, W Leach, A I Arieff
Glucocorticosteroid therapy results in an increase in the number of circulating neutrophils and a decrease in the number of eosinophils. Utilizing the double layer soft agar technique, we examined the effect of physiologic to pharmacologic concentrations of hydrocortisone on the proliferation of human neutrophil progenitors and eosinophil progenitors from peripheral blood and bone marrow. When peripheral blood cultures were studied, eosinophil proliferation was inhibited in a dose-responsive fashion with 10(-8) - 10(-5) M hydrocortisone succinate, and comprised 49 +/- 4% of the colonies in control cultures and only 4 +/- 1% (P less than 0.01) at pharmacologic levels of hydrocortisone (10(-5) M). The number of neutrophil colonies, on the other hand, increased by 31% when 10(-5) M hydrocortisone was added to cultures. In order for corticosteroids to exert this effect, it was necessary to add them within 24 h of the initiation of culture. The effect of hydrocortisone on granulocyte proliferation could not be blocked by progesterone, a structurally analogous steroid. To determine whether hydrocortisone was acting directly on the progenitor cell or via an effector cell, its effect on modulating cell populations and stimulating-factor production was studied. Removal of E-rosetting cells and/or adherent cells did not affect the inhibition of eosinophil colony growth or the enhancement of neutrophil colony growth. Furthermore, addition of the potent inhibitor of T cell function, cyclosporin A, failed to affect eosinophil colony frequency, suggesting that inhibition of T cell function was an unlikely explanation for the observed hydrocortisone effect. Leukocyte conditioned media (LCM), derived from peripheral blood mononuclear cells incubated with hydrocortisone, was devoid of both neutrophil and eosinophil colony-stimulating activity, whereas a control LCM stimulated both neutrophil and eosinophil proliferation. The data suggest that the observed hydrocortisone effect on granulocyte colony formation is unlikely to be mediated by an intermediary, and that hydrocortisone acts directly on progenitor cells.
B H Bjornson, J M Harvey, L Rose
The protective effect of dietary protein restriction on the development and expression of immune-mediated interstitial nephritis was evaluated in Brown Norway rats with anti-tubular basement membrane disease. In the first series of experiments, pair-fed rats received low protein (LP) (3% casein) or normal protein (NP) (27% casein), normocaloric diets. After 6 wk, each group was immunized with renal tubular antigen in adjuvant to produce anti-tubular basement membrane antibody (alpha TBM-Ab) and tubulointerstitial nephritis. The kidneys harvested from NP rats after four more weeks on the diet had histologically more severe interstitial disease than the LP rats (histologic severity; NP = 3.1 +/- 0.2 vs. LP = 1.1 +/- 0.3; P less than 0.001), and serum creatinine values were concordantly different (NP = 1.34 +/- 0.02 vs. LP = 0.82 +/- 0.03). Titers of alpha TBM-Ab were similar in both groups, while the T cell-mediated immune response, as measured by delayed-type hypersensitivity (DTH), was nonspecifically impaired in LP rats when compared with the NP group. Admixture cotransfers of LP plus NP cells failed to demonstrate active suppression as an explanation for the depressed DTH in LP rats. The therapeutic role of dietary protein restriction was also examined in rats with established alpha TBM disease. In these experiments, rats were first immunized and fed NP diets for 4 wk (histologic severity = 3.0 +/- 0.2; creatinine = 1.78 +/- 0.02), and then were divided into two groups and followed for six more weeks on either LP or NP diets. LP rats, under these conditions, developed less disease than those fed NP diet (histologic severity; NP = 3.2 +/- 0.3 vs. LP = 1.4 +/- 0.2; P less than 0.001), and serum creatinine values were concordantly different (NP = 1.92 +/- 0.05 vs. LP = 0.97 +/- 0.02). Again, the titers of alpha TBM-Ab in both LP and NP groups were similar. These data collectively suggest that LP diet has a protective effect both on the development and extent of tubulointerstitial nephritis that is perhaps, in part, related to the selective abrogation of effector T cell immunity.
D Agus, R Mann, D Cohn, L Michaud, C Kelly, M Clayman, E G Neilson
Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on Western blotting stained with an apparent intensity 5-8% that of Factor VII. This glycoprotein, tentatively called VII*, has a molecular weight 4,500 D less than Factor VII, lacks detectable Factor VII functional activity, does not bind to barium citrate, and is not recognized by a monoclonal antibody that recognizes Factor VII but not alpha-chymotrypsin-treated Factor VII. VII* was not proteolytically produced from Factor VII during in vitro coagulation or after infusion of human Factor VII into rabbits. As determined by Western blotting, the human hepatoma cell line, HepG2, cultured in the presence of vitamin K, secreted relatively greater levels of VII* in proportion to VII (75%) than that found in human plasma. Warfarin treatment of HepG2 cells decreased the quantity of VII secreted by 77%, whereas it only inhibited the secretion of VII* by 14%. Immunologic studies of the plasmas from a patient on chronic warfarin therapy and an individual given a short course of high dose warfarin therapy corroborated the in vitro synthetic studies obtained with HepG2 cells. The data are consistent with the production of VII* by posttranslational, proteolytic, modification of VII, that, at least in the HepG2 cells studied, occurs intracellularly. However, other mechanisms for the production of VII*, in particular, alternative RNA splicing of the transcript from a single gene, cannot be excluded.
G J Broze Jr, S Hickman, J P Miletich
Human and rat placental homogenates convert L-thyroxine (T4) to 3,5,3'-L-triiodothyronine (T3) via a pathway termed type II iodothyronine deiodination. To study regulation of this pathway, cell dispersions were prepared from human placental chorionic-decidual membrane. Dispersed cells deiodinated T4 and 3,3',5'-triiodothyronine (rT3), but not T3, at the 5' position. The reaction was only slightly inhibited by 1 mM 6-n-propylthiouracil, enhanced by dithiothreitol, and substantially inhibited by 50 nM iopanoic acid. Incubation of the cells in thyroid hormone-depleted medium induced a near doubling of T4 5'-deiodination in 36-48 h, with a significant rise seen as early as 12 h. Addition of T4, rT3, or T3 to hormone-depleted medium impaired the rise in type II deiodination in a dose-dependent fashion. T4 and rT3 were equipotent in this regard, and T3 was 2-3 times less potent. T4 was effective in physiological concentrations, 6.5-13 nM in medium containing 10% calf serum, and the effect of T4 was not due to its conversion to either T3 or rT3. In cells with deiodinase activity raised by 48 h incubation in thyroid hormone-depleted medium, addition of T4, T3, or rT3 reversed the increase in 8-24 h. Secretion of prolactin and beta hCG by the dispersed cells was not substantially affected by thyroid hormone deprivation. The increase in type II deiodination during thyroid hormone deprivation appears to depend on a signal from the thyroxine molecule, per se, and could potentially defend intracellular, and/or circulating, T3 pools in pathological states of mild-to-moderate hypothyroxinemia.
J T Hidal, M M Kaplan
Human neutrophils (PMN), when stimulated with such chemotaxins as phorbol myristate acetate (PMA), destroy erythrocytes and other targets. Cytotoxicity depends on PMN-generated reactive oxygen metabolites, yet the exact toxic specie and its mode of production is a matter of some dispute. Using 51Cr-labeled erythrocytes as targets, we compared various reactive-O2 generating systems for their abilities to lyse erythrocytes as well as to oxidize hemoglobin to methemoglobin. PMA-activated PMNs or xanthine oxidase plus acetaldehyde were added to target erythrocytes in amounts that provided similar levels of superoxide. PMNs lysed 68.3 +/- 2.9% (SEM) of targets, whereas the xanthine oxidase system was virtually impotent (2.3 +/- 0.8%). In contrast, methemoglobin formation by xanthine oxidase plus acetaldehyde was significantly greater than that caused by stimulated PMNs (P less than 0.001). A similar dichotomy was noted with added reagent H2O2 or the H2O2-generating system, glucose plus glucose oxidase; neither of these caused 51Cr release, but induced 10-70% methemoglobin formation. Thus, although O2- and H2O2 can cross the erythrocyte membrane and rapidly oxidize hemoglobin, they do so evidently without damaging the cell membrane. That a granule constituent of PMNs is required to promote target cell lysis was suggested by the fact that agranular PMN cytoplasts (neutroplasts), although added to generate equal amounts of O2- as intact PMNs, were significantly less lytic to target erythrocytes (P less than 0.01). Iron was shown to be directly involved in lytic efficiency by supplementation studies with 2 microM iron citrate; such supplementation increased PMN cytotoxicity by approximately 30%, but had much less effect on erythrocyte lysis by neutroplasts (approximately 3% increase), and no effect on lysis in the enzymatic oxygen radical-generating systems. These results suggest a critical role for an iron-liganding moiety that is abundantly present in PMN, marginally so in neutroplasts, and not at all in purified enzymatic systems--a moiety that we presume catalyzes very toxic O2 specie generation in the vicinity of juxtaposed erythrocyte targets. The obvious candidate is lactoferrin (LF), and indeed, antilactoferrin IgG, but not nonspecific IgG, reduced PMN cytotoxicity by greater than 85%. Re-adding 10(-8) M pure LF to neutroplasts increased their ability to promote hemolysis by 48.4 +/- 0.9%--to a level near that of intact PMNs. We conclude that O-2 and H2O2 are not sufficient to mediate target cell lysis, but require iron bound to LF, which, in turn, probably generates and focuses toxic O2 radicals, such as OH, to target membrane sites.
G M Vercellotti, B S van Asbeck, H S Jacob
Medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCADH; EC 18.104.22.168) deficiency (MCD) is an inborn error of beta-oxidation. We measured 3H2O formed by the dehydrogenation of [2,3-3H]acyl-CoAs in a 3H-release assay. Short-chain acyl-CoA dehydrogenase (SCADH; EC 22.214.171.124), MCADH, and isovaleryl-CoA dehydrogenase (IVDH; EC 126.96.36.199) activities were assayed with 100 microM [2,3-3H]butyryl-, -octanoyl-, and -isovaleryl-CoAs, respectively, in fibroblasts cultured from normal controls and MCD patients. Without the artificial electron acceptor phenazine methosulfate (PMS), MCADH activity in fibroblast mitochondrial sonic supernatants (MS) was 54% of control in two MCD cell lines (P less than 0.05). Addition of 10 mM PMS raised control acyl-CoA dehydrogenase activities 16-fold and revealed MCADH and SCADH activities to be 5 (P less than 0.01) and 73% (P greater than 0.1) of control, respectively. Thus, the catalytic defect in MCD involves substrate binding and/or dehydrogenation by MCADH and not the subsequent reoxidation of reduced MCADH by electron acceptors. 20 microM flavin adenine dinucleotide (FAD) did not stimulate MCD MCADH activity in either the 3H-release or electron-transfer(ring) flavoprotein-linked dye-reduction assays. Mixing experiments revealed no MCADH inhibitor in MCD MS; IVDH activities were identical in both control and MCD MS. In postmortem liver MS from another MCD patient, 3H2O formation from [2,3-3H]octanoyl-CoA was 15% of control. When 3H2O formation was assayed with 200 microM [2,3-3H]acyl-CoAs, 15 mM PMS, and 20 microM FAD in fibroblast sonic supernatants from seven MCD cell lines, SCADH, MCADH, and IVDH activities were 72-112% (P greater than 0.1), 4-9% (P less than 0.01), and 86-135% (P greater than 0.1) of control, respectively, revealing no significant biochemical heterogeneity among these patients.
B A Amendt, W J Rhead
The present study explores the interactions between lymphocytes and monocytes that are required for expression of procoagulant activity (PCA) by monocytes in response to purified protein derivative of the tubercle bacillus (PPD) or tularemia antigen. The PCA response was antigen specific: peripheral blood mononuclear cells (PBM) from donors sensitive to PPD or tularemia showed an increase in PCA only in response to the sensitizing antigen. The PCA was tissue factorlike in that Factors VII and X were required for expression of the activity, whereas Factor VIII was not. Maximum PCA developed only after 36 to 72 h. Fractionation of PBM into lymphocytes and monocytes after antigenic stimulation demonstrated that greater than 90% of the PCA was associated with monocytes. Isolated monocytes or lymphocytes incubated with sensitizing antigen had the same PCA as control cells. Purified lymphocytes that had been pulsed with antigen were unable to elicit a PCA response from monocytes to which they were added. However, adherent monocytes incubated with antigen, then washed free of unbound protein, were able to trigger lymphocytes to become stimulatory for PCA toward responding monocytes. The development of antigen-specific PCA in PBM could be blocked by including a monoclonal antibody to HLA-DR antigen in the incubation. The antibody had no effect on the clotting assay, on preformed PCA, cell viability, or on stimulatory antigen itself. These results indicate that elaboration of PCA by mononuclear cells may be an intrinsic part of the classical immune response to antigen, and may explain the presence of fibrin in immune lesions, as well as the occurrence of thrombotic complications in many immune disorders.
B S Schwartz
Rabbit renal papillary collecting tubule cells were isolated as a homogeneous population and grown in primary culture. These cells were maintained in fully defined medium to inhibit fibroblast overgrowth and to facilitate labeling of endogenous inositol phospholipids with myo-[2-3H]inositol with high specific activity. These cells demonstrated the morphology, cyclic AMP responsiveness, and prostaglandin E2 (PGE2) elaboration, consistent with previous published characterizations. When cells labeled with myo-[2-3H]inositol were stimulated by bradykinin at 10(-7) M, time-dependent and reversible changes in the distribution of inositol polyphosphates were observed. Inositol 1,4,5-triphosphate and inositol 1,4-diphosphate showed time-dependent and dose-dependent increases to maximal levels of 225 and 223% of control, respectively. These data indicate that the elaboration of inositol polyphosphates is a biochemical correlate to bradykinin stimulation and may play a role in PGE2 release in renal papillary collecting tubule cells.
J A Shayman, A R Morrison
The third component of complement (C3) is a plasma glycoprotein with a variety of biologic functions in the initiation and maintenance of host response to infectious agents. While the hepatocyte is the primary source of plasma C3, mononuclear phagocytes contribute to the regulation of tissue availability of C3. Lipopolysaccharide (LPS), a constituent of cell walls of gram-negative bacteria, consists of a polysaccharide moiety (core polysaccharide and O antigen) covalently linked to a lipid portion (lipid A). Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis, we examined the effects of LPS on synthesis of C3 by human mononuclear phagocytes as well as synthesis of the second component of complement (C2), factor B, lysozyme, and total protein. LPS increased C3 synthesis 5-30-fold without affecting the kinetics of secretion of C3 or the synthesis of C2, lysozyme, or total protein. Factor B synthesis was consistently increased by LPS. Experiments with lipid A-inactivated LPS (alkaline treated), LPS from a polysaccharide mutant strain, and lipid X (a lipid A precursor) indicated that the lipid A portion is the structural element required for this effect. Northern blot analysis demonstrated at least a fivefold increase in C3 mRNA in LPS-treated monolayers, which suggests that the regulation of the increase in C3 synthesis is pretranslational. C2 mRNA and factor B mRNA were increased approximately twofold. The availability of specific gene products in human mononuclear phagocytes that respond to LPS should permit understanding of the molecular regulation of more complex functions of these cells elicited by LPS in which multiple gene products are coordinately expressed.
R C Strunk, A S Whitehead, F S Cole
We used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. None of the monoclonal autoantibodies appeared to bind to a significant percentage of cells of relatively small cell size, either before or after culture. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Further experiments, including those using aggregated Ig to block antibody binding, strongly indicated that anti-histone antibody binding was not Fc receptor mediated. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations (0.25 micrograms/ml) of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.
V M Holers, B L Kotzin
Since the discovery of prostacyclin (PGI2) in 1976, there has been great interest in its vascular effects and potential clinical applications. High infusion rates of PGI2 markedly depress arterial blood pressure both in animal studies and in clinical trials. This fall in pressure may result entirely from a decrease in arterial resistance. However, it is possible that the administration of PGI2 may decrease ventricular filling due to an increase in vascular capacity. To investigate whether or not PGI2 affects vascular capacity, we infused PGI2 intraarterially at both 10 and 25 micrograms/min into 15 dogs on total cardiopulmonary bypass. These infusions were associated with a 25 +/- 3 mmHg decrease in arterial pressure and an increase in vascular capacity of 155 +/- 29 ml (SE, P less than 0.005). This increase in capacity was greater (P less than 0.02) than the increase of 23 +/- 42 ml resulting from infusions of nitroglycerin into eight dogs at 2 mg/min, which produced a decrease in arterial pressure of 23 +/- 4 mmHg, which was the maximal effect that could be achieved. Neither bilateral cervical vagotomy nor beta adrenergic blockade with propranolol significantly diminished the increase in vascular capacity associated with infusions of PGI2. The results from studies in four eviscerated dogs indicated that PGI2 acts on both splanchnic and extrasplanchnic capacity vasculature. To compare the direct effects of PGI2 with those of nitroglycerin and nitroprusside on venous tone, we used an isolated canine spleen preparation. Infusions of PGI2 (100 mcg/min) increased spleen weight in this preparation by 9.0 +/- 2.4% (n = 10, P less than 0.001); this increase was significantly greater than increases of 3.6 +/- 2.2% (P less than 0.001) and 3.5 +/- 2.3% (P less than 0.001) caused by high dose infusions of nitroglycerin (1 mg/min) and nitroprusside (400 micrograms/min), respectively. Thus, PGI2 substantially increases vascular capacity by a mechanism that appears to involve a direct action on vascular smooth muscle. Furthermore, these results suggest that PGI2 might be useful in clinical conditions in which an increase in vascular capacity is indicated.
T G Fulghum, J P DiMarco, E W Supple, I Nash, J Gendlerman, D F Eton, J B Newell, R M Zusman, W J Powell Jr
When large volumes of air are inhaled at rapid rates of ventilation, substantial segments of the tracheobronchial tree become involved in the conditioning process and the inspirate does not reach body conditions of temperature and humidity until it passes well into the peripheral bronchi. To determine if the manner in which ventilation is elevated is an important factor in producing this response, we measured the temperature of the airstream at six points in the tracheobronchial tree from the pharynx to the subsegmental bronchi during 5 min of exercise and voluntary hyperventilation in seven normal subjects while they inhaled frigid air. Minute ventilation and respiratory frequency were recorded at minute intervals and intrathoracic temperatures were measured continuously. With both forms of hyperpnea, airway temperature fell dramatically, and there were no significant differences between exercise and hyperventilation. These results demonstrate that the thermal events that occur within the lung during short, moderately intense degrees of exercise can be readily simulated by voluntary hyperventilation when ventilation and inspired air conditions are matched. Our data also indicate that this form of exercise does not result in an increase in airstream temperature and raise the possibility that the bronchial blood supply may be determined by the local thermal needs of the airways to recover heat and water independent of, at least moderate, increases in cardiac output.
E R McFadden Jr, B M Pichurko
We have examined the urinary excretion of stable immunoreactive eicosanoids in 23 female patients with systemic lupus erythematosus (SLE), 16 patients with chronic glomerular disease (CGD), and 20 healthy women. SLE patients had significantly higher urinary thromboxane B2 (TXB2) and prostaglandin (PG) E2 excretion and significantly lower 6-keto-PGF1 alpha than did healthy women. In contrast, CGD patients only differed from controls for having reduced 6-keto-PGF1 alpha excretion. The group of SLE patients with active renal lesions differed significantly from the group with inactive lesions for having a lower creatinine clearance and urinary 6-keto-PGF1 alpha and higher urinary TXB2. Higher urinary TXB2 excretion was associated with comparable platelet TXB2 production in whole blood, undetectable TXB2 in peripheral venous blood, and unchanged urinary excretion of 2,3-dinor-TXB2. A significant inverse correlation was found between urinary TXB2 and creatinine clearance rate (CCr). In contrast, the urinary excretion of 6-keto-PGF1 alpha showed a significant linear correlation with both CCr and para-aminohippurate clearance rate (CPAH). In four SLE and seven CGD patients, inhibition of renal cyclooxygenase activity by ibuprofen was associated with a significant reduction in urinary 6-keto-PGF1 alpha and TXB2 and in both CCr and CPAH. However, the average decrease in both clearances was 50% lower in SLE patients than in CGD patients, when fractionated by the reduction in urinary 6-keto-PGF1 alpha or PGE2 excretion. We conclude that the intrarenal synthesis of PGI2 and TXA2 is specifically altered in SLE. Such biochemical alterations are associated with changes in glomerular hemodynamics and may play a role in the progression of SLE nephropathy.
C Patrono, G Ciabattoni, G Remuzzi, E Gotti, S Bombardieri, O Di Munno, G Tartarelli, G A Cinotti, B M Simonetti, A Pierucci
To study the effect of dietary fat on postprandial substrate utilization and nutrient balance, respiratory exchange was determined in seven young men for 1 h before and 9 h after the ingestion of one of three different breakfasts: i.e., bread, jam, and dried meat (482 kcal: 27% protein, 62% carbohydrate, and 11% fat); bread, jam, and dried meat plus 50 g of margarine containing long-chain triglycerides (LCT); or bread, jam, and dried meat plus 40 g medium-chain triglycerides (MCT) and 10 g LCT margarine (858 kcal: 15% protein, 35% carbohydrate, and 50% fat). Plasma glucose concentrations peaked 45 min after the start of the meals. When compared with the low fat meal, the LCT margarine supplement had no effect at any time on circulating glucose and insulin concentrations, nor on the respiratory quotient. When MCTs were consumed, plasma glucose and insulin concentrations remained lower and plasma FFA concentrations higher during the first 2 h. 9 h after the breakfasts, the amounts of substrates oxidized were similar in each case, i.e., approximately 320, 355, and 125 kcal for carbohydrate, fat, and protein, respectively. This resulted in comparable carbohydrate (mean +/- SD = -22 +/- 32, -22 +/- 37, and -24 +/- 22 kcal) and protein balances (-7 +/- 9, +7 +/- 7, and -8 +/- 11 kcal) after the low fat, LCT- and MCT-supplemented test meals, respectively. However, after the low fat meal, the lipid balance was negative (-287 +/- 60 kcal), which differed significantly (P less than 0.001) from the fat balances after the LCT- and MCT-supplemented meals, i.e., +60 +/- 33 and +57 +/- 25 kcal, respectively. The results demonstrate that the rates of fat and of carbohydrate oxidation are not influenced by the fat content of a meal.
J P Flatt, E Ravussin, K J Acheson, E Jéquier
5-Nitrofurans have been used in the study of chemical carcinogenesis. There is substantial evidence that N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) is deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) in the process of FANFT-induced bladder cancer. Paradoxically, ANFT is less potent as a uroepithelial carcinogen than FANFT when fed to rats. Feeding aspirin with FANFT to rats decreases the incidence of bladder cancer. Isolated kidneys were perfused with 5-nitrofurans to determine renal clearances and whether aspirin acts to decrease urinary excretion of the carcinogen. In FANFT-perfused kidneys, FANFT was deformylated to ANFT and excreted (1.06 +/- 0.22 nmol/min) at a rate eightfold higher than excretion of FANFT. In kidneys perfused with equimolar ANFT, excretion of ANFT was 0.25 +/- 0.05 nmol/min, which suggests a coupling of renal deformylation of FANFT to excretion of ANFT in FANFT-perfused kidneys. Neither aspirin nor probenecid altered the urinary excretion or half-life of FANFT or ANFT. In rats fed 0.2% FANFT as part of their diet, coadministration of aspirin (0.5%) increased urinary excretion of ANFT during a 12-wk feeding study, which suggests decreased tissue binding or metabolism of ANFT. Kidney perfusion with acetylated ANFT (NFTA), a much less potent uroepithelial carcinogen, resulted in no ANFT excretion or accumulation, which indicates the specificity of renal deformylase. Renal deformylase activity was found in broken cell preparations of rat and human kidney. These data describe a unique renal metabolic/excretory coupling for these compounds that appears to explain the differential carcinogenic potential of the 5-nitrofurans tested. These results are consistent with the hypothesis that aspirin decreases activation of ANFT by inhibiting prostaglandin H synthase.
L A Spry, T V Zenser, S M Cohen, B B Davis
We recently reported the biological activity and some of the biochemical characteristics of a factor produced by a human T cell hybrid clone able to block hematopoietic progenitor cell proliferation. This 85-kD protein factor, which we have termed colony-inhibiting lymphokine (CIL), has growth regulatory activity on bone marrow precursors bearing Ia (class II) antigens of either granulocytic-monocytic (CFU-GM) or erythroid lineage (BFU-E and CFU-E). Experiments aimed to investigate the specificity of the inhibitory effect on hematopoietic progenitor cell growth suggested that the expression of HLA-DR surface antigens was required on the target cells. We describe in this communication how DR+ cell lines ceased dividing after a few days of culture in the presence of CIL, whereas DR- cell lines were completely unaffected. The increased DR expression on the ML3 cell surface, mediated by the activity of the gamma interferon (IFN gamma), increases the sensitivity to the growth inhibition factor of the ML3 cell line. To verify the hypothesis that the DR antigens might serve as receptors for the factor, enabling it also to interfere in the immune response, we tested CIL in a mixed lymphocyte reaction (MLR), one of the best known in vitro Ia antigen-dependent T cell-mediated immune responses. CIL is able to block major histocompatibility complex-allogeneic MLR both in human and mouse systems. The data indicate that CIL recognizes a nonpolymorphic structure (presumably on all Ia molecules) presented by stimulator cells of either species, and thereby interferes with specific interactions between stimulator and responder cells. Blocking of the alloantigen stimulation stage is also indicated, since CIL is effective only if added to the culture medium during the first 48 h of the MLR. Finally, mouse monoclonal anti-DR antibodies are able to sharply reduce CIL activity on sensitive DR+ cell lines. CIL may act physiologically as a multifunctional mediator in a complex network that links regulation of bone marrow differentiation and the generation of immune responses.
M Trucco, S Shaw, R Korngold
Systemic administration of an aqueous suspension of group A streptococcal cell wall fragments to susceptible rats induces acute and chronic polyarthritis, as well as noncaseating hepatic granulomas. To gain insight into the role of the thymus in the pathogenesis of this experimental model, pathologic responses and cell wall tissue distribution were compared in congenitally athymic rats (rnu/rnu) and their euthymic littermates (NIH/rnu). Within 24 h, both rat strains developed acute arthritis, characterized by polymorphonuclear leukocytic exudate in the synovium and joint spaces. This acute process was maximal at day 3 and gradually subsided. Beginning 2-3 wk after injection, the euthymic, but not the athymic, rats developed the typical exacerbation of arthritis, characterized by synovial cell hyperplasia with villus formation and T helper/inducer lymphocyte-rich mononuclear cell infiltration. This process eventually resulted in marginal erosions and destruction of periarticular bone and cartilage. Parallel development of acute and chronic hepatic lesions was observed. Bacterial cell wall antigen distribution and persistence were similar in the athymic and euthymic rats. Cell wall antigens were demonstrated in the cytoplasm of cells within subchondral bone marrow, synovium, liver, and spleen, coincident with the development of the acute lesions, and persisted in these sites, although in decreasing amounts, for the duration of the experiment. Our findings provide evidence that the acute and chronic phases of the experimental model are mechanistically distinct. The thymus and functional thymus derived-lymphocytes appear not to be required for the development of the acute exudative disease but are essential for the development of chronic proliferative and erosive disease. Induction of disease is dependent upon cell wall dissemination to and persistence in the affected tissues.
J B Allen, D G Malone, S M Wahl, G B Calandra, R L Wilder
alpha-Globin is encoded by the two adjacent genes, alpha 1 and alpha 2. Although it is clearly established that both alpha-globin genes are expressed, their relative contributions to alpha-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in alpha-globin gene activity secondarily to the loss of function of one or more of these genes (alpha-thalassemia [Thal]) have not been directly investigated. This study further defines the expression of the two human alpha-globin genes by determining the relative levels of alpha 1 and alpha 2 mRNA in the reticulocytes of normal individuals and in individuals heterozygous for the common 3.7-kilobase deletion within the alpha-globin gene cluster that removes the alpha 2-globin gene (the rightward type alpha-Thal-2 deletion). To quantitate accurately the ratio of the two alpha-globin mRNAs, we have modified a previously reported S1 nuclease assay to include the use of 32P end-labeled probes isolated from alpha 1- and alpha 2-globin complementary DNA recombinant plasmids. In individuals with a normal alpha-globin genotype (as determined by Southern blot analysis [alpha alpha/alpha alpha]), alpha 2-globin mRNA is present at an average 2.8-fold excess to alpha 1. In individuals heterozygous for the rightward type alpha-Thal-2 deletion (-alpha/alpha alpha) the alpha 2/alpha 1 mRNA ratio is 1:1. These results suggest that the loss of the alpha 2-globin gene in the alpha-Thal-2 deletion is associated with a 1.8-fold compensatory increase alpha 1-globin gene expression.
S A Liebhaber, F E Cash, D M Main
Potassium secretion and sodium-potassium adenosine triphosphatase (Na-K-ATPase) activity in the distal nephron segments are known to be influenced by the dietary intake of K+. This has been attributed to a change in the plasma aldosterone level, which also influences K+ secretion and Na-K-ATPase activity in the distal nephron. To investigate whether or not dietary K+ can modulate Na-K-ATPase activity in the distal nephron independently of aldosterone, we determined Na-K-ATPase activity in four distinct nephron segments of adrenalectomized (adx) rabbits given four specific diets for 1 wk before experimentation. Na-K-ATPase activity was determined by a fluorometric microassay in which ATP hydrolysis is coupled to NADH oxidation. The nephron segments examined were the distal convoluted tubule (DCT), the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct (MCD). All diets were similar in composition except for their K+ contents, which were 100, 300, 500, and 700 meq/kg in groups 1-4, respectively. In these adx animals, Na-K-ATPase activity increased greater than 200% in the CCD as the dietary intake of K+ increased. There was a linear relationship between K+ excretion and the enzyme activity in this segment. There was a 50% increase in Na-K-ATPase activity in the CNT as the dietary intake of K+ increased in adx animals. However, there were no significant differences in Na-K-ATPase activities in the DCT and MCD among the four treatment groups. It is concluded that dietary K+ intake can influence Na-K-ATPase activity in the CCD and CNT independently of plasma aldosterone levels.
L C Garg, N Narang
The action of vasopressin (AVP) in transporting epithelia is mediated by cyclic AMP(cAMP), whereas its effects in hepatocytes are mediated by calcium and phosphoinositides. Based on our recent observation that AVP stimulates phosphoinositide turnover in toad bladder, we examined the role of calcium-phospholipid-dependent kinase (protein kinase C) as a modulator of AVP's hydroosmotic effect. Phorbol myristate acetate (PMA), which can substitute for diglyceride as an activator of protein kinase C, the diglyceride dioctanoylglycerol, and RHC-80267, a glyceride lipase inhibitor that should increase diglyceride levels, inhibited AVP-stimulated water flow, but not water flow stimulated by cAMP, suggesting inhibition of cyclic AMP production. Both the dioctanoylglycerol and RHC-80267, but not PMA, also decreased water flow in response to 8-bromo cAMP indicating a potential inhibition at post-cAMP events as well. PMA increased prostaglandin synthesis; however, inhibition of water flow persisted even when prostaglandin synthesis was completely blocked by incubation with naproxen. Furthermore, water flow was not inhibited by incubation with the inactive diglyceride substitute phorbol didecanoate, supporting the specificity of the PMA inhibition. Consistent with the site of action at adenylate cyclase suggested by the transport experiments, PMA and RHC-80237 decreased both cell cAMP content and the cyclic AMP-dependent kinase ratio (-cAMP/+cAMP), an index of intracellular cyclic AMP effect. Assay for protein kinase C activity in toad bladder epithelial cell supernatant demonstrated that the toad bladder indeed contains a kinase stimulable by phospholipid, calcium, and PMA. As an apparently independent effect, we found that addition of PMA, but not dioctanoylglycerol or RHC-80267, to the mucosal bath increased both water permeability and the frequency of granular cell luminal membrane aggregates in the absence of vasopressin, consistent with stimulation of fusion events at the luminal membrane. Our data suggest that protein kinase C can modulate AVP-stimulated water flow in toad bladder by inhibiting cAMP generation, and perhaps post-cAMP steps as well, and support the hypothesis that AVP-stimulated turnover of membrane phosphoinositides antagonize the effects of AVP via changes in diglyceride, calcium, and protein kinase C.
D Schlondorff, S D Levine
The relationship of intracellular pH (pHi) to superoxide radical (O2-) generation was investigated in chemotactic factor-stimulated human neutrophils. Exposure of cells to 100 nM N-formylmethionyl-leucyl-phenylalanine (FMLP) caused activation of Na/H exchange which, in 140 mM Na medium (pH0 7.40), led to a rise in pHi from 7.22 to 7.80. This pHi change was sensitive to amiloride (apparent Ki 78 microM), an inhibitor of Na/H countertransport. The time course of the alkalinization was similar to that of FMLP-stimulated O2- production, which was complete by 5 min. In the presence of 1 mM amiloride, which nearly blocked the pHi transient elicited by FMLP, or in the absence of external Na, where intracellular acidification was observed in FMLP-stimulated cells, O2- release was still roughly 25-45% of normal. Thus, an alkalinization cannot be an obligatory requirement for O2- generation. By independently varying either pH0, pHi, or the internal or external concentrations of Na, both the direction and magnitude of the FMLP-induced pHi transients could be altered. In each instance, the amount of O2- release correlated directly with pHi and was enhanced by intracellular alkalinization. In the absence of FMLP, a rise in pHi to 7.7-7.8 by exposure of cells to 30 mM NH4Cl, 10 microM monensin (a Na/H exchanging ionophore), or after a prepulse with 18% CO2 did not result in O2- generation. Thus, these results imply that an alkalinization per se is not a sufficient trigger. Neutrophils exposed to 4 nM FMLP exhibited a threefold slower rate of alkalinization (reaching pHi approximately 7.80 by 20-30 min) as compared to that obtained with 100 nM FMLP and did not release significant amounts of O2- under normal incubation conditions. However, these cells could be induced to generate O2- when the degree of alkalinization was enhanced by internal Na depletion or by pretreatment with 18% CO2. Together, these results indicate a modulating effect of pHi on O2- production and suggest that other functional responses of neutrophils may be regulated by their pHi.
A DNA sequence polymorphism, revealed by digestion of human DNA with the restriction endonuclease Sst-1 and hybridization with an apolipoprotein A-I complementary DNA clone, has been shown to be located in or close to the 3' noncoding region of the apolipoprotein C-III gene. This polymorphism is found in significantly increased prevalence (P less than 0.001) in Caucasian hypertriglyceridemic subjects compared with race-matched controls, and its distribution in normal individuals of differing racial origins is reported. Furthermore, no alteration of high density lipoprotein or apolipoprotein A-I and apolipoprotein C-III phenotypes was observed in individuals with or without the polymorphism.
A Rees, J Stocks, C R Sharpe, M A Vella, C C Shoulders, J Katz, N I Jowett, F E Baralle, D J Galton
We investigated, in 36 healthy volunteers, the effects of prednisone and ketotifen on recovery of lymphocyte beta 2-adrenoceptor density (determined by (-)-125iodocyanopindolol binding) and responsiveness (assessed by lymphocyte cyclic AMP [cAMP] responses to 10 microM (-)-isoprenaline) after desensitization by the beta 2-agonist terbutaline. Terbutaline (3 X 5 mg/d) decreased lymphocyte beta 2-adrenoceptor density by approximately 40-50%; concomitantly, lymphocyte cAMP responses to 10 microM (-)-isoprenaline were significantly reduced. After withdrawal of terbutaline beta 2-adrenoceptor, density and responsiveness gradually increased, reaching predrug levels after 4 d. Prednisone (1 X 100 mg orally) accelerated beta 2-adrenoceptor recovery; only 8-10 h after administration of the steroid beta 2-adrenoceptor density and cAMP responses to (-)-isoprenaline had reached values not significantly different from pretreatment levels. Similar effects were obtained with ketotifen (2 mg; thereafter 2 X 1 mg/d for 4 d): 24 h after application of the drug beta 2-adrenoceptor density and cAMP responses to (-)-isoprenaline had reached pretreatment levels. Furthermore, ketotifen simultaneously applied with terbutaline completely prevented terbutaline-induced decrease in lymphocyte beta 2-adrenoceptor density and responsiveness. Prednisone (1 X 100 mg orally) or ketotifen (2 mg; thereafter 2 X 1 mg/d for 2 d) had no significant influence on lymphocyte beta 2-adrenoceptor density in healthy volunteers not pretreated with terbutaline, but shifted the ratio high-to-low affinity state of the lymphocyte beta 2-adrenoceptor toward high affinity state. We conclude that glucocorticoids as well as ketotifen can accelerate recovery of density and responsiveness of lymphocyte beta 2-adrenoceptors desensitized by long-term treatment with beta 2-agonists. Such an effect may have clinical implications for preventing tachyphylaxis of asthmatic patients against therapy with beta 2-agonists.
O E Brodde, M Brinkmann, R Schemuth, N O'Hara, A Daul
Immunofluorescence staining of buffy coat smears from a patient with chronic myelogenous leukemia in accelerated phase showed that approximately 13% of all nucleated cells contained von Willebrand protein and, therefore, appeared to be of megakaryocytic origin. This was confirmed by positive staining with antisera against platelet factor 4 and platelet glycoproteins. Short-term cultures of the buffy coat, which lacked endothelial cells, were metabolically labeled with [35S]methionine, and von Willebrand protein was immunopurified from cell lysates and culture medium. Cultures from this patient synthesized and secreted von Willebrand protein, in contrast with cultures from other patients with leukemia, who lacked circulating megakaryocytes, and from normal volunteers. The subunit composition of the megakaryocytic von Willebrand protein was very similar to that of human umbilical vein endothelial cells. The size of the processed subunit (220 kD) and of the cellular (260 kD) and secreted (275 kD) precursors from the two cell types were indistinguishable by gel electrophoresis. Furthermore, the ratio of precursor to processed subunit and the pattern of cellular and secreted nonreduced multimers were very similar. It appears, therefore, that the processing steps in biosynthesis of von Willebrand protein used by the megakaryocytes are very similar to those of umbilical vein endothelial cells.
L A Sporn, S I Chavin, V J Marder, D D Wagner
The thermic effect of food at rest, during 30 min of cycle ergometer exercise, and after exercise was studied in eight lean (mean +/- SEM, 10 +/- 1% body fat, hydrostatically-determined) and eight obese men (30 +/- 2% body fat). The lean and obese mean were matched with respect to age, height, weight, and body mass index (BMI) to determine the relationship between thermogenesis and body composition, independent of body weight. All men were overweight, defined as a BMI between 26-34, but the obese had three times more body fat and significantly less lean body mass than the lean men. Metabolic rate was measured by indirect calorimetry under four conditions on separate mornings, in randomized order, after an overnight fast: 3 h of rest in the postabsorptive state; 3 h of rest after a 750-kcal mixed meal (14% protein, 31.5% fat, and 54.5% carbohydrate); during 30 min of cycling and for 3 h post exercise in the postabsorptive state; and during 30 min of cycling performed 30 min after the test meal and for 3 h post exercise. The thermic effect of food, which is the difference between postabsorptive and postprandial energy expenditure, was significantly higher for the lean than the obese men under the rest, post exercise, and exercise conditions: the increments in metabolic rate for the lean and obese men, respectively, were 48 +/- 7 vs. 28 +/- 4 kcal over 3 h rest (P less than 0.05); 44 +/- 7 vs. 16 +/- 5 kcal over 3 h post exercise (P less than 0.05); and 19 +/- 3 vs. 6 +/- 3 kcal over 30 min of exercise (P less than 0.05). The thermic effect of food was significantly negatively related to body fat content under the rest (r = -0.55), post exercise (r = -0.66), and exercise (r = -0.58) conditions. The results of this study indicate that for men of similar total body weight and BMI, body composition is a significant determinant of postprandial thermogenesis; the responses of obese are significantly blunted compared with those of lean men.
K R Segal, B Gutin, A M Nyman, F X Pi-Sunyer
Receptor-independent low density lipoprotein (LDL) transport plays a critical role in the regulation of plasma cholesterol levels; hence, these studies were done to characterize this process in the tissues of the rat. High rates of receptor-independent clearance were found in the spleen, but other organs, like liver, gastrointestinal tract, and endocrine glands manifested lower clearance rates that varied from 3 to 9 microliter/h per g, while the rates in nervous tissue, muscle, and adipose tissue were less than 1 microliter/h per g. Receptor-dependent uptake was much higher in liver (85 microliter/h per g) and adrenal gland (219 microliter/h per g), but was also low in most other tissues. At normal plasma LDL concentrations, 67% of the receptor-dependent transport in the whole animal was accounted for by LDL uptake in the liver. In contrast, the receptor-independent uptake found in the whole animal took place in many organs, including skeletal muscle (20%), liver (16%), small bowel (15%), skin (10%), and spleen (7%). Furthermore, in liver, the rate of cholesterol synthesis could be varied 11-fold, yet the rate of receptor-independent LDL clearance remained constant at approximately 8 microliter/h per g. When the circulating levels of LDL were systematically increased, receptor-independent LDL clearance also remained constant, so that hepatic LDL-cholesterol uptake by this mechanism increased linearly, from 1 to 20 micrograms/h per g, as the plasma LDL-cholesterol level was increased from 10 to 250 mg/dl. Finally, when equal amounts of LDL-cholesterol were delivered into the liver by either the receptor-dependent or receptor-independent mechanism, there was significant suppression of cholesterol synthesis and an increase in cholesteryl esters. Thus, in any situation in which receptor-dependent LDL degradation is lost, cholesterol balance in the whole animal and across individual organs is maintained by receptor-independent mechanisms, although when the new steady state is achieved, circulating levels of LDL must necessarily be very much increased.
D K Spady, S D Turley, J M Dietschy
Cortical collecting ducts (CCD) from rabbits treated with deoxycorticosterone (DOC) actively secrete bicarbonate at high rates. To investigate the mechanism of bicarbonate secretion, we measured bicarbonate and chloride transport in CCD from rabbits treated with DOC for 9-24 d. Removal of chloride (replaced with gluconate) from both perfusate and bath inhibited bicarbonate secretion without changing transepithelial voltage. Removal of chloride only from the bath increased bicarbonate secretion, while removal of chloride only from the perfusate inhibited secretion. In contrast to the effect of removing chloride, removal of sodium from both the perfusate and bath (replacement with N-methyl-D-glucamine) did not change the rate of bicarbonate secretion. The rate of bicarbonate secretion equaled the rate of chloride absorption in tubules bathed with 0.1 mM ouabain to inhibit any cation-dependent chloride transport. Under these conditions, chloride absorption occurred against an electrochemical gradient. Removal of bicarbonate from both the perfusate and bath inhibited chloride absorption. Removal of bicarbonate only from the bath inhibited chloride absorption, while removal of bicarbonate from the lumen stimulated chloride absorption. We conclude that CCD from DOC-treated rabbits actively secrete bicarbonate and actively absorb chloride by an electroneutral mechanism involving 1:1 chloride/bicarbonate exchange. The process is independent of sodium.
R A Star, M B Burg, M A Knepper
Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells.
I U Schraufstätter, D B Hinshaw, P A Hyslop, R G Spragg, C G Cochrane
17 thymomas were studied by indirect immunofluorescence for the presence of thymic hormones and antigens of the major histocompatibility complex (MHC). The thymoma epithelial cells (specifically identified by their keratin content) contained thymic hormones (thymulin and thymosin alpha 1), a finding corroborated by the observation of elevated thymulin serum levels. In contrast with normal or hyperplastic thymuses, thymoma epithelial cells did not express HLA-DR and HLA-DC antigens as assessed by immunofluorescence as well as immunoblot analyses. Conversely, MHC class I antigens (HLA-ABC) were normally expressed. Thus, we conclude that thymoma epithelial cells are endocrinologically active but are defective for the expression of some MHC products (class II molecules) known to play an essential role in intrathymic T cell differentiation.
W Savino, G Manganella, J M Verley, A Wolff, S Berrih, P Levasseur, J P Binet, M Dardenne, J F Bach
The blood pressure of the spontaneously hypertensive rat (SHR) is influenced by the Ca2+ content of its diet. As the SHR's greater dependence on dietary calcium may reflect a defect in intestinal calcium absorption, we measured in vitro unidirectional Ca2+ flux (J) in the duodenum-jejunum (four segments each) of the SHR (n = 6) and the normotensive Wistar-Kyoto rat (WKY; n = 6) by a modified Ussing apparatus. Because of the known and postulated interactions between Ca2+ and Na+ in both intestinal and vascular tissue, we assessed in vivo the influence of a concurrent manipulation of Na+ intake (three levels: 0.25%, 0.45%, and 1.0%) on the blood pressure development of SHRs (n = 35) and WKYs (n = 35), between 6 and 20 wk of age, exposed to three levels of dietary calcium (0.1, 1.0, and 2%). Net calcium flux (Jnet) (mean +/- SEM) was significantly (P less than 0.01) lower in the SHR (-2.8 +/- 6.3 nmol/cm2 X h) than in the WKY (34.6 +/- 8.8 nmol/cm2 X h). The SHR's decreased Jnet resulted from a significantly (P less than 0.03) lower mucosa-to-serosa flux (Jm-s) in the SHR (41.0 +/- 5.6 nmol/cm2 X h) compared with the Jm-s of the WKY (70.1 +/- 9.1 nmol/cm2 X h). Serosa-to-mucosa flux for calcium did not differ between the SHR (43.8 +/- 6.6 nmol/cm2 X h) and the WKY (35.5 +/- 8.0 nmol/cm2 X h). The SHR's decreased (P less than 0.002) Jm-s was confirmed by additional measurements in SHRs and WKYs. Jm-s was 36.2 +/- 3.7 nmol/cm2 X h in the SHRs (n = 11) and 64.4 +/- 6.7 nmol/cm2 X h in the WKYs (n = 9). The provision of an increased dietary Ca2+ (2% by weight) and increased Na+ (1%) to the SHR prevented the emergence of hypertension (P less than 0.001) (mean +/- SEM systolic blood pressure at 20 wk of age; 135 +/- 5 mmHg for the 2% Ca2+, 1% Na+ SHR vs. 164 +/- 2 mmHg for the control diet SHR). Ca2+ (0.1%) and Na+ (0.25%) restriction accelerated the SHR's hypertension (192 +/- 2 mmHg) (P less than 0.001) and was associated with higher pressures in the WKY (146 +/- 4 mmHg in the restricted WKY vs. 134 +/- 4 mmHg in the control WKY). In a parallel group of 24 SHRs and 24 WKYs fed one of three diets (2% Ca2+/1% Na+; 1% Ca2+/0.45% Na+; or 0.1% Ca2+/0.25% Na+), the heart (P < 0.05) and kidney (P = 0.08) weight of the SHRs varied depending on the diet at 20 wk of age. Low Ca2+ and Na+ intake was associated with increased heart weight (1.6+/-0.9 g) compared with the normal diet for SHR (1.51+/-0.07 g). Increased Ca2+ and Na+ intake was associated with a significantly (P = 0.05) lower heart weight in the SHR (1.37+/-0.03 g) and in the WKY (1.35+/-0.06 g) compared with their normal diet controls. These findings show one mechanism for the SHR's depressor response to supplemental dietary Ca2+ and, in part, explain the sodium dependence of calcium's cardiovascular protective effect.
D A McCarron, P A Lucas, R J Shneidman, B LaCour, T Drüeke
Inflammation of epithelia is an important step in the pathophysiology of a wide variety of diseases. Because reactive oxygen metabolites are important effector molecules of acute inflammation, we examined the effect of oxidants on the barrier function of a cultured epithelium, Madin Darby Canine Kidney cells, by measuring the transepithelial electrical conductance, Gt, of monolayers grown on permeable supports. We found that H2O2, added directly or generated with glucose oxidase, increased Gt. Similar effects were observed with addition of xanthine and xanthine oxidase, a system which enzymatically generates superoxide radical O2-. The oxidant-induced increase in Gt was reversible if the exposure to oxidants was not prolonged (less than 20 min), and if the concentration of H2O2 was less than 5 X 10(-3) M. The increase in Gt suggested that oxidants increase the permeability of the paracellular pathway, a suggestion supported by an oxidant-induced increase in the permeability to 14C-mannitol, which primarily crosses epithelia via the extracellular route. In addition to functional changes in the epithelial monolayer, oxidants changed the cell morphology; after H2O2 exposure, the cells tended to pull apart, most prominently at their basolateral surfaces. These changes were heterogeneous with most areas showing no changes. Some of the morphologic changes could be reversed if the exposure to H2O2 was limited. We also observed a disruption of the normal pattern of the actin-cytoskeleton, particularly in the area of cell to cell junctions, as demonstrated by fluorescent staining of f-actin with rhodamine phallicidin. These functional and structural findings indicate that oxidants increase the permeability of the paracellular pathway in a cultured epithelium. The changes can be reversible, and are accompanied by alterations in organization of the cell cytoskeleton. These studies demonstrate the dynamic nature of the interaction between epithelial cells and oxygen metabolites.
M J Welsh, D M Shasby, R M Husted
We encountered an abnormal hemoglobin (Rahere), with a threonine residue replacing the beta 82 (EF6) lysine residue at the binding site of 2,3-diphosphoglycerate, which was responsible for overt erythrocytosis in two individuals of a Japanese family. Hemoglobin Rahere shows a lower oxygen affinity on the binding of 2,3-diphosphoglycerate or chloride ions than hemoglobin A. Although a decrease in the positive charge density at the binding sites of 2,3-diphosphoglycerate in hemoglobin Rahere apparently shifts the allosteric equilibrium toward the low affinity state, it greatly diminishes the cofactor effects by anions. The oxygen affinity of the patient's erythrocytes is substantially lowered by the presence of bezafibrate, which combines with sites different from those of 2,3-diphosphoglycerate in either hemoglobin Rahere or hemoglobin A.
J Sugihara, T Imamura, S Nagafuchi, J Bonaventura, C Bonaventura, R Cashon
Previous studies comparing the effects of oral, intraportal, and peripheral venous administration of glucose in conscious dogs demonstrated a significant increase in hepatic extraction of insulin only after oral glucose, but similar hepatic uptake of glucose after oral and intraportal glucose, which was greater than that after peripheral intravenous glucose infusion. This study evaluated the effect of atropine blockade of the parasympathetic nervous system on the increased fractional hepatic extraction of insulin and the role of gastric inhibitory polypeptide (GIP) on augmented hepatic uptake of oral glucose in conscious dogs with chronically implanted Doppler flow probes on the portal vein and hepatic artery, and catheters in the portal and hepatic veins and carotid artery. Since atropine infusion decreased absorption of glucose, and in order to achieve comparable portal vein levels of glucose and insulin, the dogs receiving atropine were given 1.9 +/- 0.1 g/kg glucose, compared with the control dogs who received 1.1 +/- 0.1 g/kg. The percentage of the glucose load that was absorbed was greater in the dogs not given atropine (80 +/- 4 vs. 44 +/- 7%), but because of the different loads, the absolute amount of glucose absorbed was similar in both groups (20.2 +/- 1.6 vs. 21.7 +/- 4.1 g). Although delayed by atropine, the peak portal vein glucose and insulin concentrations and the amounts presented to the liver were similar in both groups. However, the increased portal vein plasma flow and fractional hepatic extraction of insulin observed after oral glucose was not observed in the dogs infused with atropine. The net hepatic glucose uptake after oral glucose was significantly less at 10, 20, and 45 min in the atropine-treated dogs, and the area under the curve over the 180-min period was 44% less. However, the latter was not statistically significant. Infusion of GIP with peripheral intravenous glucose did not increase hepatic uptake of glucose or the fractional hepatic extraction of insulin compared with peripheral intravenous glucose alone. These results indicate an important role for parasympathetic innervation in the augmented fractional hepatic extraction of insulin, and increased portal vein plasma flow after oral glucose. Although a relationship between the augmented fractional extraction of insulin and the net hepatic glucose uptake may exist, it does not necessarily indicate that the former is required for the latter. Such parasympathetic innervation may be involved in the greater removal of glucose by the liver after oral compared with peripheral glucose administration. The augmented hepatic uptake of glucose and fractional hepatic extraction of insulin after oral glucose doesn not appear to be mediated by gastric inhibitory polypeptide.
Z Chap, T Ishida, J Chou, R Lewis, C Hartley, M Entman, J B Field
Inflammatory pulmonary injury was induced in Macaca mulatta rhesus monkeys by the intrabronchial instillation of the formylated peptide norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA). Indicators of pulmonary injury included an increase in mean protein content of bronchoalveolar lavage (BAL) fluid from 0.51 mg/ml in untreated animals to 3.74 mg/ml and 6.64 mg/ml in FNLP- and PMA-treated animals, respectively, the appearance of a diffuse pulmonary infiltrate in chest roentgenograms, and histologic evidence of a predominantly neutrophilic leukocytic infiltration. Concomitant with the appearance of pulmonary injury was the generation of proteases and oxidants in the BAL fluids. Neutrophil elastase, bound to alpha 1-protease inhibitor (alpha 1-PI), was found to increase from 0.47 micrograms/ml in untreated monkeys to 0.99 micrograms/ml in FNLP-treated animals and 1.23 micrograms/ml in monkeys receiving PMA. Radioiodinated human prekallikrein, instilled for 2 min into the inflammatory site and retrieved by lavaging, was found to have undergone proteolytic cleavage; this cleavage was not consistently inhibitable with the inclusion of antibody to elastase. BAL fluids were shown to contain an amidolytic activity when tested on the synthetic substrate H-D-pro-phe-arg-pNA. This activity was partially inhibitable with known inhibitors of active Hageman factor and kallikrein. beta-Glucuronidase levels in the BAL fluids increased from 0.85 U/ml to 4.36 U/ml and 8.25 U/ml in FNLP- and PMA-treated animals, respectively. Myeloperoxidase (MPO) levels also increased from 1.37 OD U/ml X min to 16.59 and 30.47 OD U/ml X min in the same groups of animals. Oxidant generation was also assessed in several different ways. The specific activity of the oxidant-sensitive inhibitor alpha 1-PI recovered in the BAL fluid decreased from 0.80 in control samples to 0.57 and 0.65 in FNLP- and PMA-treated animals. That this inactivation was due to oxidant injury of the molecule was confirmed by the return to full activity of four out of five BAL samples after their incubation with the reducing agent dithiothreitol in the presence of methionine sulfoxide peptide reductase. The specific activity of catalase in the BAL fluids of animals given 3-amino, 1,2,4 triazole (AT) 1 h before lavaging showed drops from 0.97 in untreated monkeys to 0.04 in FNLP-treated and 0.49 in PMA-treated monkeys. MPO levels also fell in the AT-treated injured animals from 16.59 to 0.85 delta OD/min X ml in FNLP animals in the absence and presence of AT, and 30.47 to 0.60 delta OD/min X ml in PMA-treated animals. Inhibition of MPO by AT was shown in vitro to be H2O2 dependent. Total glutathione levels in the BAL fluids did not change appreciably after FNLP or PMA treatment. These studies present substantial evidence of the generation of both proteases and oxidants during the establishment of acute pulmonary inflammatory injury in an experimental primate model.
S D Revak, C L Rice, I U Schraufstätter, W A Halsey Jr, B P Bohl, R M Clancy, C G Cochrane
The utility of freshly isolated suspensions of rabbit tubules enriched in proximal segments for studying the pathogenesis of oxygen deprivation-induced renal tubular cell injury was evaluated. Oxygenated control preparations exhibited very good stability of critical cell injury-related metabolic parameters including oxygen consumption, cell cation homeostasis, and adenine nucleotide metabolism for periods in excess of 2 h. Highly reproducible models of oxygen deprivation-induced injury and recovery were developed and alterations of injury-related metabolic parameters in these models were characterized in detail. When oxygen deprivation was produced under hypoxic conditions, tubules sustained widespread lethal cell injury and associated metabolic alterations within 15-30 min. However, when oxygen deprivation was produced under simulated ischemic conditions, tubules tolerated 30-60 min with only moderate amounts of lethal cell injury occurring, a situation similar to that seen with ischemia in vivo. Like ischemia in vivo, simulated ischemia in vitro was characterized by a fall in pH during oxygen deprivation. No such fall in pH occurred in the hypoxic model. To test whether this fall in pH could contribute to the protection seen during simulated ischemia in vitro, tubules were subjected to hypoxia at medium pHs ranging from 7.45 to 6.41. Striking protection from hypoxic injury was seen as pH was reduced with maximal protection occurring in tubules made hypoxic at pHs below 7.0. Measurements of injury-associated metabolic parameters suggested that the protective effect of reduced pH may be mediated by pH-induced alterations of tubule cell Ca++ metabolism. This study has, thus, defined and characterized in detail a new and extremely versatile model system for the study of oxygen deprivation-induced cell injury in the kidney and has established that pH alterations play a major role in modulating such injury.
J M Weinberg
Hepatic cirrhosis with portal hypertension and gastroesophageal hemorrhage is a disease complex that continues to be treated by surgical portasystemic shunts. Whether or not a reduction or diversion of portal blood flow to the liver adversely affects the ability of the liver to maintain fuel homeostasis via gluconeogenesis, glycogenolysis, and ketogenesis is unknown. 11 patients with biopsy-proven severe hepatic cirrhosis were studied before and after distal splenorenal or mesocaval shunts. Hepatic, portal, and renal blood flow rates and glucose, lactate, pyruvate, glycerol, amino acids, ketone bodies, free fatty acids, and triglyceride arteriovenous concentration differences were determined to calculate net precursor-product exchange rates across the liver, gut, and kidney. The study showed that hepatic contribution of glucose and ketone bodies and the caloric equivalents of these fuels delivered to the blood was not adversely affected by either a distal splenorenal or mesocaval shunt. In addition to these general observations, isolated findings emerged. Mesocaval shunts reversed portal venous blood and functionally converted this venous avenue into hepatic venous blood. The ability of the kidney to make a substantial net contribution of ketone bodies to the blood was also observed.
O E Owen, M A Mozzoli, F A Reichle, T H Kreulen, R S Owen, G Boden, M Polansky
Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.
S Shak, I M Goldstein
In order to quantitate the pathways by which liver glycogen is repleted, we administered [1-13C]glucose by gavage into awake 24-h fasted rats and examined the labeling pattern of 13C in hepatic glycogen. Two doses of [1-13C]glucose, 1 and 6 mg/g body wt, were given to examine whether differences in the plasma glucose concentration altered the metabolic pathways via which liver glycogen was replenished. After 1 and 3 h (high-dose group) and after 1 and 2 h (low-dose group), the animals were anesthetized and the liver was quickly freeze-clamped. Liver glycogen was extracted and the purified glycogen hydrolyzed to glucose with amyloglucosidase. The distribution of the 13C-label was subsequently determined by 13C-nuclear magnetic resonance spectroscopy. The percent 13C enrichment of the glucosyl units in glycogen was: 15.1 +/- 0.8%(C-1), 1.5 +/- 0.1%(C-2), 1.2 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 1.6 +/- 0.1%(C-5), and 2.2 +/- 0.1%(C-6) for the high-dose study (n = 4, at 3 h); 16.5 +/- 0.5%(C-1), 2.0 +/- 0.1%(C-2), 1.3 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 2.2 +/- 0.1%(C-5), and 2.4 +/- 0.1%(C-6) in the low-dose study (n = 4, at 2 h). The average 13C-enrichment of C-1 glucose in the portal vein was found to be 43 +/- 1 and 40 +/- 2% in the high- and low-dose groups, respectively. Therefore, the amount of glycogen that was synthesized from the direct pathway (i.e., glucose----glucose-6-phosphate----glucose-1-phosphate----UDP-glucose---- glycogen) was calculated to be 31 and 36% in the high- and low-dose groups, respectively. The 13C-enrichments of portal vein lactate and alanine were 14 and 14%, respectively, in the high-dose group and 11 and 8%, respectively, in the low-dose group. From these enrichments, the minimum contribution of these gluconeogenic precursors to glycogen repletion can be calculated to be 7 and 20% in the high- and low-dose groups, respectively. The maximum contribution of glucose recycling at the triose isomerase step to glycogen synthesis (i.e., glucose----triose-phosphates----glycogen) was estimated to be 3 and 1% in the high- and low-dose groups, respectively. In conclusion, our results demonstrate that (a) only one-third of liver glycogen repletion occurs via the direct conversion of glucose to glycogen, and that (b) only a very small amount of glycogen synthesis can be accounted for by the conversion of glucose to triose phosphates and back to glycogen; this suggests that futile cycling between fructose-6-phosphate and fructose-1,6-diphosphate under these conditions is minimal. Our results also show that (c) alanine and lactate account for a minimum of between 7 and 20% of the glycogen synthesized, and that (d) the three pathways through which the labeled flux is measured account for a total of only 50% of the total glycogen synthesized. These results suggest that either there is a sizeable amount of glycogen synthesis via pathway(s) that were not examined in the present experiment or that there is a much greater dilution of labeled alanine/lactate in the oxaloacetate pool than previously appreciated, or some combination of these two explanations.
G I Shulman, D L Rothman, D Smith, C M Johnson, J B Blair, R G Shulman, R A DeFronzo
To examine the influence of insulin and insulinlike growth factor (IGF) on erythropoiesis, we tested their effects in human bone marrow cultures prepared with biochemically defined medium or a platelet-poor plasma-derived serum (PDS) that was depleted of hormones by adsorption to activated charcoal. Erythroid colony formation was enhanced two- to threefold by 10 ng/ml of electrophoretically pure IGF-II and 100 ng/ml of highly purified insulin (P less than 0.05). Dose-response curves for IGF-II were parallel to and shifted by one to two orders of magnitude to the left relative to those for insulin. When added together to culture, IGF-II and insulin expressed additive activities. In contrast, their activities were synergistic with those of erythropoietin and burst-promoting activity. The erythropoietic actions of IGF-II and insulin were similar in PDS and whole blood serum (WBS) containing cultures. Furthermore, when added to cultures with electrophoretically pure platelet-derived growth factor, their respective activities were synergistic. We conclude that insulin and IGF-II potentiate human marrow erythropoiesis in vitro. Their activities appear to be mediated by a similar receptor or postreceptor system.
N Dainiak, S Kreczko
Precursors of plasma cells were studied in the bone marrow of 28 patients with multiple myeloma, plasma cell leukemia, and benign monoclonal gammopathy. Pre-B and B cell populations were analyzed with anti-B monoclonal antibodies corresponding to the clusters standardized at the Leucocyte Typing Workshops in Paris and Boston (CD9, CD10, CD19-22, CD24). In advanced forms of plasma cell malignancies, such as cases of multiple myeloma in stages II and III and of plasma cell leukemia, some cells of lymphoid morphology expressed common acute lymphoblastic leukemia antigen (CALLA, CD10) and HLA-DR, but contained no detectable terminal deoxynucleotidyl transferase enzyme. These CALLA+ cells were absent in benign monoclonal gammopathies. In multiple myeloma, the CALLA+ cells were negative for surface and cytoplasmic immunoglobulins (Ig), and, unlike CALLA+, terminal deoxynucleotidyl transferase (TdT+) pre-B cells in the normal bone marrow also failed to react with antibodies to B cell-associated antigens such as CD9, CD19, CD22, and CD24. The CALLA+, Ig- cells could be regarded as preplasmacytic since, after having been separated and stimulated with the phorbol ester 12-0-tetradecanoyl-phorbol-13 acetate in vitro, they transformed into plasma cells and synthesized the same heavy and light chains as myeloma cells.
F Caligaris-Cappio, L Bergui, L Tesio, G Pizzolo, F Malavasi, M Chilosi, D Campana, B van Camp, G Janossy
To investigate the unique distribution in plasma of apolipoprotein A-IV (apo A-IV) we have determined, in a series of in vitro and in vivo studies, the redistribution among lipoproteins of 125I-apo A-IV. Free 125I-apo A-IV associated predominantly with high density lipoprotein (HDL) (72 +/- 3.5%) in incubations with plasma, and with triglyceride-rich lipoproteins (TRL) (65 +/- 3.0%) in incubations with lymph, rather than with the lipoprotein-deficient fraction (LDF) where greater than 90% of apo A-IV resides. Incubations with 125I-apo A-IV (incorporated within HDL or TRL) also resulted in similar redistributions of label. Specific radioactivities of apo A-IV in HDL and in TRL were of a similar order and 15-fold higher than those in LDF. However, when 125I-apo A-IV in LDF was incubated with plasma, 57 +/- 2.6% of label remained in the LDF, though the specific radioactivity of apo A-IV in HDL was 15-fold higher than in LDF. Thus, apo A-IV apparently exchanges freely between TRL, HDL, and a part of apo A-IV in LDF, but most of apo A-IV in LDF is refractive to free exchange or transfer. In vivo experiments carried out in five subjects, in which 125I-apo A-IV was injected within TRL, HDL, or LDF, were consistent with the in vitro data in showing rapid exchange of label among plasma apo A-IV containing fractions with much higher specific radioactivities in HDL than in LDF (10-30-fold). However, the small fraction of apo A-IV in LDF that did become labeled was removed from plasma in a biexponential fashion and at the same rate as from HDL. Thus, only a small fraction of the bulk of apo A-IV in plasma LDF exchanges freely with apo A-IV in TRL and HDL, suggesting that apo A-IV in LDF exists in at least two pools. This is consistent with our previous findings that apo A-IV in plasma is present in two distinct complexes with lipids and other peptides.
T Ohta, N H Fidge, P J Nestel
The purpose of the present study was to develop immunotoxins directed against human ovarian carcinoma cells. Four monoclonal antibodies (260F9, 454C11, 280D11, and 245E7) were chosen because they were found to bind to various ovarian carcinoma cell lines. These antibodies were covalently linked to either Pseudomonas exotoxin (PE) or ricin A chain (RTA), and the conjugates were tested against five ovarian cancer cell lines (OVCAR-2, -3, -4, -5; A1847). The ability of the immunotoxins to inhibit both protein synthesis and colony formation was evaluated. Qualitatively similar results were obtained for both types of assays. Usually, PE conjugates were more toxic than their corresponding RTA conjugates. 454C11-PE was very toxic for all ovarian carcinoma lines, whereas 454C11-RTA had low activity. Both 260F9-PE and 260F9-RTA were active in all OVCAR cell lines but not in A1847 cells. 280D11-PE was toxic for OVCAR-4; otherwise, 280D11-PE and RTA conjugates of both 280D11 and 245E7 had little activity. Specificity of immunotoxin action was shown by competition by excess antibody, nontoxicity in nontarget cells, and inactivity of an irrelevant immunotoxin. To investigate the basis of antibody-dependent differences in activity of the various immunotoxins, antibody uptake was studied in OVCAR-2 cells, and the results indicate that antibody internalization is one important factor in the activity of immunotoxins.
R Pirker, D J FitzGerald, T C Hamilton, R F Ozols, W Laird, A E Frankel, M C Willingham, I Pastan
The thermic effect of glucose was investigated in nine obese and six lean subjects in whom the same rate of glucose uptake was imposed. Continuous indirect calorimetry was performed for 240 min on the supine subject. After 45 min, 20% glucose was infused (609 mg/min) for 195 min and normoglycemia was maintained by adjusting the insulin infusion rate. At 2 h, propranolol was infused (bolus 100 micrograms/kg; 1 microgram/kg X min) for the remaining 75 min. To maintain the same glucose uptake (0.624 g/min), it was necessary to infuse insulin at 3.0 +/- 0.6 (leans) and 6.6 +/- 1.2 mU/kg X min (obese) (P less than 0.02). At this time, glucose oxidation was 0.248 +/- 0.019 (leans) and 0.253 +/- 0.022 g/min (obese) (NS), and nonoxidative glucose disposal was 0.375 +/- 0.011 and 0.372 +/- 0.029 g/min, respectively. Resting metabolic rate (RMR) rose significantly by 0.13 +/- 0.02 kcal/min in both groups, resulting in similar thermic effects, i.e., 5.5 +/- 0.7% (leans) 5.4 +/- 0.9% (obese) (NS) and energy costs of glucose storage 0.35 +/- 0.06 and 0.39 +/- 0.09 kcal/g (NS), respectively. With propranolol, glucose uptake and storage remained the same, while RMR fell significantly in both groups, with corresponding decreases (P less than 0.05) in the thermic effects of glucose to 3.7 +/- 0.6% and 2.9 +/- 0.8% (NS) and the energy costs of glucose storage 0.23 +/- 0.04 and 0.17 +/- 0.05 kcal/g (NS) in the lean and obese subjects, respectively. These results suggest that the defect in the thermic effect of glucose observed in obese subjects is due to their insulin resistance, which is responsible for a lower rate of glucose uptake and hence decreased rate of glucose storage, which is an energy-requiring process.
E Ravussin, K J Acheson, O Vernet, E Danforth, E Jéquier
A patient with a lymphoproliferative disorder developed bleeding associated with a prolonged bleeding time and a selective defect of platelet aggregation in response to ristocetin. The patient's purified IgG was shown to inhibit aggregation of washed normal platelets by ristocetin and von Willebrand factor (F VIII:vWF). By Western blotting, it was shown that antibody bound specifically to an antigen of Mr 210,000 present on normal platelets but missing on platelets from patients with congenital Bernard-Soulier syndrome (BSS). Binding was effected by the F(ab)2 portion of the IgG, indicating the presence of an autoantibody rather than an immune complex. These results suggest that the 210,000-Mr protein is involved in the interaction of F VIII:vWF with platelets. Furthermore, we have demonstrated the apparent absence of an additional protein on congenital BSS platelets. Heat-aggregated IgG was also shown to bind to the 210,000-Mr protein, suggesting that this protein may function as an Fc receptor on platelets. The relationship of the 210,000-Mr protein to glycoprotein Ib and the precise role of this protein in the interaction of platelets with F VIII:vWF need to be characterized.
R B Stricker, D Wong, S R Saks, L Corash, M A Shuman
A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.
B S Schwartz, P J Reitnauer, J A Hank, P M Sondel
Abnormal T cell function is a feature of a spectrum of inherited and acquired diseases. We have detected a frequent restriction fragment length polymorphism in the human T cell antigen receptor beta-chain locus that may aid in the analysis of these disorders. A study of a panel of 18 normal individuals, testing for the presence of the polymorphism, showed it to account for 36% of the alleles in that group. In view of the fact that the T cell receptor beta-chain locus has been mapped to chromosome 7, and that the disease ataxia telangiectasia (AT) is associated both with abnormal T cell function and with chromosomal abnormalities of the same region of chromosome 7, we investigated the possibility that the polymorphism could demonstrate linkage of the T cell receptor locus to the gene for that disease. We demonstrated that the mutation causing AT did not lie within the beta-chain locus itself, and that there was preliminary evidence that the two loci were not closely linked. This polymorphism may provide a useful tool for the study of other genetic disorders associated with abnormalities of T cell function, as well as disorders associated with inherited or acquired abnormalities of chromosome 7.
N Berliner, A D Duby, C C Morton, P Leder, J G Seidman
To facilitate the direct study of progenitor cell biology, we have developed a simple and efficient procedure based upon negative selection by panning to purify large numbers of committed erythroid and myeloid progenitors from human fetal liver. The nonadherent, panned cells constitute a highly enriched population of progenitor cells, containing 30.4 +/- 13.1% erythrocyte burst forming units (BFU-E), 5.5 +/- 1.9% granulocyte-macrophage colony forming units (CFU-GM), and 1.4 +/- 0.7% granulocyte-erythroid-macrophage-megakaryocyte colony forming units (CFU-GEMM) as assayed in methylcellulose cultures. These cells are morphologically immature blasts with prominent Golgi. This preparative method recovers 60-100% of the committed progenitors detectable in unfractionated fetal liver and yields 2-30 X 10(6) progenitors from each fetal liver sample, and thus provides sufficient numbers of enriched progenitors to allow direct biochemical and immunologic manipulation. Using this technique, a purified recombinant protein previously thought to have only granulocyte-macrophage colony stimulating activity (GM-CSA) is shown to have both burst promoting activity and multipotential colony stimulating activity. Progenitor purification by panning thus appears to be a simple, efficient method that should facilitate the direct study of committed hematopoietic progenitors and their differentiation.
S G Emerson, C A Sieff, E A Wang, G G Wong, S C Clark, D G Nathan