The Effects of the Immunologic Release of Histamine upon Human Lung Cyclic Nucleotide Levels and Prostaglandin Generation

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Abstract

The effect of the antigen-induced, immunoglobulin (Ig)E-dependent release of mediators from human lung tissue was analyzed for coincident changes in the tissue levels of cyclic nucleotides. Simultaneously with the appearance of mediators, lung cyclic guanosine 3′,5′-monophosphate (GMP) increased from 0.9±0.2 to 12.63±4.5 pmol/mg protein and cyclic AMP increased threefold from the initial levels of 5.1±1.4 pmol/mg protein. The release of histamine and prostaglandin (PG)F2α, as well as the associated increases in cyclic nucleotides, peaked within 10 min of anaphylaxis. Antagonists of histamine's H-1 receptor prevented anaphylaxis-associated increases in cyclic GMP, whereas H-2 antagonists prevented the cyclic AMP response. Neither of these antagonists influenced the pattern or quantity of histamine or slow-reacting substance of anaphylaxis release. Prevention of PGF2α synthesis with acetylsalicylic acid failed to influence histamine or slow-reacting substance of anaphylaxis release or the concomitant increases in cyclic nucleotides. Histamine, added exogenously, produced a prompt increase in the cyclic AMP and cyclic GMP levels of human lung. As was seen after anaphylaxis, H-1 anatagonists prevented the cyclic GMP response to histamine, whereas H-2 antagonists prevented the cyclic AMP response.

Authors

Leslie F. Platshon, Michael Kaliner

The Micellar Sink: A QUANTITATIVE ASSESSMENT OF THE ASSOCIATION OF ORGANIC ANIONS WITH MIXED MICELLES AND OTHER MACROMOLECULAR AGGREGATES IN RAT BILE

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Abstract

Although the importance of mixed micelles in the solubilization and biliary excretion of lipids is established, little is known about a possible role of mixed micelles in the excretion of other biliary solutes. Ultrafiltration and ultracentrifugation techniques were used to investigate the interaction between substances that are excreted in bile and biliary mixed micelles. Substances (urea, erythritol, sucrose) excreted in bile at concentrations equal to, or less than, that in plasma did not show an association with mixed micelles, whereas substances (indocyanine green, iopanoic acid, rose bengal, unconjugated and conjugated sulfobromophthalein, and conjugated bilirubin) excreted in bile at high concentration relative to plasma did. The percentage of these latter substances in bile associated with micelles varied from 26 to 93% and was relatively independent of concentration. In addition to their association with mixed micelles, these test solutes formed self-aggregates that were stabilized primarily by ionic bonds, and only a small percentage (range = 0-5%) of these solutes were present in bile in the form of monomer or complexes small enough to pass a 5,000-mol wt membrane.

Authors

Bruce F. Scharschmidt, Rudi Schmid

Acquired Cell-Mediated Immunodepression in Acute Chagas' Disease

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In this study two groups of patients with acute Chagas' disease were identified. Group one consisted of five patients with apparent acute Chagas' disease. These patients showed symptoms and signals of an acute illness, such as high fever and enlarged spleen. One of these patients developed severe myocarditis and heart failure. Group two consisted of seven patients with inapparent acute Chagas' disease. This was a nonclinical entity, not perceived by the patient who did not seek medical care. The diagnosis was made by the shift of a serologic test which indicates the presence of immunoglobulin M antibodies to Trypanosoma cruzi.

Authors

Antonio R. L. Teixeira, Glória Teixeira, Vanize Macêdo, Aluizio Prata

Antiherpesvirus Activity in Human Sera and Urines after Administration of Adenine Arabinoside: IN VITRO AND IN VIVO SYNERGY OF ADENINE ARABINOSIDE AND ARABINOSYLHYPOXANTHINE IN COMBINATION

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The minimum inhibitory concentration (MIC) of adenine arabinoside (ara-A) in rabbit kidney microtiter tissue cultures (RK-13) to a prototype strain of herpes simplex virus, type 1 (E115) based upon inhibition of cytopathic effects is 1.5 μg/ml. In this system, the MIC of arabinosylhypoxanthine (ara-Hx), the major in vivo metabolic derivative of ara-A, is 75 μg/ml. Inhibition of cytopathic effects of herpes simplex virus, type 1 (HSV-1) in microtiter wells of RK-13 cells varies directly with the concentrations of ara-A or ara-Hx, and inversely with residual HSV-1. The MIC of ara-A for HSV-1 in RK-13 cells is 5-20 times lower than similar measures with vero renal, mouse embryo, or human foreskin cultures. With RK-13 tissue cultures in microtiter plates, an assay for “ara-A equivalents” in human body fluids was developed which compares in sensitivity with high pressure liquid chromatography and has the advantage of simultaneously measuring combined antiherpesvirus effects of ara-A and its major metabolic derivative, ara-Hx.

Authors

Keith J. Champney, Carl B. Lauter, Elizabeth J. Bailey, A. Martin Lerner

Immunoregulation in humans: control of antitetanus toxoid antibody production after booster immunization.

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Abstract

Booster immunization of normal individuals with soluble tetanus toxoid resulted in the ability of the individuals' peripheral blood lymphocytes to synthesize immunoglobulin (Ig)G antitetanus toxoid antibody in vitro when stimulated by pokeweed mitogen. The capacity for this in vitro antitetanus toxoid antibody response developed within 14 days after booster immunization, reached a peak between days 36--50, and disappeared by day 60. The inability of pokeweed mitogen to stimulate antitetanus toxoid antibody synthesis in vitro before booster immunization was not due to excess suppression by thymus-derived (T) lymphocytes but reflected insufficient numbers of functionally specific helper T lymphocytes and bone marrow-derived (B) lymphocytes. Antigen-specific T-lymphocyte suppression and decreased B-lymphocyte function were associated with the observed reduction of in vitro synthesis of antitetanus toxoid antibody from 20--60 days post-immunization. The in vitro kinetics of antitetanus toxoid antibody synthesis paralleled the synthesis of total IgG in that stimulation by pokeweed mitogen was required and that antibody secretion into the medium initiated by day 4 and increased through day 9.

Authors

R H Stevens, A Saxon

Gonococcal interactions with polymorphonuclear neutrophils: importance of the phagosome for bactericidal activity.

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Gonococci are capable of attaching to the surface of polymorphonuclear leukocytes (PMN). In this location they resist phagocytosis and are not killed by PMN. To delineate the factors involved in the survival of these gonococci, we investigated the interaction of virulent gonococci, which adhere to cells and resist phagocytosis, and avirulent gonococci, which are phagocytized and killed by PMN. In the presence of serum, both virulent and avirulent gonococci associate equally well with PMN and stimulate increases in oxidative metabolism. In the absence of serum virulent gonococci attached to PMN and stimulated PMN oxidative metabolism to a greater extent than avirulent gonococci which did not attach to PMN (P = 0.0009). Therefore, the survival of virulent gonococci attached to the PMN surface is not a result of failure to activate oxidative and bactericidal mechanisms. Both virulent and avirulent gonococci stimulated equivalent PMN specific granule release as measured by the appearance of lactoferrin in the media. Phagocytosis of avirulent gonococci stimulated significantly greater beta-glucuronidase release (P = 0.01) and myeloperoxidase-mediated iodination of protein (P = 0.001) by PMN than attachment of virulent gonococci. In the absence of serum neither type of gonococci stimulated beta-glocuronidase release or protein iodination by PMN. Thus, virulent gonococci fail to stimulate primary granule release by PMN. To further assess the role of attachment versus ingestion on the survival of gonococci, PMN were treated with cytochalasin B to block ingestion. Cytochalasin B-treated PMN were unable to kill either virulent or avirulent gonococci despite normal degranulation stimulated by the latter. The failure of PMN to kill surface-attached gonococci appears to be a consequence of the failure of PMN to enclose the virulent gonococci within a phagosome. The phagocytic vacuole thus plays a critical role in normal PMN bactericidal activity by providing a closed space in which the proper concentration of substances may be achieved to generate microbicidal activity.

Authors

P Densen, G L Mandell

Human Monocyte Antibody-Dependent Cell-Mediated Cytotoxicity to Tumor Cells

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Abstract

Previous investigations of mononuclear cell antibody-dependent cell-mediated cytotoxicity (ADCC) toward tumor cells suggest that K lymphocytes and not monocytes are active in this cytotoxic reaction. This report, however, demonstrates that human monocytes are able to carry out ADCC toward three different human tumor cell lines (CEM T lymphoblasts, Raji bone marrow-derived (B) lymphoblasts, and HeLa cells). The cytolytic event was found to be temperature dependent and rapid, with most of the lysis occurring in the first 4 h of incubation. The extent of lysis was directly related to the number of monocytes (effector cells) and to the degree of antibody sensitization of the target cells. The antibody-dependent cell contact-mediated nature of the cytolytic event was confirmed by inhibition with competing nonspecific monomeric immunoglobulin and by the ability of monocytes in “innocent bystander” experiments to lyse antibody-coated targets but not nonantibody-coated target cells. Evidence that monocytes were clearly the effector cells in the monocyte preparations included the observation that preincubation of effector cells with opsonized zymosan particles abolished ADCC by monocytes, but had little effect on lymphocyte ADCC. Furthermore, no evidence for Fc receptor K lymphocyte contamination of the monocyte preparations was found using antibody-coated target cells that were selectively lysed by lymphocytes but not monocytes. We suggest that ADCC toward tumor cell targets may prove to be a useful assay of monocyte function in normal and disease states.

Authors

George M. Shaw, Paul C. Levy, Albert F. Lobuglio

Serum Amyloid A: EVIDENCE FOR ITS ORIGIN IN POLYMORPHONUCLEAR LEUKOCYTES

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In this study the presence of an amyloid A, antigenically related material was determined in four subpopulations of human leukocytes. Monocytes, granulocytes, thymus-derived lymphocytes, and bone marrow-derived and null lymphocytes were isolated from the peripheral blood of five apparently normal subjects, two patients with secondary amyloidosis, three patients with acute infections, and seven patients with metastatic cancer. Mononuclear leukocytes, isolated from the interface of a Ficoll-Hypaque gradient, were separated into monocytes, thymus-derived lymphocytes, and bone marrow-derived plus null lymphocytes by glass adherence and depletion of sheep erythrocyte rosette-forming lymphocytes. Granulocytes were isolated by sedimentation in 2% methyl cellulose from the erythrocyte-rich pellet formed at the bottom of the Ficoll-Hypaque gradient. The four isolated leukocyte subpopulations were cultured and, at varying intervals, the amyloid A content of the culture medium and of sonicated, 2 × 106 cells was determined by radioimmunoassay. Our results indicated a 2-14 times greater amount of amyloid A-related material in the sonicated granulocytes compared with the individuals' serum amyloid A levels. The mononuclear subpopulations showed a low or negligible amyloid A content. The amount of amyloid A antigenic material was further found to increase in cultured granulocytes, reaching a peak value between the 16th and 30th h of culture. The granulocytes of only two out of eight individuals tested released amyloid A antigenically related material into the culture medium. This release was found to be blocked by the presence of colchicine, vincristine, puromycin, or cycloheximide in the culture medium. In contrast, only the presence of puromycin or cycloheximide was shown to significantly inhibit the intracellular increase of amyloid A in the cultured granulocytes. Thus, it appears that among the circulating blood cells, the granulocytes produce amyloid A antigenically related material and could release it under conditions that remain to be further defined.

Authors

C. Julian Rosenthal, Lee Sullivan

Properties of endogenous somatostatin-like immunoreactivity and synthetic somatostatin in dog plasma.

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Somatostatin-like immunoreactivity (SLI) in the peripheral venous plasma of dogs and in their pancreatic and gastric venous effluents was characterized and compared with synthetic somatostatin. Both endogenuous plasma SLI and somatostatin added to plasma were eluted from Sephadex gels at pH 8.8 in the 150,000--200,000-mol wt region but at pH 2.5 both appeared in the 1,500--2,000-mol wt region. The SLI released from the isolated dog pancreas perfused with plasma-free buffer was eluted entirely as a 1,600-dalton polypeptide, but when the pancreas was perfused with plasma, SLI was eluted in the 150,000--200,000-mol wt zone. Affinity chromatography of plasma samples on immobilized antibodies directed against the central portion of the somatostatin molecule (residues 5--9 and 11) removed approximately equal to 90% of both endogenous SLI and somatostatin added to plasma, but neither was removed by affinity chromatography on antibodies directed against the NH2-terminal region of somatostatin (residues 1--4). The SLI from plasma and from pancreas perfusate isolated by affinity chromatography was identical in molecular size, charge, and immunometric properties to synthetic somatostatin. It is concluded that endogenous SLI is secreted by the pancreas and stomach in a form not distinguishable from synthetic somatostatin, but circulates in plasma bound to large molecular weight components; the NH2-terminal residues of somatostatin appear to be important in this binding.

Authors

J M Conlon, C B Srikant, E Ipp, V Schusdziarra, W Vale, R H Unger

Reversible depression in myocardial performance in dogs with experimental phosphorus deficiency.

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Abstract

The effects of phosphorus depletion on cardiac muscle function in six awake dogs were evaluated with surgically implanted transducers to serially measure ascending aortic root blood flow and high fidelity left ventricular pressure. After the animals recovered from surgery, phosphorus depletion was induced by feeding them a synthetic phosphorus-deficient diet plus aluminum carbonate gel for 35 days, followed by the same diet with phosphorus supplementation for 21 days. In addition to the cardiac studies, sequential measurements of phosphorus content in skeletal muscle and phosphorus in serum were obtained to ascertain the level of phosphorus depletion. Serum inorganic phosphorus concentration (mg/100 ml) decreased from 5.1 +/- 0.1 on day 0 to 0.9 +/- 0.1 on day 35 (P less than 0.01), and total muscle phosphorus (content mmul/100 g fat-free dry weight) decreased from 28.0 +/- on day 0 to 22.6 +/- 0.5 on day 35 (P less than 0.01). During the period of phosphorus depletion, there was no significant change in heart rate; however, stroke volume (milliliter) and peak blood flow velocity (centimeter per second) declined from 24 +/- 2 to 17 +/- 2 (P less than 0.01) and 121 +/- 12 to 98 +/- 7 (P less than 0.01), respectively. Maximum ascending aortic blood flow acceleration (centimeter per second square) and maximum left ventricular time rate of change of pressure (mm Hg per second) also decreased from 4,630 +/- 313 to 3,817 +/0 346 (P less than 0.01) and 2,582 +/- 347 to 2,120 +/- 297 (P less than 0.01) during phosphorus depletion. After repletion all values returned to control values. These results indicate that moderate diet-induced phosphorus depletion can depress myocardial performance. With repletion of phosphorus, myocardial performance improves.

Authors

T J Fuller, W W Nichols, B J Brenner, J C Peterson

Circulating immune complexes in sera of children with neuroblastoma: correlation with stage of disease.

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Abstract

The presence of circulating immune complexes (ICS) in freshly drawn sera of 67 children with neuroblastoma was studied by the Raji cell radioimmunoassay of Theofilopoulos et al. (J. Clin. Invest. 57: 169--182), with particular emphasis on the correlation of levels of ICS with stage of disease and changes attributable to treatment. There was a close correlation between amount of complexes and stage of disease and treatment. Levels of ICS increased as the stage of the disease advanced, and were significantly higher (P less than 0.005) in stage IV than in all other stages combined. When patients with stage IV disease were subdivided into "before," "during," and "after" treatment groups, there was a significant decrease in ICS levels as treatment progressed. Studies of complement and complement components did not give such a clear relationship. A significant decrease of hemolytic C1 values was found in patients with "active disease" compared to normal age-matched controls. Some high C3 levels, determined immunochemically, were associated with low hemolytic levels of C3, which were attributed to C3 cleavage detected by immunoelectrophoresis. Based on our survival data, ICS, which were significantly different in 20 patients now decreased when compared to those of other patients, are very valuable in the prognosis of neuroblastoma.

Authors

W E Brandeis, L Helson, Y Wang, R A Good, N K Day

Effects of osmolality and oxygen availability on soluble cyclic AMP-dependent protein kinase activity of rat renal inner medulla.

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Abstract

The renal inner medulla is ordinarily exposed to osmolalities that are much higher and to O2 tensions that are lower than those in other tissues. The effects of media osmolality and O2 availability on basal and arginine vasopressin(AVP)-responsive soluble cyclic (c)AMP-dependent protein kinase activity were examined in slices of rat inner medulla. Increasing total media osmolality from 305 to 750 or 1,650 mosM by addition of urea plas NaCl to standard Krebs-Ringer bicarbonate buffer significantly reduced basal cAMP content and protein kinase activity ratios. This occurred in the presence or absence of O2. Incubation of slices in high osmolality buffer also blunted increases in inner medullary slice cAMP and protein kinase activity ratios induced by O2. These changes reflected predominantly an action of the urea rather than the NaCl content of high osmolality buffers. In contrast to effects on basal activity, high media osmolality significantly enhanced activation of inner medullary protein kinase by AVP. Conversely, increases in media O2 content suppressed AVP stimulation of enzyme activity. This inhibitory effect of O2 was best expressed at low osmolality. Naproxen and ibuprofen, inhibitors of prostaglandin biosynthesis, reduced basal kinase activity ratios and increased AVP responsiveness in the presence, but not in the absence, of O2. Exogenous prostaglandins (PG) modestly increased (PGE2 and PGE1) or did not change (PGF2alpha) cAMP and protein kinase activity ratios in O2-deprived inner medullary slices. Protein kinase activation by PGE2 was not observed in oxygenated inner medulla with high basal activity ratios. The stimulatory effects of PGE2 and PGE1 on protein kinase activity observed in O2-deprived slices were additive with those of submaximal or maximal AVP. PGE2, PGE1, and PGF2alpha all failed to suppress AVP activation of protein kinase. Thus, enhanced endogenous PGE production may contribute to the higher basal protein kinase activity ratios induced by O2. However, the results do not support a role for PGE2, PGE1, or PGF2alpha in O2-mediated inhibition of AVP responsiveness. The present data indicate that both solute content and O2 availability can alter the expression of AVP action on cAMP-dependent protein kinase activity in inner medulla. AVP activation of protein kinase is best expressed when osmolality is high and O2 availability is low, conditions that pertain in inner medulla during hydropenia.

Authors

F R DeRubertis, P A Craven

Altered activity of the nucleotide regulatory site in the parathyroid hormone-sensitive adenylate cyclase from the renal cortex of a patient with pseudohypoparathyroidism.

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Abstract

A series of clinical studies suggest that the primary defect underlying pseudohypoparathyroidism is an abnormality of the parathyroid hormone-receptor-adenylate cyclase complex of the renal cortical cell plasma membrane. In the present study we compared parathyroid hormone-stimulated adenylate cyclase activity in membrane preparations from the renal cortex of three controls and a patient with pseudohypoparathyroidism. In the pseudohypoparathyroid preparation the Km for ATP was significantly greater and parathyroid hormone elicited markedly diminished adenylate cyclase activity at a subsaturating concentration of ATP. In contrast, the dose-response effect of enzyme activity to parathyroid hormone was the same in the control preparations, and that of the pseudohypoparathyroidism kidney, at a saturating concentration of ATP. The apparent alteration in enzyme kinetics, however, was normalized upon addition of guanosine 5'-triphosphate to the reaction mixtures. These results indicate that the defect in the parathyroid hormone-receptor-adenylate cyclase complex of the renal cell membranes, in our patient with pseudohypoparathyroidism, is an abnormal nucleotide receptor site of decreased activity. Such a defect may result in partial uncoupling of the parathyroid hormone receptor and adenylate cyclase, rendering the organ refractory to hormonal stimulation.

Authors

M K Drezner, W M Burch Jr

Deep nephron function after release of acute unilateral ureteral obstruction in the young rat.

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Abstract

The effects of acute unilateral ureteral obstruction (UUO) of 18 h duration on deep nephron function was evaluated in 14 weanling rats with the technique of micropuncture. After release of UUO, 3.4 +/- 0.66% (SE) of the filtered water remained at the tip of the collecting duct nearly fivefold greater than in controls (0.75 +/- 0.10%). Similar differences were seen in fractional sodium that remained at this site. The ratio of tubular fluid osmolality to that of plasma was also reduced in the UUO group (1.53 +/- 0.06 vs. 4.60 +/- 0.26 in controls, P less than 0.001). Single nephron glomerular filtration rate of cortical and deep nephrons was significantly less (P less than 0.001) after release of UUO. Although the percentage of filtering nephrons was significantly reduced in both nephron populations, the decline in glomerular filtration rate was greater in cortical than in juxtamedullary nephrons (cortical:juxtamedullary nephrons = 27.6 +/- 4.5% vs. 53.3 +/- 5.2% in controls, P less than 0.005) which suggests that single nephron glomerular filtration rate is redistributed to deep nephrons after release of UUO. In contrast to cortical nephrons, the amount of tubular fluid which remains near the bend of the loop of Henle of deep nephrons was greater after release of UUO. This appeared to be the result of a decrease in the reabsorption of both water (tubular fluid:plasma inulin = 2.41 +/- 0.16 vs. 7.94 +/- 0.69 in controls, P less than 0.001) and sodium (52.3 +/- 4% vs. 40.7 +/- 2.9% of the filtered sodium in controls, P less than 0.02). It is suggested that this altered reabsorption occurs along both the proximal tubule and descending limb of the loop of Henle of juxtamedullary nephrons. Inner medullary plasma flow (IMPF), as measured with the [125I]albumin-accumulation technique, was significantly depressed before release of UUO, but exceeded control values 90 min postrelease. Such changes imply that the filtration fraction of deep nephrons is decreased and that physical factors in the proximal tubular reabsorption of sodium have been altered. When papillary solute content was measured before release of UUO it was low (428 +/- 23 vs. 1,205 +/- 106 mosmol/kg in controls, P less than 0.001) which indicates that the decline in papillary osmolality is not a consequence of the increased IMPF seen after ureteral release, but rather precedes it. In fact, the decline in papillary osmolality may contribute to the increase in IMPF after release of UUO and to the decreased reabsorption of fluid along the descending limb of the loop of Henle.

Authors

J Buerkert, D Martin, M Head, J Prasad, S Klahr

Hypophosphatemia and Rhabdomyolysis

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Clinical observations suggest that overt rhabdomyolysis may occur if severe hypophosphatemia is superimposed upon a pre-existing subclinical myopathy. To examine this possibility, a subclinical muscle cell injury was induced in 23 dogs by feeding them a phosphorus- and calorie-deficient diet until they lost 30% of their original weight. To induce acute, severe hypophosphatemia in the animals after partial starvation, 17 of the dogs were given large quantities of the same phosphorus-deficient diet in conjunction with an oral carbohydrate supplement, which together provided 140 kcal/kg per day.

Authors

J. P. Knochel, C. Barcenas, J. R. Cotton, T. J. Fuller, R. Haller, N. W. Carter

Direct release of parathyroid hormone fragments from functioning bovine parathyroid glands in vitro.

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To determine the origin of circulating parathyroid hormone fragments, hormonal peptides released from bovine parathyroid tissue in a physiologically responsive in vitro "perifusion" system were analyzed by gel exclusion chromatography and region-specific radioimmunoassays. When exposed to low Ca++, the tissue released large quantities of intact hormone (parathyroid hormone 1--84) as well as amino- and carboxyl-terminal fragments. Fragments of the hormone were also released when the tissue was exposed to high Ca++, but the carboxyl fragments comprised a much greater proportion of the hormonal peptides released. Control experiments indicated that fragmentation of the hormone occurred within the gland and not after it was secreted. These experiments provide direct evidence, therefore, that release of fragments from the parathyroid gland may contribute to the immunologic heterogeneity of the hormone in the circulation.

Authors

D A Hanley, K Takatsuki, J M Sultan, A B Schneider, L M Sherwood

Active release of human platelet factor VIII-related antigen by adenosine diphosphate, collagen, and thrombin.

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Abstract

Platelet Factor VIII-related antigen (VIIIR:Ag) represents a significant proportion of the total circulating VIIIR:Ag pool. However, its participation in the events of primary hemostasis has not been shown. We now report that platelet-contained VIIIR:Ag is released from platelets by collagen, ADP and thrombin. The concentrations of these agonists, required for VIIIR:Ag release, are the same or lower than those required for release of serotonin, lysosomal enzymes, or fibrinogen. This release has the features of an energy-dependent secretory response because it is blocked by the metabolic inhibitors, antimycin A and 2-deoxy-D-glucose. The electrophoretic characteristics of the VIIIR:Ag released by collagen and ADP are similar to those of plasma VIIIR:Ag. However, thrombin-released platelet VIIIR:Ag differs from that of plasma in that the less anodal forms are relatively depleted. These differences do not appear to be the result of proteolytic degradation of platelet-derived VIIIR:Ag, but may reflect interactions between specific molecular forms of VIIIR:Ag and the platelet membrane. These studies suggest mechanisms by which platelet-contained VIIIR:Ag may contribute to the primary events of hemostasis.

Authors

J Koutts, P N Walsh, E F Plow, J W Fenton 2nd, B N Bouma, T S Zimmerman

Characterization of the molecular defect in infantile and adult acid alpha-glucosidase deficiency fibroblasts.

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Abstract

Different clinical expressions of acid alpha-glucosidase deficiency have been described. The present study was undertaken to investigate the basic metabolic defect in the infantile and adult forms of the disease. Acid alpha-glucosidase (EC 3.2.1.20) was purified from normal and from adult acid alpha-glucosidase deficiency fibroblasts. The pH optimum; Michaelis constant; electrophoretic mobility in starch; thermal denaturation at pH 4.0 and 7.0; and inhibition by turanose, alpha-methylglucoside and trehalose were the same in purified enzyme from normal and mutant cells. Placental acid alpha-glucosidase was purified to, or near, homogeneity. Monospecific antibodies raised against the enzyme in each of three enzyme peaks obtained from the last purification step were found to cross-react with the enzyme of all three peaks, and with purified, normal fibroblast enzyme. Cross-reacting material (CRM) also was identified in fibroblast lysates from normal subjects and from both forms of acid alpha-glucosidase deficiency. The amount of CRM in the adult form appeared to be significantly less than in normal cells or cells from the infantile form. Enzyme activity was demonstrated in the immune complexes of the normal and adult acid alpha-glucosidase deficiency fibroblasts, but not of the infantile form. Competition for antibody binding sites was observed between normal and both types of mutant enzymes. The findings indicate that this case of infantile acid alpha-glucosidase deficiency is the result of a structural gene mutation which causes the synthesis of a catalytically inactive (CRM-positive) enzyme protein. It appears that in the adult form, the mutation causes a reduction in the amount of the enzyme protein present in the cells.

Authors

N G Beratis, G U LaBadie, K Hirschhorn

Experimental glomerulonephritis in the isolated perfused rat kidney.

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Abstract

The development of immune deposits on the subepithelial surface of the glomerular capillary wall was studied in isolated rat kidneys perfused at controlled perfusion pressure, pH, temperature, and flow rates with recirculating oxygenated perfusate containing bovine serum albumin (BSA) in buffer and sheep antibody to rat proximal tubular epithelial cell brush border antigen (Fx1A). Control kidney were perfused with equal concentrations of non-antibody immunoglobulin (Ig)G. Renal function was monitored by measuring inulin clearance, sodium reabsorption, and urine flow as well as BSA excretion and fractional clearance. Perfused kidneys were studied by light, immunofluorescence, and electron microscopy. All kidneys perfused with anti-Fx1A developed diffuse, finely granular deposits of IgG along the glomerular capillary wall by immunofluorescence. Electron microscopy revealed these deposits to be localized exclusively in the subepithelial space and slit pores. Similar deposits were produced in a nonrecirculating perfusion system, thereby excluding the formation of immune complexes in the perfusate caused by renal release of tubular antigen. Control kidneys perfused with nonantibody IgG did not develop glomerular immune deposits. Renal function and BSA excretion were the same in experimental and control kidneys. Glomerular deposits in antibody perfused kidneys were indistinguishable from deposits in rats injected with anti-Fx1A or immunized with Fx1A to produce autologous immune complex nephropathy. These studies demonstrate that subepithelial immune deposits can be produced in the isolated rat kidney by perfusion with specific antibody to Fx1A in the absence of circulating immune complexes. In this model deposits result from in situ complex formation rather than circulating immune complex deposition.

Authors

W G Couser, D R Steinmuller, M M Stilmant, D J Salant, L M Lowenstein

Evidence that the Brain Participates in the Humoral Natriuretic Mechanism of Blood Volume Expansion in the Dog

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We examined the role of the central nervous system in the activation of the humoral natriuretic mechanism elicited by blood volume expansion. Studies were performed in anesthetized dogs pretreated with deoxycorticosterone acetate (15 mg/day) and sodium chloride for 12 days. An isolated dog kidney perfused with blood from the femoral artery of the volume expanded dog served as the bioassay system for the humoral natriuretic factor. In group I volume expansion of intact dogs (n = 14) with equilibrated blood promoted an increase in fractional sodium excretion (FENa) from a control level of 2.6±0.5 to 13.6±1.6%, P <0.001. In the isolated kidney FENa increased from 3.6±0.8 to 6.8±1.1%, P <0.01. The natriuresis from the isolated kidney occurred in the absence of significant changes in renal arterial pressure, glomerular filtration rate, plasma protein concentration, or packed cell volume, whereas renal blood flow decreased slightly. In group II (n = 20) the dogs were decapitated by means of a specially designed neck vise. In 10 dogs blood pressure was supported by a constant infusion of dopamine (3.8±0.7 μg/min per kg body weight). Despite the fact that in response to the same volume stimulus, decapitated dogs manifested an increase in blood volume and cardiac output similar in magnitude to that of intact dogs whereas the rise in mean arterial pressure of decapitated dogs exceeded that of intact dogs, the natriuretic response of decapitated dogs was significantly less than that of intact dogs. FENa in decapitated dogs increased 4.7±1.1 compared to 11.1±1.4% in intact dogs (p <0.01). Furthermore, volume expansion of decapitated dogs failed to elicit a natriuretic response from the isolated kidney. FENa in the isolated kidney measured 2.6±0.4 before and 2.6±0.4% after blood volume expansion. These data indicate that decapitation inhibits activation of the humoral natriuretic mechanism elicited by blood volume expansion and are consistent with the interpretation that the brain is the source of the natriuretic factor or that the brain participates in the activation of the humoral natriuretic mechanism at some other site in the body.

Authors

George J. Kaloyanides, Murgurdich B. Balabanian, Ralph L. Bowman, Philip Pool

Evidence of Incomplete Left Ventricular Relaxation in the Dog: PREDICTION FROM THE TIME CONSTANT FOR ISOVOLUMIC PRESSURE FALL

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Abstract

Although it has been proposed that incomplete relaxation explains certain increases in left ventricular end diastolic pressure relative to volume, there has been no clear demonstration that incomplete relaxation occurs in the intact working ventricle. To identify incomplete relaxation, left ventricular pressure-dimension relationships were studied in 10 canine right heart bypass preparations during ventricular pacing. The fully relaxed, exponential diastolic pressure-dimension line for each ventricle was first determined from pressure and dimension values at the end of prolonged diastoles after interruption of pacing. For 167 beats during pacing under widely varying hemodynamic conditions, diastolic pressure-dimension values encountered this line defining the fully relaxed state during the filling period indicating that relaxation was complete before end diastole. The time constant for isovolumic exponential pressure fall (T) was determined for all beats. For this exponential function, if no diastolic filling occurred, 97% of pressure fall would be complete by 3.5 T after maximal negative dP/dt. For the 167 beats the fully relaxed pressure-dimension line was always encountered before 3.5 T.

Authors

Myron L. Weisfeldt, James W. Frederiksen, Frank C. P. Yin, James L. Weiss

Viral Infection-Homograft Interactions in a Murine Model

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The effects on some host defenses of murine cytomegalovirus (MCMV) and(or) EL4, a mouse ascites homograft, were studied in mice. Assays of cellular and humoral immunity in response to either or both of these perturbations were carried out by quantitation of various immune activities.

Authors

John D. Hamilton, James F. Fitzwilliam, K. S. Cheung, John Shelburne, David J. Lang, D. B. Amos

The modification of biophysical and endotoxic properties of bacterial lipopolysaccharides by serum.

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Abstract

Normal rabbit serum reduces the buoyant density of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (d = 1.44 g/cm3) and Salmonella minnesota R595 (d = 1.38 g/cm3) to a value less than g/cm3. This density shift is associated with the inhibition of a number of endotoxic activities of the LPS; namely, the pyrogenic activity, the ability to produce an immediate neutropenia in rabbits, lethality in adrenalectomized mice, and anticomplementary activity. A qualitatively similar change in buoyant density was observed to occur after intravenous injection of the LPS into rabbits. Preliminary evidence suggests that the density shift does not occur as a result of the degradation of the glycolipid backbone of the LPS. These data suggest that the interactions of LPS with plasma (or serum) components leading to reduction in buoyant density may account for a major pathway of LPS detoxification.

Authors

R J Ulevitch, A R Johnston

Aldosterone Stimulation of Riboflavin Incorporation into Rat Renal Flavin Coenzymes and the Effect of Inhibition by Riboflavin Analogues on Sodium Reabsorption

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Abstract

This study was designed to investigate a possible relationship between the effect of aldosterone upon urinary electrolytes and the incorporation of [14C]riboflavin into renal [14C]flavin mononucleotide (FMN) and [14C]flavin adenine dinucleotide (FAD). Adrenalectomized Sprague-Dawley rats that weighed between 185 and 210 g were pretreated with 15 μg/100 g body wt dexamethasone intraperitoneally. 16 h later they were administered aldosterone (1.5 μg/100 g body wt) and [14C]riboflavin (5.0 μCi/200 g body wt). The urethra of each rat was ligated, and the rats were sacrificed by decapitation 3 h later. The urine was aspirated from the bladders of each rat and analyzed for total Na+ and K+ excretion while the kidneys were removed and the formation of [14C]FMN and [14C]FAD was determined for each kidney. There was a significant increase in the formation of renal [14C]FMN and [14C]FAD (27.3 and 14.4%, respectively) after aldosterone treatment. Aldosterone significantly decreased the excretion of Na+ by 50%, and increased that of K+ by 55%.

Authors

Daniel Trachewsky

Modulation of Cyclic Nucleotides in Isolated Rat Glomeruli: ROLE OF HISTAMINE, CARBAMYLCHOLINE, PARATHYROID HORMONE, AND ANGIOTENSIN-II

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Abstract

Because glomerular functions are modulated by numerous humoral agents, probably acting through cyclic nucleotides, the effects of some polypeptide hormones and biogenic amines on cyclic AMP (cAMP) and cyclic 3′,5′-guanosine monophosphate (cGMP) were studied in glomeruli isolated from rat renal cortex. Glomeruli and cortical tubules were prepared by a combination of sieving and density-gradient centrifugation. Under basal conditions, the contents of cAMP and cGMP in glomeruli were significantly higher than in tubules and unfractionated renal cortical tissue.

Authors

Vicente E. Torres, Thomas E. Northrup, Richard M. Edwards, Sudhir V. Shah, Thomas P. Dousa

Mechanism of Inhibition of Porcine Elastase by Human Alpha-1-Antitrypsin

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Abstract

The interaction of the human plasma protein, alpha-1-antitrypsin, with porcine pancreatic elastase was studied by isolating and characterizing their reaction products. Native alpha-1-antitrypsin has a mass ratio (Mr) of 54,000, an amino-terminal glx, and a carboxy-terminal lys residue. The elastase used has an Mr of 26,400 and an amino-terminal val residue. When the two proteins are combined at inhibitor excess, two major products result. One of the products is a complex of the enzyme and inhibitor with amino-terminal ser and val residues, which indicates that a peptide has been removed from the amino-terminal end of the inhibitor. The second product is a modified form of alpha-1-antitrypsin with an Mr of 51,300, an aminoterminal glx residue and a carboxy-terminal thr-leu dipeptide. It has no inhibitory activity against elastase. The components of the isolated complex can be split at high pH in the presence of diisopropyl fluorophosphate, which results in a catalytically inactive enzyme with the same Mr and amino-terminal residue as the native enzyme, and a large fragment of alpha-1-antitrypsin (alpha-1-antitrypsin*). This fragment has an Mr of 50,100, an amino-terminal ser residue and a carboxy-terminal thr-leu dipeptide. Based on these data, the following hypothesis is proposed. Elastase can attack alpha-1-antitrypsin at either of two major sites. If it attacks first at the carboxy side of the thr-leu dipeptide, located in the carboxy-terminal portion of the inhibitor, the alpha-1-antitrypsin is cleaved into two fragments with loss of inhibitory activity and absence of complex formation. If, however, the elastase first attacks an x-ser bond near the amino-terminal end of the inhibitor, the elastase then reacts with alpha-1-antitrypsin at the same leu moiety to form a stable complex with complete inhibition of the enzyme.

Authors

Harold L. James, Allen B. Cohen

Studies on the Structure of Low Density Lipoproteins Isolated from Macaca Fascicularis Fed an Atherogenic Diet

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Abstract

Cynomolgus monkeys, Macaca fascicularis, fed cholesterol-containing saturated-fat diets develop increased levels of high molecular weight plasma low density lipoproteins (LDL), associated with accelerated atherosclerosis. To study the composition and structure of these abnormal particles, LDL from monkeys, fed atherogenic and control diets, were characterized chemically and examined by differential scanning calorimetry and low-angle X-ray scattering. LDL from animals on the experimental diet showed an increase in molecular weight (4.0 to 7.0 × 106, experimental diet compared with 3.0 to 3.7 × 106, control diet) associated with a large increase in cholesterol ester content and concomitant smaller increases in protein, phospholipid, and free cholesterol. There was a strong positive correlation between molecular weight and the number of saturated and monounsaturated cholesterol esters in the particle. In contrast, particle content of polyunsaturated cholesterol esters remained constant despite large changes in total particle cholesterol esters.

Authors

Alan R. Tall, Donald M. Small, David Atkinson

Role of secretory events in modulating human neutrophil chemotaxis.

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Abstract

The relationship between neutrophil polymorphonuclear leukocyte (PMN) locomotion and the exocytosis of neutrophil cytoplasmic granules was studied by assessing these processes in cells migrating through micropore filters and by measuring the effects of degranulating stimuli on PMN chemotaxis, orientation, adhesiveness, and ability to bind the chemoattractant f-Met-Leu-[3H]Phe. Studies of cells migrating through cellulose nitrate filters indicated that concentrations of f-Met-Leu-Phe optimal for exocytosis were greater than those optimal for chemotaxis and actually inhibited cell migration. In other studies incubation of PMNs with concentrations of secretagogues causing exocytosis of 30% or greater PMN lysozyme increased cell adhesiveness and inhibited chemotaxis. PMNs that had secreted more than 30% lysozyme appeared round, did not orient in a gradient of chemoattractant, and were capable of significantly less f-Met-Leu-[3H]Phe binding than were control cells. The decreased binding of f-Met-Leu-Phe was not associated with hydrolysis of chemotactic peptide by washed cells, although peptide hydrolysis was caused by cell products secreted extracellularly after vigorous exocytosis. In contrast, when only 10--15% cellular lysozyme was released f-Met-Leu-Phe binding was enhanced significantly and there was no depression of chemotaxis. The data indicate limited exocytosis of intracellular granule contents is associated with increased availability of PMN cehmotactic factor receptors. Vigorous exocytosis is associated with inactivation of chemotactic responsiveness related to increase cell adhesiveness, decreased PMN binding of chemotactic factors, and to hydrolysis of chemoattractants by factors secreted extracellularly.

Authors

J I Gallin, D G Wright, E Schiffmann

Bile formation in the rat: the role of the paracellular shunt pathway.

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Abstract

Transepithelial movement of water and solute occurs both through the cell membrane as well as across the intercellular junctional complex (paracellular shunt pathways). Permeability of paracellular shunt pathways is increase by transmucosal osmotic gradients, and in certain epithelia these changes are associated with bullous-like deformations (blisters) of the zonula occludens and localization of lanthanum within junctional complexes. Although bile acids increase biliary secretion by osmotic forces, the source of this water movement into bile is not known. In the present studies we examined whether a choleretic infusion of sodium dehydrocholic acid (DHC) or its taurine conjugate, taurodehydrocholate, altered the solute permeability characteristics and morphologic appearance of the junctional complexes of rat hepatocytes. Animals were continuously infused for 1 hr with 1% albumin--0.9% NaCl alone or 120 mumol of DHC and bile flow and biliary clearance of [14C]sucrose, an indirect marker of biliary permeability were measured. The number of intercellular blisters adjacent to the bile canaliculus were counted in an unbiased manner from photographs obtained with scanning electron microscopy. Bile flow and the biliary sucrose clearance remained unchanged in control animals whereas DHC infusions resulted in a progressive increase in the biliary clearance of [14C]sucrose during the 60 min of infusion even though the choleretic response to DHC was stable during the final 30 min of infusion. DHC infusions produced surface invaginations, or blisters, (0.1--0.7 micrometer in diameter) which were located immediately adjacent to the hemi-bile canaliculus and occurred with a frequency of 1.62 +/- 0.08 per hepatocyte surface, which was fivefold greater than observed in controls. In separate groups of animals 5 mM ionic lanthanum chloride was perfused intraportally after taurodehydrocholate infusions, and the number of junctional complexes that contained the electron dense marker were quantitated by transmission electron microscopy. Localization of lanthanum in the junctional complexes of fasted control animals was not observed, whereas approximately equal to 50% of the zonula occludens in DHC-infused animals contained lanthanum which was also occasionally identified within the lumen of the bile canaliculus. These results indicate that infusions of DHC cause blisters adjacent to the junctional complex of rat hepatocytes in association with changes in solute conductivity of the zonula occludens to cations such as ionic lanthanum chloride, and presumably to larger solutes such as sucrose. Qualitatively similar morphologic findings were also observed during the infusion of sodium taurocholate at physiologic rate (40 mumol/h). These studies suggest that the paracellular shunt pathway in the liver is an important site for bile acid-induced water and solute movement into bile.

Authors

T J Layden, E Elias, J L Boyer

Adenosine deaminase deficiency: disappearance of adenine deoxynucleotides from a patient's erythrocytes after successful marrow transplantation.

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Abstract

Accumulation of adenine deoxynucleotides (dATP and dADP) in the erythrocytes of a patient with adenosine deaminase (ADA) deficiency was confirmed. The patient, now 18 mo old, was treated with a bone marrow transplantation from his HLA identical sister at 7 mo of age. Before and after the transplant, his erythrocyte and lymphocyte ADA activities, as well as his erythrocyte nucleotide profiles, were measured. 10 wk after the marrow transplant, no ADA activity could be detected in his erythrocytes, whereas there was a mixture of donor and patient lymphocytes as measured by ADA assays and karyotyping. At the same time, both dATP and dADP had disappeared from his erythrocytes, which were entirely of patient origin. These findings indicate that partial engraftment of donor lymphocytes into an ADA-deficient patient is capable of "correcting" alterations of deoxynucleotide concentrations in the patient's ADA-deficient erythrocytes.

Authors

S H Chen, H D Ochs, C R Scott, E R Giblett, A J Tingle

Decreased circulating thymus-derived cells with receptors for the Fc portion of immunoglobulin G in systemic lupus erythematosus.

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Abstract

Thymus-derived cells with receptors for the Fc portion of immunoglobulin G (Fcgamma+ T cells) have recently been found to have a suppressor function, a function that is decreased in systemic lupus erythematosus (SLE). Fcgamma+ T cells were found significantly diminished in 21 untreated SLE patients, particularly in the 7 patients who had active disease. Most Fcgamma+ T cells were separated with a subpopulation of T cells with low affinity for sheep erythrocytes. Decrease of this subpopulation was dependent on the decrease in Fcgamma+ T cells. Non-T cells with Fcgamma receptors were also diminished in SLE patients, but their decrease did not correlate with disease activity. The decrease in suppressor-cell function in SLE may be a result of loss, rather than of dysfunction, of the suppressor Fcgamma+ T cells.

Authors

D Alarcón-Segovia, A Ruíz-Argüelles

Presence of immunoreactive beta-endorphin in normal human plasma: a concomitant release of beta-endorphin with adrenocorticotropin after metyrapone administration.

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Abstract

To elucidate whether or not beta-endorphin exists in plasma of normal subjects, plasma extracts obtained before and after metyrapone administration were subjected to gel exclusion chromatography, and fractions obtained were assayed by a sensitive radioimmunoassay for beta-endorphin. The basal plasma level of beta-endorphin was 5.8 +/- 1.1 pg/ml (mean +/- SE, n = 5), which rose significantly to the level of 48.9 +/- 3.8 pg/ml after a single oral dose (30 mg/kg of body wt) of metyrapone administration (P less than 0.001). Plasma ACTH levels also increased from the mean basal level of 73 +/- 4 pg/ml to 269 +/- 41 pg/ml after metyrapone administration. These results indicate that beta-endorphin, distinct from beta-lipotropin, exists in normal human plasma and that it is released from the pituitary concomitantly with ACTH.

Authors

K Nakao, Y Nakai, S Oki, K Horii, H Imura