CORRECTION

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Abstract

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Effect of growth hormone-release inhibiting hormone on hormones stimulating exocrine pancreatic secretion.

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The nature and extent of growth hormone-release inhibiting hormone (GH-RIH, somatostatin)-induced inhibition of pancreatic secretion of bicarbonate and protein, an index of enzyme secretion, were studied by administration of exogenous secretin or cholecystokinin (CCK) and of a number of stimulants for endogenous release of these hormones in fasted pancreatic fistula dogs with and without an infusion of GH-RIH. The results of this study show that GH-RIH inhibits the pancreatic fluid and bicarbonate secretion induced by duodenal acidification and exogenous secretion. The kinetic analysis shows that the interaction between GH-RIH and secretin affecting pancreatic bicarbonate secretion possesses the characteristics of competitive inhibition. GH-RIH does not change the pancreatic protein response to exogenous CCK, but profoundly inhibits pancreatic response to a variety of the endogenous stimulants of CCK release, including duodenal perfusion of sodium oleate, amino acid mixture, or feeding of a peptone meal. We conclude that GH-RIH is a very potent inhibitor of the endogenous release of CCK from the intestinal mucosa and inhibits competitively the action of secretin but not CCK on the exocrine pancreatic secretion.

Authors

S J Konturek, J Tasler, W Obtulowicz, D H Coy, A V Schally

The role of adrenergic mechanisms in the substrate and hormonal response to insulin-induced hypoglycemia in man.

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Sequential determinations of glucose outflow and inflow, and rates of gluconeogenesis from alanine, before, during and after insulin-induced hypoglycemia were obtained in relation to alterations in circulating epinephrine, norepinephrine, glucagon, cortisol, and growth hormone in six normal subjects. Insulin decreased the mean (+/-SEM) plasma glucose from 89+/-3 to 39+/-2 mg/dl 25 min after injection, but this decline ceased despite serum insulin levels of 153+/-22 mul/ml. Before insulin, glucose inflow and outflow were constant averaging 125.3+/-7.1 mg/kg per h. 15 min after insulin, mean glucose outflow increased threefold, but then decreased at 25 min, reaching a rate 15% less than the preinsulin rate. Glucose inflow decreased 80% 15 min after insulin, but increased at 25 min, reaching a maximum of twice the basal rate. Gluconeogenesis from alanine decreased 68% 15 min after insulin, but returned to preinsulin rates at 25 min, and remained constant for the next 25 min, after which it increased linearly. A fourfold increase in mean plasma epinephrine was found 20 min after insulin, with maximal levels 50 times basal. Plasma norepinephrine concentrations first increased significantly at 25 min after insulin, whereas significantly increased levels of cortisol and glucagon occurred at 30 min, and growth hormone at 40 min after insulin. Thus, insulin-induced hypoglycemia in man results from both a decrease in glucose production and an increase in glucose utilization. Accelerated glycogenolysis produced much of the initial, posthypoglycemic increment in glucose production. The contribution of glycogenolysis decreased with time, while that of gluconeogenesis from alanine increased. Of the hormones studied, only the increments in plasma catecholamines preceded or coincided with the measured increase in glucose production after hypoglycemia. It therefore seems probable that adrenergic mechanisms play a major role in the initiation of counter-regulatory responses to insulin-induced hypoglycemia in man.

Authors

A J Garber, P E Cryer, J V Santiago, M W Haymond, A S Pagliara, D M Kipnis

A comparative analysis of the C1-binding ability of fragments derived from complement-fixing and noncomplement-fixing IgM proteins.

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The purpose of this study was to examine the molecular parameters necessary for initiation of complement fixation by IgM proteins. To determine why some IgM molecules are capable of complement fixation while others are not, several different Waldenström IgM proteins were examined for their ability to fix total hemolytic complement in the CH(50) assay. Subsequently, the C1 fixing ability of a 56-residue fragment derived from the Cmu4 domain of each of these IgM molecules was studied with C1 fixation assay. One of the three Waldenström IgM proteins (Gr) used in the present study was found unable to consume complement in a CH(50) assay when tested at the same concentration as the two complement-consuming IgM molecules (Dau and Bus). However, when the 56-residue C(H)4 fragment from the Cmu4 domain of each IgM molecule was tested for C1-fixing ability, all three were found to bind C1. On the basis of these observations, it is proposed that a C1 binding site exists within the Cmu4 domain of both complement-fixing and noncomplement-fixing IgM molecules. Presumably, the latter molecules are unable to interact in their native state with C1 in the manner required for initiation of the classical complement pathway, possibly due to the configurational inaccessibility of the entire C1 binding site.

Authors

M M Hurst, J E Volanakis, R M Stroud, J C Bennett

Defective neutrophil chemotaxis in bone marrow transplant patients.

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Infection is a frequent cause of death in patients receiving bone marrow transplants. Although lymphocyte dysfunction has been observed in a few such patients, no systematic study of neutrophil function has yet been reported. Neutrophil chemotaxis was evaluated by a 51Cr-radioassay after bone marrow transplantation in 34 patients with acute leukemia or aplastic anemia. The response to a chemotactic stimulus (C5a) was severely depressed (less than 35% of normal) in 18 patients, moderately depressed (35-65% of normal) in an additional 6, and normal in 10 subjects. The mean response in the absence of graft vs. host disease and antithymocyte globulin administration was 73.3+/-9.2% (SE) in contrast to 29.7+/-9.6% (P is less than 0.01) in patients with graft vs. host disease treated with antithymocyte golbulin. Both graft vs. host disease and antithymocyte globulin were implicated since the presence of either factor alone was associated with depressed chemotaxis (31.1+/-4.9% for graft vs. host disease, P is less than 0.01; 17.0+/-7.8% for antithymocyte globulin, P is less than 0.02). When normal neutrophils were incubated with antithymocyte globulin in vitro, their chemotactic response was markedly suppressed in the absence of a cytotoxic effect. Transplant patients with defective chemotaxis experienced significantly more infections than those with normal chemotaxis, and analysis of specific etiologic agents showed that this was predominantly related to bacterial pathogens. Chemotactic inhibitors were detected in the sera of seven patients and elevated IgE levels were found in nine subjects, eight of whom had graft vs. host disease. Generation of chemotactic activity by endotoxin activation of serum was reduced in five patients. The results demonstrate a severe defect in neutrophil chemotaxis in some bone marrow transplant patients and suggest that neutrophil dysfunction may predispose to infection in such patients.

Authors

R A Clark, F L Johnson, S J Klebanoff, E D Thomas

An assessment of daily production and significance of thyroidal secretion of 3, 3', 5'-triiodothyronine (reverse T3) in man.

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While 3, 3', 5'-triiodothyronine (reverse T3, rT3) has been detected both in human serum and in thyroglobulin, no quantitative assessment of its metabolic clearance rate (MCR), production rate (PR), or secretion by the thyroid is yet available. This study examines this information in euthyroid subjects and evaluates it in light of similar information about two other iodothyronines in the thyroid: 3, 5, 3'-triiodothyronine (T3) and thyroxine (T4). Thus, it was noted that rT3 is cleared from human serum at a much faster rate than are T3 and T4; the mean (+/-SE) MCR of rT3 was 76.7+/-5.4 liters/day in 10 subjects, whereas MCR-T3 and MCR-T4 in 8 of them were 26.0+/-2.2 liters/day and 1.02+/-0.06 liters/day, respectively. Therefore, even though the mean serum concentration of rT3, 48+/-2.8 ng/100 ml, was much lower than that (128+/-6.7 ng/100 ml) of T3, the mean PR-rT3 (36.5+/-2.8 mug/day) and the mean PR-T3 (33.5+/-3.7 mug/day) were similar; in comparison, the mean serum concentration and PR of T4 were 8.6+/-0.5 mug/100 ml and 87.0+/-3.9 mug/day, respecitvely. These data and those on the relative proportion of rT3, T3, and T4 in 10 thyroid glands were used to assess the significance of the contribution of thyroidal secretion to PR-rT3 and PR-T3. It was estimated that whereas thyroidal secretion may account for about 23.8% of serum T3 (or PR-T3), it may account for only about 2.5% of serum rT3 (or PR-rT3). Since peripheral metabolism of T4 is the only known source of rT3 and T3 other than the thyroidal secretion, it could be calculated that as much as 73.0 mug or 84% of daily PR-T4 may normally be metabolized by monodeiodination either to T3 or to rT3. MCR and PR of various iodothyronines were also examined in five cases with hepatic cirrhosis, where, as documented previously, serum rT3 may be elevated while serum T3 is diminished. The mean MCR-rT3 in these cases (41.0 liters/day) was clearly (P is less than 0.005) less than that (76.7 liters/day) in normal subjects. This was the case at a time when the mean MCR-T3 (26.7 liters/day) and the mean MCR-T4 (1.19 liters/day) did not differ from those (vide supra) in normal subjects. Distinct from changes in MCRs, the mean PR-rT3 (33.0 mug/day) was similar to, and the mean PR-T3 (10.1 mug/day) and the mean PR-T4 (66.4 mug/day) were much less than, the corresponding value in normal subjects. Furthermore, while the ratio of PR-rT3 and PR-T4 (rT3/T4) in individual patients was either supranormal or normal, the ratio of PR-T3 and PR-T4 (T3/T4) was clearly subnormal. The various data suggest that: (a) just as in the case of T3, the thyroid gland is a relatively minor source of rT3; peripheral metabolism of T4 is apparently its major source; (b) the bulk of T4 metabolized daily is monodeiodinated to T3 or to rT3; (c) monodeiodination may be an obligatory step in metabolism of T4; (d) monodeiodination of T4 to rT3 is maintained normal or is increased in hepatic cirrhosis at a time when monodeiodination of T4 to T3 is decreased.

Authors

I J Chopra

Disturbed islet-cell function related to endogenous gastrin release. Studies on insulin secretion and glucose tolerance in pernicious anemia.

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The insulin and gastrin response to oral glucose, intravenous glucose, or a protein-rich meal were measured in 44 nondiabetic patients with pernicious anemia (PA) and in 44 control subjects. 36 of the PA-patients had hypergastrinemia, while serum gastrin concentrations in the remaining eight patients were below normal. Three hypergastrinemic PA-patients were in addition studied during an oral glucose loading with synchronous intravenous infusion of gastrin-17. During both oral and intravenous glucose tests blood glucose concentrations were similar in patients and in controls. After ingestion of protein blood glucose concentrations in PA-patients with hypergastrinemia were above those of the controls (P less than 0.05). Parenteral infusion of gastrin-17 during oral glucose loading also increased blood glucose concentrations above the levels observed after glucose alone. In PA-patients with hypergastrinemia the insulin response was augmented in all tests. In patients with hypogastrinemia serum insulin concentrations were lower than normal in the fasting state and during stimulation with glucose intravenously (P less than 0.01). In hypergastrinemic patients serum gastrin concentrations decreased after oral as well as intravenous glucose administration. The decrease was larger during the oral test. In hypogastrinemia oral glucose induced, as in controls, a small initial rise followed by a slow fall in serum gastrin concentrations. No variations were seen in these patients during the intravenous glucose infusion. Gel filtration of serum from hypergastrinemic patients disclosed a decrease in the concentrations of all four main components of gastrin during the glucose loadings. Taken together with earlier studies on the effect of exogenous gastrin the results suggest that endogenous hypergastrinemia induces hyperglycemia and potentiates insulin secretion. In contrast hypogastrinemia is associated with hypoinsulinism.

Authors

J F Rehfeld

Chemiluminescence and superoxide production by myeloperoxidase-deficient leukocytes.

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The role of superoxide anion- and myeloperoxidase-dependent reactions in the light emission by phagocytosing polymorphonuclear leukocytes has been investigated using leukocytes that lack myeloperoxidase, inhibitors (azide, superoxide dismutase), and model systems. Our earlier finding that oxygen consumption, glucose C-1 oxidation, and formate oxidation are greater in polymorphonuclear leukocytes that lack myeloperoxidase than in normal cells during phagocytosis has been confirmed with leukocytes from two newly described myeloperoxidase-deficient siblings. Although the maximal rate of superoxide anion production by myeloperoxidase-deficient leukocytes is not significantly different from that of normal cells, superoxide production falls off less rapidly with time so that with prolonged incubation, it is greater in myeloperoxidase-deficient than in normal cells. Chemiluminescence by myeloperoxidase-deficient leukocytes during the early postphagocytic period however is decreased. Light emission by normal leukocytes is strongly inhibited by both superoxide dismutase and azide, whereas that of myeloperoxidase-deficient leukocytes, while still strongly inhibited by superoxide dismutase is considerably less sensitive to azide. Zymosan, the phagocytic particle employed in the intact cell system, considerably increased the chemiluminescence of a cell-free superoxide-H2O2 generating system (xanthine-xanthine oxidase) and a system containing myeloperoxidase, H2O2, and chloride. Light emission by the xanthine oxidase model system is strongly inhibited by superoxide dismutase and is not inhibited by azide, whereas the myeloperoxidase-dependent model system is strongly inhibited by azide but only slightly inhibited by superoxide dismutase. These findings suggest that light emission by phagocytosing polymorphonuclear leukocytes is dependent on both myeloperoxidase-catalyzed reactions and the superoxide anion, and involves in part the excitation of the ingested particle. These studies are discussed in relation to the role of the superoxide anion and chemiluminescence in the microbicidal activity of the polymorphonuclear leukocyte.

Authors

H Rosen, S J Klebanoff

Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 alpha, 25-dihydroxyvitamin D2/D3.

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A competitive protein binding assay for measurement of the plasma concentration of 1 alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin D3 (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely. 25-hydroxyvitamin D2 (25-OHD2) and 1alpha, 25-dihydroxyvitamin D2 [1alpha, 25-(OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1alpha, 25-(OH)2D2 as well as 1alpha, 25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1alpha, 25-(OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1alpha, 25-(OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1alpha, 25-(OH)2D2 was generated in an in vitro kidney homogenate system using 25-OHD2 as substrate. Comparison of this sterol with 1alpha, 25-(OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of 1alpha, 25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and 1alpha, 25-(OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (greater than 90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15-fold increase in the plasma 25-OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1alpha, 25-(OH)2D was not substantially enhanced in vitamin D-intoxicated patients. We therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1alpha, 25-(OH)2D but may be cuased by an excessive circulating concentration of 25-OHD.

Authors

M R Hughes, D J Baylink, P G Jones, M R Haussler

Phosphaturic effect of dopamine in dogs. Possible role of intrarenally produced dopamine in phosphate regulation.

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A possible role for dopamine in phosphate handling by the dog kidney was investigated by intrarenal artery infusions of dopamine. Dopamine increased fractional phosphate excretion both in the presence and absence of control of parathyroid hormone and calcitonin. In addition, dopamine increased both renal blood flow and sodium excretion, however, the phosphaturia was independent of these changes; since 30 min after completion of dopamine infusion, renal blood flow and sodium excretion returned to control levels and phosphate excretion remained elevated. For comparison, the vasodilator isoproterenol increased renal blood flow and sodium excretion without a significant change in fractional phosphate excretion. Thus, the phosphaturic effect of dopamine is probably independent of its vasodilator effect. The phosphaturic effect of dopamine could not be accounted for by subsequent conversion to norepinephrine, since norepinephrine was antiphosphaturic in the dog. The effect of endogenous dopamine on renal phosphate excretion was investigated by intrarenal infusion of the precursor dopa. Dopa was phosphaturic both in the presence and absence of parathyroid hormone and calcitonin. In dogs pretreated with carbidopa, which blocks conversion of dopa to dopamine, dopa was no longer phosphaturic, although the kidney remained responsive to dopamine. It is postulated that dopamine may play a role in the intrarenal regulation of phosphate excretion.

Authors

J L Cuche, G R Marchand, R F Greger, R C Lang, F G Knox

The effect of hyperventilation on distal nephron hydrogen ion secretion.

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This study was designed to determine the effect of acute hyperventilation on distal nephron hydrogen ion secretion. The blood PCO2 declined and stabilized rapidly when bicarbonate loaded rats were hyperventilated. In contrast, the urine PCO2 declined slowly, resulting in an early increase in the urine minus blood (U-B) PCO2 which could not be obliterated by carbonic anhydrase infusion. Within approximately 50 min, the U-B PCO2 in the hyperventilated and carbonic anhydrase infused rats approached zero. Consequently, equilibrium between collecting duct urine and arterial blood PCO2 was then presumed to exist. This provided the basis for the subsequent studies on a series of rats. The U-B PCO2 decreased from a control of 22+/-1 mm Hg (mean+/-SEM) to 11+/-2 mm Hg (mean+/-SEM) with hypocapnia, and rose again to its control value when the blood PCO2 returned to prehyperventilation values. This decline in U-B PCO2 with acute hyperventilation could not be attributed to changes in urine flow, phosphate, or bicarbonate excretion, suggesting, therefore, a decrease in distal nephron (probably collecting duct) hydrogen ion secretion with acute hyperventilation. Possible pitfalls in the interpretation of the UB PCO2 are illustrated.

Authors

R A Giammarco, M B Goldstein, M L Halperin, B J Stinebaugh

The effects of glucose and insulin on renal electrolyte transport.

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The effects of hyperglycemia and hyperinsulinemia on renal handling of sodium, calcium, and phosphate were studied in dogs employing the recollection micropuncture technique. Subthreshold sustained hyperglycemia resulted in an isonatric inhibition of proximal tubular sodium, fluid, calcium, and phosphate reabsorption by 8-14%. Fractional excretion of sodium and phosphate, however, fell (P is less than 0.01) indicating that the increased delivery of these ions was reabsorbed in portions of the nephron distal to the site of puncture and in addition net sodium and phosphate transport was enhanced resulting in a significant antinatriuresis and antiphosphaturia. The creation of a steady state plateau of hyperinsulinemia while maintaining the blood glucose concentration of euglycemic levels mimicked the effects of hyperglycemia on proximal tubular transport and fractional excretion of sodium and calcium. Tubular fluid to plasma insulin ratio fell, similar to the hyperglycemic studies. These results suggest that the effects of hyperglycemia on renal handling of sodium and calcium may be mediated through changes in plasma insulin concentration. In contrast to hyperglycemia, however, hyperinsulinemia cuased a significant fall in tubular fluid to plasma phosphate ratio with enhanced proximal tubular phosphate reabsorption (P is less than 0.02). This occurred concomitantly with a significant inhibition of proximal tubular sodium transport. These data indicate that insulin has a direct effect on proximal tubular phosphate reabsorption, and this effect of insulin is masked by the presence of increased amounts of unreabsorbed glucose in the tubule that ensues when hyperinsulinemia occurs secondary to hyperglycemia. Fractional excretion of phosphate fell significantly during insulin infusion but unlike the hyperglycemic studies, the fall in phosphate excretion could be entirely accounted for by enhanced proximal reabsorption.

Authors

R A DeFronzo, M Goldberg, Z S Agus

Intestinal myoelectric activity in response to live Vibrio cholerae and cholera enterotoxin.

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The myoelectric response of the rabbit ileum was studied in response to live Vibrio cholerae culture, a whole cell lysate of cholera, and the purified enterotoxin. Each cholera preparation produced a series of highly organized migrating action potential complexes (MAPC). An MAPC was defined as action potential discharge with a duration of 2.5 s or longer, followed by similar activity on at least one other consecutive electrode site. The mean and modal onset time of MAPC activity occurred 4 h after the infection with live Vibrio cholerae culture, the freeze-dried whole cell lysate preparation, or the purified enterotoxin. After the onset of activity this pattern persisted for the duration of the recording period (up to 12 h). The MAPC had a mean propagation velocity of 0.85+/-0.07 cm/s (mean+/-SEM), which remained constant with time. Direct visual observation of the loop revealed that the MAPC's resulted in contractions that propelled intraluminal contents in an aborad direction. The mean fluid output from the 12-cm ileal loops was 6.4+/-1.1 ml/h (mean+/-SEM). Control experiments consisted of recordings from: (a) a ligated ileal loop into which nothing was placed; (b) a ligated ileal loop into which either uninfected culture broth or 0.9% NaCl solution was injected; (c) a ligated ileal loop infused with 0.9% NaCl solution at a rate of 11.2 ml/h, and (d) rapid injection of 1.0, 2.5, 5.0, or 10.0-ml boluses of 0.9% NaCl into the proximal catheter. MAPC activity was not observed in any of the control experiments. These studies indicate that in addition to a secretory component to cholera, there exists a highly organized MAPC that results in contractions that propel intraluminal contents in an aborad direction.

Authors

J R Mathias, G M Carlson, A J DiMarino, G Bertiger, H E Morton, S Cohen

The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell.

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Abstract

Studies were undertaken to define the mechanism whereby bile acid facilitates fatty acid and cholesterol uptake into the intestinal mucosal cell. Initial studies showed that the rate of uptake (Jd) of several fatty acids and cholesterol was a linear function of the concentration of these molecules in the bulk phase if the concentration of bile acid was kept constant. In contrast, Jd decreased markedly when the concentration of bile acid was increased relative to that of the probe molecule but remained essentially constant when the concentration of both the bile acid and probe molecule was increased in parallel. In other studies Jd for lauric acid measured from solutions containing either 0 or 20 mM taurodeoxycholate and saturated with the fatty acid equaled 79.8+/-5.2 and 120.8+/-9.4 nmol.min(-1).100 mg(-1), respectively: after correction for unstirred layer resistance, however, the former value equaled 113.5+/-7.1 nmol.min(-1).100 mg(-1). Maximum values of Jd for the saturated fatty acids with 12, 16, and 18 carbons equaled 120.8+/-9.4, 24.1+/-3.2, and 13.6+/-1.1 nmol.min(-1).100 mg(-1), respectively. These values essentially equaled those derived by multiplying the maximum solubility times the passive permeability coefficients appropriate for each of these compounds. The theoretical equations were then derived that define the expected behavior of Jd for the various lipids under these different experimental circumstances where the mechanism of absorption was assumed to occur either by uptake of the whole micelle, during interaction of the micelle with an infinite number of sites on the microvillus membrane or through a monomer phase of lipid molecules in equilibrium with the micelle. The experimental results were consistent both qualitatively and quantitatively with the third model indicating that the principle role of the micelle in facilitating lipid absorption is to overcome unstirred layer resistance while the actual process of fatty acid and cholesterol absorption occurs through a monomer phase in equilibrium with the micelle.

Authors

H Westergaard, J M Dietschy

Suppression of B-cell differentiation by leukocytes from hypogammaglobulinemic patients.

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Abstract

Leukocytes from patients with several forms of immunodeficiency characterized by apparently differing defects in B-lymphocyte maturation produced few or no plasmacytoid cells in vitro, and were capable of suppressing the generation of plasma cells in co-culture with cells of normal persons, in the presence of pokeweed mitogen. Such inhibition was commonly observed in cultures which included cells from patients with primary immunoglobulin deficiency, but was not seen to a significant degree in identical co-cultures of cells from normals. The suppression observed was not dependent upon mixed leukocyte culture reactivity. Both sheep erythrocyte-rosetting lymphocytes and adherent cells appeared to participate in these effects in some patients. In one patient with common variable immunodeficiency, but not in several others, removal of suppressing cells permitted the patient's remaining cells to differentiate into plasma cells in vitro. Because of the diverse syndromes in which suppression was observed, it is likely that, in at least some hypogammaglobulinemic patients, the suppression is secondary to the disease process rather than being the primary pathogenic mechanism.

Authors

F P Siegal, M Siegal, R A Good

Characterization of the protease activity in the chemotactic factor inactivator.

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Abstract

The chemotactic factor inactivator (CFI) isolated from human serum contains a kininase activity that causes extensive hydrolysis of bradykinin. The highly chemotactic synthetic peptide Met-Leu-Phe was completely hydrolyzed by CFI preparations. The release of the constituent amino acids from this peptide coincided with a loss of its chemotactic activity. The N-formyl, but not the amide derivative, of the leukotactic peptide Met-Leu-Phe was resistant to the action of CFI, as evidenced by chemotactic and biochemical assays. Examination of the specificity of CFI proteolysis revealed that short polypeptide substrates are degraded sequentially from the amino terminus. Larger peptides are less extensively hydrolyzed, and the patterns of hydrolysis are more complex. Inactivation of the bacterial chemotactic factors by CFI was overcome by the addition of high concentrations of peptides which were substrated for CFI. CFI preparations readily hydrolyzed the peptide Arg-Phe-Ala. The constituent amino acids were conveniently identified by thin-layer chromatography method. This procedure afforded a rapid assay for measuring CFI activity in the whole human serum as well as in fractions throughout the purification steps. Moreover, CFI also hydrolyzed L-leucyl-beta-napthylamide at rates comparable to peptides. Thus, L-leucyl-beta-napthylamide served as a useful substrate for estimating CFI activity in preparations at various stages of purification. This substrate was also useful in kinetic studies. The results from these studies indicate an aminopeptidase activity is the mechanism whereby CFI inhibits the activity of chemotactic substrates.

Authors

P A Ward, J Ozols

Heterogeneity of lymphocyte subpopulations in severe combined immunodeficiency. Evidence against a stem cell defect.

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Surface markers typical of T and B lymphocytes were present on varying proportions of peripheral blood lymphocytes from three infants with severe combined immunodeficiency disease. Despite this, functions mediated by T and B cells were either absent or very minimal in all three, including cell-mediated responses in vivo; the in vitro proliferative response to mitogens, allogeneic cells, or antigens; effector cell function in lymphocyte-antibody lymphocytolytic interaction assays; and in vitro synthesis of IgG, IgA, and IgM. In contrast, mononuclear cells from one of the infants were tested and found capable of lysing both human and chicken antibody-coated erythrocyte targets normally. Co-cultivation experiments with unrelated normal control lymphocytes failed to demonstrate suppressor cell activity for immunoglobulin synthesis in these infants. Augmentations of immunoglobulin production from 310 to 560% over that expected on the basis of individual culture data were noted in co-cultures of one of the infants' cells with two different unrelated normal control cells. These findings suggest that that infant may have had a T helper cell defect or that his T cells were unable to produce soluble factors necessary for B cell differentiation. The finding of cells with differentiation markers characteristic of T and B lymphocytes in each of these patients, though in variable quantities, is further evidence for heterogeneity among patients with the clinical syndrome of severe combined immunodeficiency and argues against the concept that their immunodeficiency was due to a stem cell defect.

Authors

R H Buckley, R B Gilbertsen, R I Schiff, E Ferreira, S O Sanal, T A Waldmann

Effect of salicylates on histamine and L-histidine metabolism. Inhibition of imidazoleacetate phosphoribosyl transferase.

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Abstract

In man and other animals, urinary excretion of the histidine and histamine metabolite, imidazoleacetate, is increased and that of its conjugated metabolite, ribosylimidazoleacetate, decreased by salicylates. Imidazoleacetate has been reported to produce analgesia and narcosis. Its accumulation as a result of transferase inhibition could play a part in the therapeutic effects of salicylates. To determine the locus of salicylate action, we have investigated the effect of anti-inflammatory drugs on imidazoleacetate phosphoribosyl transferase, the enzyme that catalyzes the ATP-dependent conjugation of imidazoleacetate with phosphoribosylpyrophosphate. As little as 0.2 mM aspirin produced 50% inhibition of the rat liver transferase. In vivo, a 30% decrease in the urinary excretion of ribosylimidazoleacetate has been observed with plasma salicylate concentrations of 0.4 mM. The enzyme was also inhibited by sodium salicylate but not by salicylamide, sodium gentisate, aminopyrine, phenacetin, phenylbutazone, or indomethacin. The last four drugs have been shown previously not to alter the excretion of ribosylimidazoleacetate when administered in vivo. Since both the drug specificity and inhibitory concentrations are similar in vivo and in vitro, it seems probable that the effect of salicylates on imidazoleacetate conjugation results from inhibition of imidazoleacetate phosphoribosyl transferase.

Authors

J Moss, M C De Mello, M Vaughan, M A Beaven

Fibrinopeptide A in plasma of normal subjects and patients with disseminated intravascular coagulation and systemic lupus erythematosus.

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Abstract

A radioimmunoassay for fibrinopeptide A (FPA) has been developed. This assay uses rabbit antibodies induced by injection of native FPA-human serum albumin conjugates and 125I introduced into tyrosine-FPA synthesized in out laboratory. Plasma FPA is separated from fibrinogen by TCA extraction. The assay is capable of detecting as little as 50 pg/ml of FPA. In 20 normal donors this assay revealed a mean concentration of 0.9 ng/ml (0.3 SD). In five patients with disseminated intravascular coagulation, FPA concentrations ranged from 13.0 to 346 ng/ml. Two groups of patients with systemic lupus erythematosus (SLE) whose disease had achieved complete remission were studied; one consisted of four patients with no history of lupus nephritis and another with a history of nephritis. Mean FPA concentrations of 1.5 ng/ml (range, 0.7-1.8 ng/ml) and 2.7 ng/ml (range, 1.1-5.6 ng/ml) were found in these two groups, respectively. Another group of nine patients with active SLE, but without evidence of lupus nephritis, had a mean FPA concentration of 4.5 ng/ml (range, 2.4-7.8 ng/ml). Finally, a group of seven patients with active SLE, including active nephritis, had a mean FPA concentration of 10.2 ng/ml (range, 5.3-17.0 ng/ml). A positive correlation was found between the concentration of plasma FPA and serum DNA-binding activity and an inverse correlation was found between plasma FPA and the concentration of serum C3. No correlation existed between plasma FPA and concentration of serum creatinine. Several possibilities for the origin of plasma FPA in patients with SLE were considered; at present it seems most likely that FPA arises through the action of thrombin on fibrinogen.

Authors

M Cronlund, J Hardin, J Burton, L Lee, E Haber, K J Bloch

Exaggerated phosphaturic response to circulating parathyroid hormone in patients with familial X-linked hypophosphatemic rickets.

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Abstract

To determine whether the phosphaturic response to circulating parathyroid hormone (PTH) is exaggerated in patients with familial x-linked hypophosphatemic vitamin D-resistant rickets (FHR), we examined the phosphaturic response to parathyroid extract (PTE) (administered intravenously in the posthypercalcemic state) in two unrelated adult hemizygotes with FHR. In these two patients whose plasma concentration of PTH was normal (determined by radioimmunoassay). neither vitamin D nor phosphate therapy had been given during the past 10 yr. Two normal men and a hypophosphatemic man with intestinal malabsorption, hypocalcemia, and osteomalacia served as control subjects. In all subjects, calcium gluconate was adminstered intravenously from 6 p.m. to 12 midnight at a rate that maintained the concentration of serum calcium at 13-15 mg/100 ml during the administration of calcium. When normocalcemia had recurred the next morning, and the plasma PTH concentration and urinary excretion of cyclic 3', 5'-AMP were reduced. PTE was administered intravenously at successively increasing rates of 0.1, 0.4, and 0.8 U/kg per h, each rate lasting 90 min. Minutes after the initiation of PTE in the affected hemizygotes, fractional excretion of filtered phosphate increased from negligible values to values strikingly greater than those of similarly studied control subjects and plateaued at strikingly greater values throughout further administration of PTE. This phenomenon of exaggerated phosphaturia could not be attributed to volume expansion, decreases in serum concentration of calcium during the study, differences in percent of administered calcium retained, or hemodynamic changes. Only the phosphaturic response to PTE appeared to be exaggerated. At any cumulative dose of PTE, urinary excretion of cyclic 3', 5'-AMP in the hemizogytes was indistinguishable from that of control subjects. The findings in this study suggest that in patients with FHR, circulating PTH is required for the genetically transmitted abnormality to be physiologically expressed as a reduction in net renal reabsorption of phosphate, and that this physiological expression of the genetic abnormality is expressed fully at normal or nearly normal circulating levels of PTH.

Authors

E Short, R C Morris Jr, A Sebastian, M Spencer

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects.

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Abstract

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.

Authors

K Tanaka, R Mandell, V E Shih

Antibody-dependent cell-mediated cytotoxicity in selected autoimmune diseases.

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Abstract

Antibody-dependent cell-mediated cytotoxicity mediated by peripheral blood lymphocytes was studied in patients with systemic lupus erythematosus, polyarteritis nodosa. Sjogren's syndrome, and rheumatoid arthritis. The target cells were chicken erythrocytes coated with rabbit anti-chicken erythrocyte antibody. Antibody-dependent cell-mediated cytotoxic activity was normal in Sjogren's syndrome and rheumatoid arthritis but significantly decreased (P is less than 0.001) in active systemic lupus erythematosus and in two patients with polyarteritis nodosa. A partial regeneration of antibody-dependent cell-mediated cytotoxic activity was obtained by treatment with pronase and DNase followed by overnight incubation. Sera from patients with systemic lupus erythematosus inhibited antibody-dependent cell-mediated cytotoxic activity of normal lymphocytes. The inhibitory activity was studied by specific immunoadsorption and sucrose density geadient ultracentrifugation. Removal of IgG but not IgM greatly reduced inhibition. Inhibitory factors were present in 7S and heavier fractions containing IgG. Five systemic lupus erythematosus patients were studied serially to determine if improvement in clinical status could be correlated with a decrease in serum inhibitory factors as studied by inhibition of normal antibody-dependent cell-mediated cytotoxicity. Indeed, a greater serum inhibitory capacity was found in each patient during periods of greater disease activity.

Authors

J L Feldmann, M J Becker, H Moutsopoulos, K Fye, M Blackman, W V Epstein, N Talal

Cystinosis. Intracellular cystine depletion by aminothiols in vitro and in vivo.

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Abstract

Certain aminothiols rapidly deplete cultured cystinotic skin fibroblasts of their abnormally high free (nonprotein) cystine pool. The free cystine content of these cells if reduced by over 90% in 1 h with 0.1 mM cysteamine. This is more rapid than previously known methods of removing free cystine from cystinotic fibroblasts. The disulfide, cystamine, is also able to deplete cystinotic cells of free cystine. A patient with nephropathic cystinosis and end-stage renal disease was treated with cysteamine, both intravenously and orally. Both methods of administration rapidly lowered the free cystine content of the patient's peripheral leukocytes. Study of the patient's urinary sulfur excretion did not conclusively determine the effect of this therapy on the total body cystine pool. Her renal status remained at end stage after 1 mo of oral cysteamine, when an episode of grand mal seizures prompted cessation of the study. Determination of the proper place of aminothiol therapy in this disease will depend upon further clinical trial with patients whose kidney function has not deteriorated to the point of irreversible change, accompanied by careful monitoring of plasma aminothiol levels.

Authors

J G Thoene, R G Oshima, J C Crawhall, D L Olson, J A Schneider

Neutrophil function in gram-negative rod bacteremia. The interaction between phagocytic cells, infecting organisms, and humoral factors.

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Abstract

To assess the phagocytic and bactericidal function of neutrophils in the acute stages of gram-negative rod bacteremia, cells from 30 nonleukopenic patients were studied in a test system utilizing plasma obtained simultaneously with culture-positive blood, the autologous infecting strain, and two laboratory test strains of Staphylococcus aureus and Pseudomonas aeruginosa. Results were compared to those obtained with normal neutrophils and plasma. Patient and control plasma were simultaneously tested with each source of phagocytic cells to localize any abnormalities. Four patients had a defect against their infecting strain, 33% of the inoculum phagocytized and killed versus 80% by controls. In these cases differences were localized to the patients' plasma, as normal plasma tested with patients' cells reversed the defect. Thus, four patients had impaired opsonization when compared to normal controls, but we also observed that 11 of 30 bacteremic isolates, all Escherichia coli, showed absolute or relative resistance to phagocytosis in the patient and control assay system. No intrinsic granulocyte killing abnormalities were noted. There was poor correlation between results obtained with infecting strains compared to laboratory test organisms. We conclude that in patients without evidence of an inherited neutrophil bactericidal disorder, recurrent infection, or treatment with cytotoxic drugs, intrinsic bactericidal defects are uncommon at the onset of gram-negative bacteremia, and impaired opsonization is the most commonly encountered cause of neutrophil dysfunction.

Authors

R J Weinstein, L S Young

Physical chemistry of the lipids of human atherosclerotic lesions. Demonstration of a lesion intermediate between fatty streaks and advanced plaques.

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Abstract

95 individual human atherosclerotic lesions from 26 persons were classified into three groups under the dissecting microscope: fatty streaks, fibrous plaques, and gruel (atheromatous) plaques. Each lesion was isolated by microdissection, its lipid composition determined by chromatography, and the physical states of the lipids identified by polarizing microscopy and in some cases by X-ray diffraction. The composition of each lesion was plotted on the in vitro phase diagram of the major lipids of plaques: cholesterol, cholesterol ester, and phospholipid. The observed physical states were compared with those predicted by the location of the lipid composition on the phase diagram. The most severe lesions (gruel plaques) had an average lipid composition of cholesterol 31.5+/-1.9%, cholesterol ester 47.2+/-2.3%, and phospholipid 15.3+/-0.5%. Their compositions fell within the three-phase zone of the phase diagram, predicting the lipids to be separated into a cholesterol crystal phase, a cholesterol ester oily phase and a phospholipid liquid crystalline phase. In addition to the phospholipid liquid crystalline phase of membranes and myelin-like figures demonstrable by electron microscopy, polarizing microscopy revealed the other two predicted phases, isotropic cholesterol ester-rich droplets and cholesterol crystals. X-ray diffraction studies verified the identity of the crystals as cholesterol monohydrate. Fibrous plaques also had an average lipid composition within the three-phase zone of the phase diagram. Polarizing microscopy revealed the presence of cholesterol monohydrate crystals and lipid droplets in all of these lesions; the droplets were predominately isotropic in 28 of the 31 fibrous plaques. Although these lesions had less free cholesterol and more cholesterol ester than gruel plaques, they were otherwise similar. Fatty streaks had compositions within both the two- and three-phase zones of the phase diagram. Compared with gruel plaques, the fatty streaks within the two-phase zone, defined as "ordinary," had more cholesterol ester, less free cholesterol, a higher cholesteryl oleate/cholesteryl linoleate ratio, a lower sphingomyelin/lecithin ratio, more anisotropic lipid droplets, and rare or no cholesterol crystals. Those lesions within the three-phase zone had many chemical and physical features intermediate between ordinary fatty streaks and gruel plaques. Moreover, 68% of these "intermediate" lesions had no cholesterol crystals by polarizing microscopy in spite of their compositions being within the three-phase zone, indicating the cholesterol ester oily phase or the phospholipid phase or both were supersaturated with cholesterol. Identification of this group of intermediate lesions provides evidence that some fatty streaks may be precursors of advanced plaques.

Authors

S S Katz, G G Shipley, D M Small

Alterations in T and B lymphocytes in heart transplant patients early and late postoperatively.

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Abstract

Alterations in the percent and absolute number of thymus-derived (T) and bursa-equivalent (B) lymphocytes in peripheral blood were followed in 10 patients treated with antithymocyte globulin, prednisone, and azathioprine after cardiac transplantation. During the 1st postoperative wk the percent of T cells dropped below 10% in almost all cases (normal range, 65-91%) with a concomitant rise in the percent of B cells. However, the absolute T- and B-cell counts were both markedly depressed (less than 200 cells/mm3). During the 7-wk postoperative period the percent of T cells rose to 45-60% and the absolute count rose from 100 to 350 cells/mm3 (normal range, 1,092-2,400 cells/mm3). Although the percent of B cells was elevated (35-50%) during this period, the absolute B-cell count remained below the range of normals (268-640 cells/mm3). Follow-up of long-term survivors (3-60 mo postoperative) showed a continued marked T (467 cells/mm3) and B (95 cells/mm3) lymphocytopenia. Chronological relationships between the percent and absolute T-cell count and episodes of graft rejection in individual patients are discussed as possible adjuncts in the prediction of rejection crises.

Authors

T H Khalaf, S Strober, G Garrelts, E B Stinson

The regulation of plasma 18-hydroxy 11-deoxycorticosterone in man.

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Abstract

18-hydroxy 11-deoxycorticosterone (18-OH DOC), a weak mineralocorticoid, was estimated by a radioimmunoassay procedure after purification in 49 patients with hypertension and 38 normal control subjects. The sensitivity of the method was 2-4 pg; there was no detectable blank, and the precision was 9-10%. In normal subjects the absolute plasma levels were similar to those of aldosterone. ACTH administration produced a 23-fold increase, and sodium restriction resulted in a 4-fold increase (5.4+/-0.7-20.5+/-3.0 ng/dl). On the other hand, the plasma levels of 18-OH DOC declined by nearly 50% with upright posture or angiotensin II infusion. During both of these procedures, plasma aldosterone levels significantly increased. Patients with normal and low renin hypertension had similar changes in plasma 18-OH DOC levels with sodium restriction. However, the mean high sodium level in the normal renin essential hypertension group (11.6+/-1.6 ng/dl) was significantly greater (P is less than 0.001) than in the control group (5.4+/-0.7 ng/dl). In addition, at least 22% and perhaps as high as 37% of the hypertensive subjects had levels greater than the upper limits of normal on a high sodium intake. Differences between the groups were less impressive in the sodium-restricted studies. There were no significant differences in age, duration of hypertension, sodium balance, serum sodium, potassium, or blood urea nitrogen in those patients who had elevated levels of plasma 18-OH DOC. Patients with primary aldosteronism had levels within the normal range on both dietary intake. However, in contrast to the other groups there were no significant changes in the plasma levels with sodium restriction. Thus, a significant number of patients with essential hypertension presumably have an alteration in 18-OH DOC secretion.

Authors

G H Williams, L M Braley, R H Underwood

Interactions of acetylcholine and epinephrine on the dynamics of insulin release in vitro.

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Abstract

An in vitro system for perifusion of rat pancreatic islets has been utilized to define the effects of epinephrine on acetylcholine-induced insulin release over varying concentrations of the two agents. Perifusion of islets with epinephrine before challenge with acetycholine produced marked enhancement of both phases of cholinergically induced insulin release; enhancement of the first phase being more marked with increase in acetylcholine concentration and the converse being observed with the second phase. Perifusion of islets with epinephrine during stimulation with acetylcholine produced inhibition of insulin release, an effect dependent upon the concentration of epinephrine and of acetylcholine. There was an order of difference in the acetycholine concentration needed to overcome significant epinephrine-mediated inhibition of the first phase of insulin release (5 X 10(-4) mug/ml) and that needed to overcome inhibition of the second phase (5 X 10(-3) mug/ml). Comparison of the effects of various concentrations of epinephrine on glucose- and acetyl-choline-induced insulin release revealed that epinephrine was a less potent inhibitor of the first phase of acetylcholine-induced insulin release than of the first phase of glucose-induced insulin release. These data provide some insight into the potential interactions between cholinergic and adrenergic autonomic systems in modifying insulin release.

Authors

I M Burr, A E Slonim, R Sharp

Lymphokine stimulation of collagen accumulation.

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Abstract

Lymphokine-rich supernates from normal human peripheral blood mononuclear cells, stimulated by the mitogen phytohemagglutinin, have been shown to cause enhanced collagen accumulation by human embryonic lung fibroblasts (WI-38), as measured by hydroxyproline content of fibroblast monolayers, [14C] proline incorporation into soluble collagen and collagenase release of radioactivity in supernates and monolayers of cultures incubated with [14C] proline. This fibroblast-stimulating activity, demonstrable by suitable dilutions of the supernates, coexisted with a number of other lymphokine activities such as lymphotoxin, proliferation inhibitory factor, and cloning inhibitory factor, which tend to reduce the numbers of function of fibroblasts. The increased content of collagen appeared to be the product of selected surviving and responding fibroblasts. The factor causing this increased collagen accumulation was nondialyzable and stable at -70 degrees C. It represents the first described lymphoid cell-derived activity capable of enhancing collagen accumulation. Fibroblast-stimulating activity may be implicated in the abnormal fibrosis seen in association with chronic inflammation in a variety of disease states. It may have special relevance to progressive systemic sclerosis.

Authors

R L Johnson, M Ziff