Infection-induced anergy is a frequent complication of bacterial, viral, and parsitic infection. A marked suppression of the thymus-derived (T) lymphocyte response to concanavalin A has been demonstrated in vitro during renal infection and the mechanisms by which suppression occurs have been investigated. In particular we have considered the possibility that suppression might result from the inhibitory effect of prostaglandins, secreted by activated macrophages with immunoregulatory potential. The experiments have shown that the T-lymphocyte effector status in experimentally-induced renal infection is determined by two suppressor cells, one infection-induced and the other naturally occurring. The inability to respond to mitogenic stimulation was reversible and restoration of immune responsiveness to splenic lymphocytes from infected animals could be achieved in two stepwise manipulations; differential centrifugation removed the infection-induced suppressor cells, and the suppressor activity of the naturally occurring suppressor cells could then be inhibited by indomethacin. Thus the two suppressor cells were distinguishable on the basis of their physical characteristics and their response to indomethacin. The dominant factor determining the immune responsiveness of splenic lymphocytes from the pyelonephritic animals was, however, the infection-induced suppressor cell. This cell has been characterized as a sedimentable cell (30 g) with suppressor activity demonstrable in co-culture experiments. Plastic-adherent cells from the sedimentable fraction of pyelonephritic animals' splenic cells were shown to have suppressor activity that was not inhibited by indomethacin. The infection-induced and naturally occurring suppressor cells can be viewed as prototypes for the equivalent cells in man and may be useful models for studying the role of these cells as determinants in the pathogenesis of infectious disease.
T Miller, E Marshall
The role of six suppressor mechanisms upon T and B cell responses was studied on 17 untreated patients with Hodgkin's disease. Proliferative hyporesponsiveness to mitogen was greatly impaired in 8 of the 13 patients. 10 of these patients had an excessive degree of suppression by cells that adhered to foreign surfaces. Suppression by adherent cells correlated with impairment of proliferative responses and, in some instances, suppression was largely inhibited with indomethacin. Likewise, adherent cells suppressed immunoglobulin synthesis. A correlation was evident between suppression of T and B cell responses by adherent mononuclear leukocytes from individual patients. This suppression coincided with elevated percentages of monocytes in the patient mononuclear cell preparations. This excess of monocytes was not the result of a circulating monocytosis. The monocyte excess may have been acquired during isopyknic cell separation. A second form of suppression was observed in 5 of the 11 patients affected by a lymphocyte that neither adhered to glass wool nor required preactivation. It did not inhibit allogeneic lymphocytes, which contrasts with the suppressor abnormality of monocytoid cells.
J J Twomey, A H Laughter, L Rice, R Ford
Quinine- or quinidine-induced thrombocytopenic purpura is caused by synthesis of an immunoglobulin (Ig)G antibody, which caused platelet damage in the presence of the offending drug. The nature of the antigenic stimulus has been examined by measuring incorporation of [3H]thymidine into DNA during lymphocyte transformation to blast cells in the presence of the drug. Although patients' lymphocytes responded normally to the nonspecific mitogen, phytohemagglutinin P, they did not respond to either drug or platelets alone. However, significant transformation occurred when patients' lymphocytes were cultured for 7 d with homologous or autologous platelets in the presence of therapeutic concentrations of the drugs (0.39-39 microM). Platelet membranes were more active than intact platelets on the basis of protein content, whereas platelets from a patient with Bernard-Soulier syndrome were inactive. Washed platelets pretreated with the drugs were inactive when cultured with lymphocytes in the absence of the drugs, whereas platelets pretreated similarly in plasma caused transformation. Control lymphocytes from 20 normal patients and 6 patients with nondrug-induced thrombocytopenia were not transformed by drugs and platelets in the presence of normal serum or serum containing drug-dependent antibody, showing that the observed response was specific for presensitized lymphocytes. Thus lymphocytes of patients with drug-induced thrombocytopenia are transformed by an antigen that forms after interaction of plasma, specific platelet membrane components and the drug.
P K Hosseinzadeh, B G Firkin, S L Pfueller
Mevalonate, an essential intermediate in cholesterol synthesis, is metabolized either to cholesterol or, by the shunt pathway, to CO2. Previous investigations have demonstrated that the kidneys are the chief site of circulating mevalonate metabolism and that sex hormones as well as insulin markedly influence circulating mevalonate metabolism. The present study examined in rats the influence of thyroid hormone status on mevalonate metabolism in vivo and in vitro. L-thyroxine administration increased renal conversion of circulating mevalonate to cholesterol, 41% in the females and 22% in the males. Conversely, hypothyroidism induced by 6 N propyl-2-thiouracil reduced renal conversion of circulatng mevalonate to cholesterol by 45% in females and 27% in males; thyroid hormone replacement in these animals returned cholesterogenesis in the kidneys to supranormal levels. Neither L-thyroxine nor hypothyroidism altered circulating mevalonate conversion to cholesterol in the liver or carcass. In vitro studies confirmed the in vivo observations. Changes in thyroid hormone produced only minor changes in the shunt pathway of mevalonate metabolism. This study demonstrates that the major effect of the thyroid hormone on the metabolism of circulating mevalonate is to alter the conversion of mevalonate to cholesterol, an effect localized solely to the kidneys.
K R Feingold, M H Wiley, G MacRae, M D Siperstein
Sera of 22 patients with active and 13 with inactive coccidioidomycosis, as well as 15 healthy subjects who were skin-test positive to coccidioidin and 39 healthy subjects who were coccidioidin skin-test negative, were assayed for immune complexes. Circulating immune complexes were measured by the Clq-binding assay, the Clq-solid phase assay, the monoclonal rheumatoid factor inhibition assay, and the monoclonal rheumatoid factor solid phase assay. An increased concentration of circulating immune complexes was detected in 73% of those with active disease by at least one assay compared with 13% of the healthy controls. Significantly increased levels of immune complexes were detected in sera of patients with active coccidioidomycosis by the Clq-binding assay (P < 0.001), the Clq-solid phase assay (P < 0.001), the monoclonal rheumatoid factor inhibition assay (P < 0.005), and the monoclonal rheumatoid solid phase assay (P < 0.05) compared with the results obtained in the 54 healthy subjects. In contrast, those with inactive disease did not show significantly increased concentrations of circulating immune complexes. Sucrose density gradient ultracentrifugation of patients' sera established that the immune complexes were of intermediate size, sedimenting between the 6.6S and 19S markers. Immune complexes were shown to contain both coccidioidin antigen and anticoccidioidin antibody. In addition, a radioimmunoassay was developed to quantitate coccidioidin antigen-containing immune complexes. The latter assay proved highly sensitive in detecting immune complexes in patients with active coccidioidomycosis.
S Yoshinoya, R A Cox, R M Pope
The decreased intestinal absorption of calcium and accelerated bone loss associated with chronic glucocorticoid excess may be mediated by changes in vitamin D metabolism, leading to decreased availability of circulating 1,25-dihydroxyvitamin D. This hypothesis was examined in 14 patients with either endogenous or exogenous glucocorticoid excess. Analysis of paired serum samples (mean +/- SE) in 13 patients during euglucocorticoidism and during hyperglucocorticoidism showed that glucocorticoid excess resulted in small decreases of plasma 25-hydroxy-vitamin D concentrations (22 +/- 2- 18 +/- 2 ng/ml; P < 0.05) but no significant changes in plasma 1,25-dihydroxyvitamin D (32 +/- 8- 23 +/- 6 pg/ml) or serum immunoreactive parathyroid hormone (21 +/- 2- 18 +/- 2 muleq/ml). Additionally, we studied plasma kinetics of [3H]1,25-dihydroxyvitamin D3 after intravenous bolus administration in 10 hyperglucocorticoid patients and in 14 normal controls. Assessment with a three-compartment model showed no significant abnormalities in production rates (hyperglucocorticoid patients 1.2 +/- 0.3 micrograms/d, controls 1.5 +/- 0.2 micrograms/d) or metabolic clearance rates (hyperglucocorticoid patients, 18 +/- 2%; controls, 14 +/- 2%) or feces (hyperglucocorticoid patients, 60 +/- 9%, controls, 54 +/- 6%). We conclude that glucocorticoid excess does not effect plasma levels, production, or degradation of 1,25(OH)2D in humans. Thus, other mechanisms must be postulated to explain satisfactorily the abnormalities of bone structure and intestinal calcium absorption that may occur after chronic glucocorticoid therapy.
E Seeman, R Kumar, G G Hunder, M Scott, H Heath 3rd, B L Riggs
The frequency of HLA-A, B, and Cw antigens as well as the antigens expressed preferentially on B cells and monocytes (DRw and Ia-like) was examined in a normal population and two related disease populations, psoriasis and psoriatic arthritis. HLA antigens distinguishing the two disease populations were found. Psoriatic patients demonstrated an increase in frequency of HLA-A1, B17, and B13. Patients with psoriatic arthritis demonstrated an increased frequency of HLA-A26, B38, and DRw4. Antigens showing a common increase in frequency in the two disease populations were HLA-Cw6, DRw7, and Ia744. These results demonstrate genetic differences as well as similarities in the two populations of patients with the common clinical feature of psoriasis. In addition to the above analysis, we examined the association of individual alloantigens elevated in frequency in the diseased population. These same alloantigens were examined for association in the normal population. This analysis revealed HLA antigen associations in the two disease groups that differed from the association of several antigens in the normal population. The results suggest that at least two genetic factors, one mapping in the HLA-A, C-B region and one mapping in the HLA-B-DRw region are associated with the disease states. Thus, multiple factors controlled by genes in the major histocompatibility complex appear to contribute to the disease entities under investigation.
C Murray, D L Mann, L N Gerber, W Barth, S Perlmann, J L Decker, T P Nigra
The antibody-dependent cell-mediated cytoxicity (ADCC) by human monocytes and neutrophils was investigated by measuring the release of 51chromate from prelabeled erythrocytes coated with immunoglobulin G. ADCC was found to be positively correlated to phagocytosis of 51Cr-labeled erythrocytes and to the postphagocytic events of the effector cells, activation of the hexose monophosphate shunt, and degranulation. Exclusion of oxygen from the incubation media halved the ADCC by both cell types without affectijg phagocytosis or degranulation. Likewise, ADCC by cells from patients suffering from chronic granulomatous disease (CGD) was only half the intensity of ADCC by cells from normals. Inhibitors of mitochondrial respiration were without depressing effect of ADCC. Azide, which in addition to its blocking action on oxydative phosphorylation also inhibits catalase and myeloperoxidase, resulted in a approximately equal to 40% stimulation of ADCC by cells from normals but was without effect of ADCC by cells from CGD patients. The hydroxyl radical scavenger, mannitol, significantly depressed ADCC by cells from normals (P < 0.01) but was without effect on cells from CGD patients. Azide and mannitol also were without effect on ADCC by normal cells when oxygen was excluded. In a xanthine-xanthine oxidase system, erythrocytes were effectively lysed. This lysis was inhibited by catalase, superoxide dismutase, and mannitol. When comparable concentrations of glucose oxidase were used no lysis was observed. H2O2 either alone or in combination with azide did not lyse erythrocytes. It is suggested that ADCC by both monocytes and neutrophils is partly dependent on the generation of hydroxyl radicals by the effector cells.
N Borregaard, K Kragballe
Patients with gyrate atrophy of the choroid and retina have 10- to 20-fold increased ornithine concentrations in body fluids and significantly reduced activity of ornithine aminotransferase in lymphocytes and cultured fibroblasts. We administered intravenous arginine to six patients and six controls to study in vivo inhibition by high ornithine concentrations of arginine-glycine transamidinase, the rate-limiting enzyme in creatine synthesis. Serum arginine concentrations curves after administration were similar for the two groups. The increment in serum ornithine was more than three times as great in patients as in controls. The mean half-times in plasma ornithine were 360 and 97 min, respectively. In the patients, the metabolic clearance of ornithine from the extracellular fluid was significantly delayed. Urinary guanidinoacetate excretion rose markedly in all controls, the excretion rate being higher in females. The patients always excreted less than the controls, the differences within the sexes being highly significant. Differences in creatine excretion after administration were less marked. We conclude that in gyrate atrophy patients, formation of guanidinoacetate, creatine, and possibly phosphocreatine is inhibited at the transaminidation step by the high concentrations of ornithine. Deficiency of the high-energy phosphates may underlie the pathogenesis of the eye and muscle atrophies.
I Sipilä, O Simell, P Arjomaa
The plasma content of B6 vitamers is governed by, among other factors, dietary supply and metabolic interconversion. This study examines the effect of pyridoxine supplementation on the plasma content of B6 vitamers and pyridoxic acid in man, and the metabolic conversion and release of B6 compounds in isolated rat hepatocytes. Six healthy human subjects were given 100 mg pyridoxine-HCl/d orally for 1--3 wk. Before pyridoxine supplementation, the mean total plasma level of B6 vitamers was 114 +/- 9 nM; and pyridoxal-P, pyridoxamine-P, pyridoxal, pyridoxine, and pyridoxamine accounted for 54, 3, 11, 27, and 5%, respectively. Plasma level of pyridoxic acid was 40 +/- 7 nM. Thus, pyridoxal-P is the principal B6 vitamer in plasma. During pyridoxine supplementation, mean plasma levels of the B6 vitamers and pyridoxic acid increased to 655 +/- 122 and 222 +/- 55 nM, respectively. The plasma content of pyridoxal-P and pyridoxic acid increased 6--7-fold and that of pyridoxal, 12-fold, but the pyridoxine level did not increase. Isolated hepatocytes, 1 g/15 ml, were incubated for 2 h with 3.33 microM [14C]pyridoxine (6 microCi/mumol). At zero time, the cells contained about 35 nmol pyridoxal-P and 25 nmol pyridoxamine-P. After 2 h incubation, the cellular content of pyridoxal-P and pyridoxamine-P did not change significantly, but the medium contained 5.9 nmol pyridoxal-P, 0.3 nmol pyridoxamine-P, 7.2 nmol pyridoxal, 26.6 nmol pyridoxine, 0.3 nmol pyridoxamine, and 7.5 nmol pyridoxic acid. Whereas the specific radioactivity of pyridoxal-P, pyridoxal, and pyridoxic acid in the medium approached that of [14C]pyridoxine, the specific radioactivity of cellular pyridoxal-P and pyridoxamine-P was only 20% of that of pyridoxine. Thus, newly synthesized pyridoxal-P is not freely exchangeable with endogenous pyridoxal-P, but is preferentially released or degraded to pyridoxal and pyridoxic acid. The latter B6 compounds are also released. These results suggest that orally ingested pyridoxine is rapidly metabolized in liver and its products are released into the circulation in the form of pyridoxal-P, pyridoxal, and pyridoxic acid.
L Lumeng, A Lui, T K Li
Bovine vascular endothelial cells maintained on dishes coated with an extracellular matrix and exposedto medium supplemented with lipoprotein-deficient serum (LPDS) require the presence of lipoprotein to proliferate optimally. High density lipoprotein (HDL) seems to be the major factor involved in the proliferation of vascular endothelial cells. This is mostly due to its lack of toxicity when added at high concentration, as well as to its nondependence on LPDS to exhibit its mitogenic properties. Therefore, HDL at physiological concentrations (1,000--1,500 microgram protein/ml) can fully replace serum. Low density lipoprotein, unlike HDL, has a biphasic effect. Although mitogenic for vascular endothelial cells when added at low concentration, once physiological concentrations are reached it becomes toxic for the cells. Moreover, and in contrast with HDL, the mitogenic effect of low density lipoprotein was found to be a function of the LPDS concentration to which cultures were exposed. The substrate upon which cultures are maintained has been found to be an important factor if a mitogenic effect of HDL is to be observed. When maintained on plastic, cells proliferate poorly in response to HDL unless fibroblast growth factor is added to the medium. In contrast, when maintained on extracellular matrix, an optimal growth rate is induced by HDL, even in the absence of fibroblast growth factor. This suggests that, in vivo, the integrity of the basement membrane upon which endothelial cells rest and migrate is an important factor in determining the cells response to lipoproteins present in plasma.
J P Tauber, J Cheng, D Gospodarowicz
Insulinlike growth Factor I (IGF I), a growth hormone-dependent peptide or somatomedin, was studied for its effects on bone formation by examining the synthesis of DNA, collagen, and noncollagen protein in cultures of 21-d fetal rat calvaria. IGF I caused a dose-dependent stimulation of the incorporation of [3H]thymidine into DNA at concentrations of 0.1--100 nM; the effect appeared after 6 h, was maximal at 12 h, and was sustained for 96 h. IGF I also increased the bone DNA content, IGF I at 0.1--3 nM had a small stimulatory effect on the incorporation of [3H]proline into collagenase-digestible protein (CDP) whereas 30 nM IGF I caused a two- to threefold increment and had a maximal effect. A smaller effect on the labeling of noncollagen protein (NCP) was also observed. The effect of CDP and NCP appeared and was maximal after 12 h and was sustained for 96 h. IGF I increased the total collagen content of bones. The IGF I stimulatory effect on the incorporation of [3H]thymidine was seen in both the periosteum and periosteum-free calvarium, whereas that on the labeling of CDP was seen only in the central, osteoblastic-rich, non-periosteal bone. Histological sections showed a 10-fold increase in the mitotic index after Colcemid arrest in IGF I-treated bones, the mitoses were equally distributed in the periosteum and central portions of the calvarium. Insulin had a stimulatory effect on the incorporation of [3H]proline into CDP and NCP and 1 nM--1 microM similar to the effect of IGF I. In contrast, high insulin concentrations (0.1 and 1 microM) were required to increase the incorporation of [3H]thymidine, and insulin did not affect DNA content. Cortisol decreased the stimulatory effect of IGF I on DNA labeling but greatly enhanced the stimulatory effect of IGF I on the incorporation of [3H]proline into CDP. Triiodothyronine and parathyroid hormone increased the incorporation of [3H]thymidine and were additive to IGF I. Triiodothyronine did not affect the labeling of CDP, but parathyroid hormone inhibited it and opposed the effect of IGF I. These studies indicate that IGF I stimulates bone DNA, collagen, and NCP synthesis in vitro. IGF I and insulin have similar effects on bone collagen synthesis but IGF I stimulates the synthesis of DNA at physiological concentrations, and insulin does not.
We used the retrograde catheter technique to investigate the effect of HeO2 on resistance to maximum expiratory flow (Vmax) in airways subsegments between alveoli and the equal pressure point (EPP), and between EPP and the flow-limiting segment (FLS). FLS were found at the same airway site in sublobar bronchi (i.d., 0.54 +/- 0.13 cm) on both air and HeO2 in the six human excised lungs studied. Static elastic recoil pressure (5 +/- 1 cm H2O) and the lateral pressure at FLS (critical transmural airway pressure -6 +/- 3 cm H2O) were not different on the two gases. delta Vmax averaged 37 +/- 8.9% and was similar to the value found in healthy subjects of similar age (66 +/- 10 yr). EPP were located on HeO2 in peripheral airways (i.d., 0.33 +/- 0.03 cm), and EPP on air were located more downstream. Resistance between EPP and FLS was highly density dependent. Resistance between alveoli and EPP behaved as if it were density independent, due in part to Poiseuille flow in the peripheral airways and in part to the consequent narrowing of peripheral airways on HeO2. This density-independent behavior in peripheral airways reduced delta Vmax on HeO2 from its predicted maximal amount of 62%. Assuming that FLS is the "choke point" these findings are consistent with wave-speed theory of flow limitation modified to include functionally density-independent pressure losses in peripheral airways. These results and conclusions are similar to those found in living dogs. They question previous interpretation of delta Vmax as an index of peripheral airway obstruction, and demonstrate the utility of the wave-speed theory in explaining complicated mechanisms of expiratory flow limitation.
S N Mink, L D Wood
In previous studies of two patients with polycythemia vera (PV) who were heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G6PD), only A type enzyme was found in nonlymphoid blood cells. However, some erythroid and granulocytic colonies grown in vitro were type B and therefore arose from presumably normal progenitors. One patient had enough type B colonies (8%) that studies of the physical characteristics of normal and PV clonal colony-forming cells could be undertaken. When marrow cells were separated by velocity sedimentation at unit gravity, most PV clonal granulocyte-macrophage progenitors (CFU-C) (type A G6PD) sedimented between 6.4 and 7.2 mm/h, whereas most residual normal, type B CFU-C sedimented less than or equal to 5.9 mm/h (P = 0.04)., When blood cells were separated over a discontinuous buoyant density gradient, PV clonal CFU-C equilibrated at densities < 1.065 g/ml, whereas residual normal CFU-C were found greater than or equal to 1.065 g/ml (P < 0.01). PV clonal and residual normal erythroid burst-forming progenitors were not separable by either method. Thus PV clonal CFU-C are larger and less dense cells than are residual normal CFU-C.
J W Singer, J W Adamson, C Ernst, N Lin, L Steinmann, S Murphy, P J Fialkow
The responses of isolated human peripheral neutrophils to either simultaneous or sequential additions of two chemotactic factors were studied. Simultaneous additions of formyl-methionyl-leucyl-phenylalanine (10-100 nM) and the fifth component of complement, C5a (1-10 microliters/ml), evoked partially additive responses of membrane depolarization as measured by the fluorescent dye 3,3'-dipropyl-thiocarbocyanine, a transient elevation of intracellular cyclic AMP (cAMP), and superoxide (O2-) generation as assessed by ferricytochrome c reduction. Preincubation of the cells with either formyl-methionyl-leucyl-phenylalanine or C5a alone caused dose-dependent inhibition of the depolarization, the cAMP increase, and O2- release induced by a subsequent exposure to an optimal dose of the same stimulus, i.e., deactivation occurred. In contrast, when cells were treated with one chemotactic factor and then exposed to the other stimulus, the cells exhibited a normal response of peak depolarization, the rise in cAMP, and O2-0 production i.e., cross-deactivation failed to occur. The results imply that deactivation of these phenomena is stimulus specific. Further, these observations are consistent with the hypothesis that cross-deactivation of chemotaxis is mediated by one or more processes that are irrelevant to O2- generation, and that occur distal to the depolarization and cAMP steps in the sequence of neutrophil activation: possibly microtubule polymerization and orientation.
L Simchowitz, J P Atkinson, I Spilberg
To determine whether vasoactive renal hormones modulate renal blood flow during alterations of sodium balance, simultaneous measurements of arterial and renal venous concentrations of norepinephrine and prostaglandin E2 (PGE2) and of plasma renin activity, as well as renal blood flow and systemic hemodynamics were carried out in 24 sodium-depleted and 28 sodium-replete anesthetized dogs. The mean arterial blood pressure of the sodium depleted dogs was not significantly different from that of the animals fed a normal sodium diet, but cardiac output was significantly lower (3.07 +/- 0.18 vs. 3.77 +/- 0.17 liters/min, mean +/- SEM; P < 0.01). Despite the higher total peripheral vascular resistance in the sodium-depleted dogs (46.1 +/- 2.9 vs. 37.0 +/- 2.1 arbitrary resistance U; P < 0.02), the renal blood flow and renal vascular resistance were not significantly different in the two groups. The arterial plasma renin activity and concentration of norepinephrine were higher in the sodium-depleted animals than in the controls; the arterial concentration of PGE2 was equal in both groups. The renal venous plasma renin activity was higher in the sodium-depleted dogs. Similarly, the renal venous norepinephrine concentration was higher in the sodium-depleted dogs than in the controls (457 +/- 44 vs. 196 +/- 25 pg/ml; P < 0.01); renal venous PGE2 concentration was also higher in the sodium depleted dogs (92 +/- 22 vs. 48 +/- 11 pg/ml; P < 0.01). Administration of indomethacin to five sodium-replete dogs had no effect on renal blood flow. In five sodium-depleted dogs indomethacin lowered renal blood flow from 243 +/- 19 to 189 +/- 30 ml/min (P < 0.05) and PGE2 in renal venous blood from 71 +/- 14 to 15 +/- 2 pg/ml (P < 0.02). The results indicate that moderate chronic sodium depletion, in addition to enhancing the activity of the renin-angiotensin system, also increases the activity of the renal adrenergic nervous system and increases renal PGE2 synthesis. In sodium-depleted dogs, inhibition of prostaglandin synthesis was associated with a significant decrease in renal blood flow. The results suggest that the renal blood flow is maintained during moderate sodium depletion by an effect of the prostaglandins to oppose the vasoconstrictor effects of angiotensin II and the renal sympathetic nervous system.
J A Oliver, J Pinto, R R Sciacca, P J Cannon
To study the effect of kallikrein on renal renin release, we superfused rat renal cortical slices with 3.5 to 140 milliesterase units (mEU)/ml of purified rat urinary kallikrein. Kallikrein was a potent stimulus of renin release. Renin rose in a dose-dependent fashion from 70 mEU/ml to 140 mEU/ml. The response to 140 mEU/ml was greater than that seen with maximal doses of prostaglandin E2 (170 +/- 43%, P < 0.05) and at least the same as isoproterenol (242 +/- 49% increase), or dibutyryl cyclic AMP (272 +/- 40%). Trypsin was ineffective under these experimental conditions. Kallikrein-stimulated renin release was completely abolished by trasylol, whereas bradykinin did not increase renin production, indicating that kallikrein's effect is not mediated via kinin generation. There was no demonstrable acid activation or kallikrein activation of the superfusate and chromatography on Sephacryl S-200 revealed a single renin peak of -40,000 mol wt, suggesting that all of the renin release was in the active form. The data suggests that urinary kallikrein acts directly on the rat kidney to release renin, possibly via proteolytic conversion of prorenin to active renin. Our results support the concept that kallikrein may be an endogenous activator of prorenin in the kidney.
S Suzuki, R Franco-Saenz, S Y Tan, P J Mulrow
We have examined the effect of in vitro hyperinsulinemia on insulin binding, glucose transport, and insulin degradation in isolated rat adipocytes. When cells were incubated with insulin for 2 or 4 h at 37 degrees C, followed by washing in insulin-free buffer to remove extracellular and receptor-bound insulin, a time and dose-dependent decrease in insulin receptors was observed, which was accompanied by a reduced ability of cells to degrade insulin. Furthermore, the quantitatively predicted rightward shift in the insulin-glucose transport dose-response curve could be demonstrated. In addition to this reduction in insulin sensitivity, a striking decrease in maximal insulin-stimulated glucose transport was observed in the 4-h insulin-treated cells, indicating an abnormality distal to the insulin receptor. Thus, in vitro insulin-induced insulin resistance in adipocytes is caused by both receptor and postreceptor abnormalities. The post-receptor defect is most likely at the level of the glucose transport system per se because the insulinlike agents, spermine and antiinsulin receptor antibodies, also had a markedly reduced ability to stimulate glucose transport in 4-h insulin-treated cells. On the other hand, when cells were incubated with 100 ng/ml insulin for up to 4 h, after which time 2-deoxy glucose uptake was measured without removing buffer insulin or allowing receptor-bound insulin to dissociate, no decrease in maximal insulin-stimulated glucose transport was found. In conclusion, (a) insulin leads to a dose-dependent loss of insulin receptors in freshly isolated adipocytes accompanied by the predicted functional consequence of decreased receptors, i.e., a rightward shift in the insulin-glucose transport dose-response curve, (b) prolonged incubation with insulin causes a marked postreceptor defect in the glucose transport system, (c) maintenance of the activated state of the glucose transport system prevents the expression of the post-receptor defect, (d) the location of the postreceptor abnormality is most likely in the glucose transport system per se, and (e) insulin-induced receptor loss is accompanied by a decrease in insulin degradation.
S Marshall, J M Olefsky
The effects of IgG in different configurations on the Fc receptor function of human monocytes were studied. Receptor function was assessed by quantitating immune adherence and/or ingestion of human erythrocytes coated with IgG anti-D antibody. Monomeric IgGl in solution inhibited the Fc receptor function of monocytes, but this function was restored completely after washing. In contrast, monomeric IgG that was adsorbed nonspecifically to a plastic surface inhibited the Fc receptor function of monocytes even after washing away unbound IgGl. This loss of function could be blocked by sodium azide and was reversed when the IgG adsorbed to plastic was degraded by trypsin, suggesting that loss of function was the reversible consequence of localized binding of most of the monocyte's receptors at the point of contact with immobilized IgGl. Fluid-phase aggregates of IgGl also reduced the Fc receptor function of monocytes as a consequence of direct binding to the monocyte surface. High concentrations of purified aggregates rapidly reduced Fc receptor function but function was reversed by trypsin even after incubation for 18 h. Lower concentrations of aggregates reduced Fc receptor function more slowly, but after 18 h of incubation, lost function was not restored by trypsin treatment. Because the transition from reversible to irreversible loss was blocked by sodium azide, an energy-dependent process of ingestion, shedding or denaturation of receptors is responsible for this irreversible loss of Fc receptor function. Rabbit IgG anti-human IgG bound to IgG adsorbed to the surface of monocytes also mediated a loss of Fc receptor function as a result of the binding of Fc receptors to the Fc portion of the rabbit IgG molecule, a process analogous to the binding of aggregated IgG. After irreversible depletion of Fc receptor function by anti-IgG, partial recovery of function was detectable within 12-24 h of incubation in vitro, and this recovery was blocked by cycloheximide, suggesting that new receptor synthesis was required for restoration of function.
R J Kurlander
Studies were carried out to compare the effects of parathyroid extract (PTE) on serum and urinary calcium (Ca) and phosphorus (P), serum 25-hydroxyvitamin D (25-OHD), serum 24,25-dihydroxyvitamin D (24,25(OH)2D), serum 1 alpha,25-dihydroxyvitamin D (1 alpha,25(OH)2D), and urinary cyclic AMP in two normal subjects, two patients with hypoparathyroidism (HP) and six patients with pseudohypoparathyroidism (PHP), some of whom were on suboptimal treatment with vitamin D. Two of the patients with PHP were studied while on long-term treatment with 1 alpha,25-(OH)2D3. Before PTE, serum 1 alpha, 25(OH)2D was at the lower limit of normal in one patient and was abnormally low in the other five patients. None of these individuals was on treatment with 1 alpha,25(OH)2D3. Serum 25-OHD and 24,25(OH)2D were either increased or at the upper limit of normal in the patients given vitamin D and were normal in the other patients. PTE lowered the serum P and increased the serum 1 alpha,25(OH)2D, serum and urinary Ca, urinary P, and urinary cyclic AMP in the normal subjects and patients with HP. In individual studies, changes in serum 1 alpha,25(OH)2D and serum Ca occurred in parallel before, during, and after PTE. In contrast, PTE had very little effect in the patients with PHP. Whereas there were highly significant positive correlations between serum 1 alpha,25(OH)2D in each of the normal subjects and patients with HP, there were significant correlations in only one of the patients with PHP. An increase in serum Ca in response to PTE was observed in one of the two patients with PHP who were on long-term treatment with 1 alpha,25(OH)2D3. In these individuals, PTE produced only slight increases in serum 1 alpha,25(OH)2D. Serum 25-OHD and 24,25(OH)2D were not changed by PTE in any of the subjects or patients. The results provide evidence that hypocalcemia in HP and PHP arises in part from low circulating 1 alpha,25-(OH)2D, and indicate that the lack of change in serum 1 alpha,25(OH)2D with PTE in patients with PHP is related to impaired renal adenylate cyclase and phosphaturic responses. These and previous results support the idea that diminished renal production of 1 alpha,25(OH)2D, because of a defect in the parathyroid hormone-responsive adenylate cyclase system, may be a contributing factor in the pathogenesis of the abnormal calcium metabolism in PHP.
P W Lambert, B W Hollis, N H Bell, S Epstein
Medullary thick ascending limbs of Henle's loop of the Swiss-Webster mouse were perfused in vitro with an isotonic perfusate and a Ringer's bathing medium. In five studies, addition of a supramaximal concentration of synthetic arginine vasopressin (AVP) to the bathing medium resulted in an increase in electrical potential difference (PD) from 5.0 +/- 1.5 mV, lumen positive, to 10.7 +/- 1.4 mV (P < 0.001). When AVP was removed, the PD returned to 2.6 +/- 0.9 mV (P < 0.001), then increased again to 6.9 +/- 1.7 mV (P < 0.01) when AVP was added a second time. A significant, but submaximal, increase in PD of 2.3 +/- 0.6 MV (P < 0.05) was observed in five medullary thick ascending limbs when AVP was added to the bathing medium at a concentration of 10 microunits/ml. This increase was approximately one-third of the response observed at a concentration of 100 microunits/ml in the same tubule. No further increment in PD was observed in five medullary thick ascending limbs when the AVP concentration was increased from 100 to 1,000 microunits/ml. In seven thick ascendcing limbs, the effect of AVP on PD was reproduced by the addition of 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate to the bathing medium at a final concentration of 0.1 mM. AVP increased unidirectional chloride flux from lumen to bath from 29.3 +/- 3.2 to 69.8 +/- 6.2 peq/cm per s (P < 0.001) in spite of an increase in the lumen positive PD from 1.6 +/- 0.5 mV to 7.0 +/- 0.6 mV (P < 0.001). Unidirectional chloride flux from bath to lumen was not affected by AVP. In another series of experiments, net chloride flux increased from 15.6 +/- 3.0 to 41.7 +/- 5.3 peq/cm per s (P < 0.05) after addition of AVP. The effect of AVP on hydraulic water permeability (Lp) was examined by adding raffinose to the bathing medium in both the presence and the absence of AVP. The calculated Lp of 16 +/- 2 nm/s per atm in the absence of AVP, although very low, was significantly different from zero (P < 0.01). However, the Lp did not increase significantly when AVP was added to the bathing medium. These results suggest that AVP has a second site of action in the kidney to increase chloride transport by the medullary thick ascending limb in addition to its well-known effect on the water permeability of the collecting tubule. The former effect would contribute to urinary concentrating ability by increasing the axial osmotic gradient in the renal medulla.
D A Hall, D M Varney
During the third trimester of human pregnancy the concentrations of deoxycorticosterone (DOC) in maternal plasma are 4-50 times those in nonpregnant women and men. It has been suggested that the increased amount of DOC in maternal plasma originates in the fetal compartment. We considered an alternate explanation for the high levels of DOC in plasma or near-term pregnant women, viz., that DOC may be derived in part from 21-hydroxylation of maternal plama progesterone. To test this hyposthesis we measured the fractional conversion of plasma progesterone to DOC from the relationship between the 3H:14C ratio of the infused tracers, [3H]progesterone and [14C]-DOC, and the 3H:14C ratio or urinary 3 alpha,21-dihydroxy-5 beta-pregnan-20-one (tetrahydro-DOC). The fractional conversion of plasma progesterone to DOC ([rho](BU)P-DOC), measured in this manner, was 0.007 +/- 0.001 (mean +/- SEM, n = 26) in the subjects of this study. The values for [rho](BU)P-DOC varied widely among subjects (0.002-0.022) but the range of values for [rho](BU)P-DOC was similar among women pregnant with an anencephalic or dead fetus, nonpregnant and adrenalectomized women, and men. The transfer constant of conversion of progesterone to DOC in plasma, [rho](BB)P-DOC, remained constant in a nonpregnant woman during the infusion of nonradiolabeled progesterone at rates of 0-14 mg/h. Based on the results of these studied, we conclude that DOC is formed by extra-adrenal 21-hydroxylation of plasma progesterone and that the rate of formation of DOC by this pathway is proportional to the concentration of progesterone in plasma.
C A Winkel, L Milewich, C R Parker Jr, N F Gant, E R Simpson, P C MacDonald
The effect of somatostatin (SRIF) on ion transport was determined in the rat colon in vitro. SRIF produced a sustained decrease in the short circuit current (Isc) (-0.8 +/- 0.1 mueq/h x cm2) and increased net Cl absorption (0.9 +/- 0.3 mueq/h x cm2). The threshold effect of SRIF on Isc was observed at 6 nM. 10 microM serotonin decreased net Na absorption (-2.6 +/- 0.4 mueq/h x cm2), net Cl absorption (-3.6 +/- 0.5 mueq/h x cm2) and increased Isc (0.7 +/- 0.1 mueq/h x cm2); these changes were totally blocked by 0.1 microM SRIF. SRIF completely blocked net Cl secretion induced by 10 mM theophylline (-2.5 +/- 0.7 to +4.1 +/- 2.0 muq/h x cm2) and partially blocked theophylline-induced inhibition of net Na absorption (0.7 +/- 0.5 to 2.1 +/- 0.4 mueq/h x cm2). SRIF also blocked prostaglandin E1 (PGE1) induced increase in potential difference and Isc (P < 0.001). Mucosal cyclic AMP levels were increased by theophylline and PGE1 but not by serotonin. SRIF had no effect on basal or theophylline- and PGE1-stimulated cyclic AMP levels. These results indicate that SRIF blocks both cyclic AMP and noncyclic AMP mediated changes in ion secretion and suggest that SRIF is acting at a step in the secretory process beyond the formation of cyclic AMP.
K Dharmsathaphorn, L Racusen, J W Dobbins
The relationship of riboflavin transport to the transport of other substances including drugs in rabbit choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, and brain cells were studied in vivo and in vitro. In vitro, the ability of rabbit choroid plexus to transport riboflavin from the medium (cerebrospinal fluid surface) through the choroid plexus epithelial cells into the extracellular and vascular spaces of the choroid plexus was documented using fluorescence microscopy. These studies provided further evidence that riboflavin is transported from cerebrospinal fluid to blood via the choroid plexus. The transport of [14C]riboflavin by the isolated choroid plexus was inhibited by thiol agents, ouabain, theophylline, various flavins (lumiflavin and lumichrome > sugar containing flavins), and cyclic organic acids including penicillin and fluorescein. Riboflavin inhibited [14C]penicillin transport competitively and the inhibition constant (K1) for riboflavin equaled the concentration of riboflavin at which the saturable transport system for riboflavin is 50% saturated (KT). These and other data suggest that riboflavin, penicillin, and possibly fluorescein are transported by the same transport system in choroid plexus. In vivo, the intra-ventricular injection or riboflavin and [14C]penicillin inhibited [14C]penicillin transport from cerebrospinal fluid. In vitro, various flavins (riboflavin > other sugar-containing flavins > lumiflavin > lumichrome) inhibited [14C]riboflavin accumulation by brain slices. These studies support the notions that: (a) riboflavin accumulation by choroid plexus (active transport) is quite different from that in brain cells (facilitated diffusion and intracellular trapping), and (b) therapeutically important cyclic organic acids (e.g., penicillin) are transported fom cerebrospinal fluid by the riboflavin transport system in choroid plexus.
In previous immunohistochemical studies, it has been found that all nuclei contain cyclic (c)GMP, which occurs in discrete aggregates and in the nucleolus. We have studied the nature of the cGMP aggregates in isolated mouse fetal nuclei using a specific immunofluorescent technique. These aggregates correspond to the areas of condensation of DNA, demonstrable by either Felugen's or acridine orange stain. Treatment with DNAase eliminated DNA and cGMP staining. Staining for RNA, with a human anti-RNA antibody, demonstrated RNA to be distributed diffusely throughout the nucleus and not preferentially in the areas of discrete cGMP aggregates. The diffuse stain for nuclear RNA was eliminated by pretreatment with RNAase but not DNAase, but aggregates of cGMP were not affected by pretreatment with RNAase. Sites of active RNA synthesis were determined by autoradiography using [3H]uridine, and did not correspond to the aggregates of cGMP. The relationship of cGMP to nucleolar function was examined in the endothelial cells of the isthmus and ampulla of the rat fallopian tube. Previous studies have shown that in proestrous, a period of increased RNA synthesis, nucleoli detectable by staining for RNA appear in the endothelial cells lining the fallopian tube. After immunofluorescent staining, we found prominent accumulation of cGMP in the nucleoli. During other phases of the cycle, there is an absence of nucleoli detectable by staining for RNA, and an absence of nucleolar cGMP. After we treated hypophysectomized or oophorectomized rats with estrogen, which is known to increase nucleolar RMA synthesis in the fallopian tube and endometrium, nucleoli in the endothelial cells of the rat fallopian tube and uterus stained strongly for cGMP. In conclusion, our studies suggest that the discrete aggregates of nuclear cGMP are associated with a fraction of DNA uninvolved in RNA synthesis. In contrast, cGMP appears in the nucleolus during a period of increased RNA synthesis, suggesting a role for cGMP in regulating nucleolar synthesis and processing of RNA.
E M Rosenberg, J G Conway, S M Tucci, E W Doucet
A small subpopulation of human peripheral blood T lymphocytes has the capacity to adhere selectively to myelinated sections of human and nonhuman brain tissue. Adherence of lymphocytes from patients with multiple sclerosis is significantly greater than adherence of control lymphocytes. Monocytes inhibit binding in controls. This function appears to be lost by multiple sclerosis monocytes.
P Dore-Duffy, V Goertz, B L Rothman
Lymphocytes with Fc receptors (FcR) for IgG active in natural cytotoxicity and antibody-dependent cellular cytotoxicity were separated into sheep erythrocyte rosetting (E+) and nonrosetting (E-) fractions, and examined for reactivity with the OK panel of hybridoma-produced monoclonal antibodies. Few cells in either the E+ FcR+ or the E- FcR+ fraction reacted with seven antibodies used to define T cells in various stages of differentiation (OK3, OKT4, OKT5, OKT6, OKT8, OKT9, OKT10). Neither fraction expressed an Ia-like antigen (detected by OKI1), but both were highly reactive with OKM1, an antibody that reacts with monocytes and granulocytes. Incubation of these cytotoxic effector cells with OKM1 plus complement abolished all cytotoxic reactivity, but incubation with a pan-T cell antibody (OKT3) plus complement had no significant effect. These cells were not monocyte precursors, because they could not be induced in vitro to develop macrophage characteristics. The data indicate that most cytotoxic effector cells in natural cytotoxicity and antibody-dependent cellular cytotoxicity are not in the T cell lineage, but have a myeloid origin.
H D Kay, D A Horwitz
The effects of vitamin D, 2.5 mg (100,000 U)/d for 4 d, on serum calcium, serum 25-hydroxyvitamin D (25-OHD), and serum 1 alpha,25-dihydroxyvitamin D [1 alpha,25(OH)2D] were compared in 17 normal subjects and 6 patients with sarcoidosis who had normocalcemia and no history of hypercalcemia. The diagnosis was confirmed histologically in each of them. Vitamin D increased mean serum 25-PHD from 30 +/- 4 to 99 +/- 15 ng/ml (P < 0.001) and did not change mean serum 1 alpha,25(OH)2D (32 +/- 3 vs. 29 +/- 3 pg/ml) or mean serum calcium (9.5 +/- 0.1 vs. 9.6 +/- 0.1 mg/dl) in the normal subjects. In contrast, vitamin D increased mean serum 25-OHD from 19 +/- 3 to 65 +/- 19 ng/ml (p < 0.05), increased mean serum 1 alpha,25(OH)2D threefold from 40 +/- 7 to 120 +/- 24 pg/ml, and increased mean serum calcium from 9.4 +/- 0.2 to 9.8 +/- 0.2 mg/dl (P < 0.01). There was a significant positive correlation between the serum 1 alpha,25(OH)2D and serum calcium in these individuals (r = 0.663, P < 0.01) but not in the normal subjects. The results (a) provide further evidence for abnormal regulation of circulating 1 alpha,25(OH)2D in sarcoidosis and (b) indicate that the abnormality may exist in patients with normal calcium metabolism. Thus, the defect in vitamin D metabolism in sarcoid apparently is more common than was previously recognized.
P H Stern, J De Olazabal, N H Bell
Incubation of a 0.5% suspension of washed normal mouse erythrocytes with ferriprotoporphyrin IX (FP) for 2.5 h at 37 degrees C and pH 7.4 results in sufficient membrane damage to produce hemolysis. A sigmoidal dose-response curve is followed with 50% hemolysis being produced by 4 microM FP. Complete hemolysis is produced by 6 microM FP. The hemolytic process has at least two phases: a lag phase of approximately 45 min, during which little hemolysis occurs, and a phase characterized by rapid hemolysis. Chloroquine, which binds tightly to FP, enhances the effect of FP by eliminating the lag phase. Under the conditions of these experiments, maximum enhancement is observed with chloroquine concentrations in the range of 5-25 microM. Since FP is produced when malaria parasites digest hemoglobin, it may mediate a chemotherapeutic effect of chloroquine by forming a complex with the drug that could enhance the toxicity of FP for biological membranes, including those of the parasite.
A C Chou, C D Fitch
Elastin-derived peptides, produced by digesting human aortic elastin and bovine ligament elastin with human neutrophil elastase, were tested for chemotactic activity. At 100 micrograms protein/ml, elastin digests were nearly as active for monocytes as saturating amounts of complement-derived chemotactic activity. Neutrophils and alveolar macrophages showed less response to elastin peptidces than did monocytes. Fractionation of the digests by gel filtration chromatography disclosed that maximal chemotactic activity eluted in fractions corresponding to 14,000-20,000 mol wt containing most of the desmosine cross-links in the digests. Whole human serum and rabbit anti-elastin immunoglobulin inhibited the chemotactic activity. Purified desmosine also showed chemotactic activity for monocytes, maximal at 10 nM. These findings suggest that elastin-degradation products enriched in cross-linking regions recruit inflammatory cells in vivo and that elastin proteolysis, characteristic of emphysema, may be a signal for recruitment of mononuclear phagocytes into the lungs.
R M Senior, G L Griffin, R P Mecham
The idiotypic determinants on IgM rheumatoid factor (RF) from a single family have been analyzed. Rabbit Fab'2 antiidiotypic antibody was prepared against purified IgM-RF from a patient with rheumatoid arthritis. As measured by radioimmunoassay, the antiidiotype reacted with at least 90% of the patient's RF, but not with non-RF immunoglobulins from the same serum, nor with 10 of 11 polyclonal and monoclonal RF from unrelated individuals. Cross-reacting idiotypes were detected on RF in four of the patients' first degree relatives, spanning three generations, without apparent relation of HLA type or clinical rheumatoid arthritis. These results suggest that IgM-RF associated idiotypes were inherited in this family.
J L Pasquali, S Fong, C Tsoukas, J H Vaughan, D A Carson