Issue published June 1, 1982

S ystemic treatment of rats with captopril (50 mg/kg body wt per os), a specific competitive inhibitor of angiotensin l-converting enzyme, significantly inhibits vascular permeability changes induced by the intradermal injection of the vasoactive mediators histamine, bradykinin, serotonin, and compound 48/80. This effect of captopril is both dose- and time-dependent with approximately 60% inhibition of edema formation observed 7 h after captopril treatment (100 mg/kg body wt per os). The inhibitory effect of captopril on edema formation is temporally unrelated to the inhibition of serum angiotensin l-converting enzyme activity or serum prostaglandin E2 levels and is not inhibited by systemic treatment of rats with indomethacin. The data suggest that captopril may have potent antiinflammatory activity through as yet undefined mechanisms.

T he receptor for Factor VIII/von Willebrand factor (F. VIIIVWF) is readily available on circulating platelets. We have found that the stimulation of platelets with traces of thrombin at concentrations that are generated physiologically (0.008 U-0.05 U/ml) induced concentration-dependent binding of 125I-labeled F. VIIIVWF in a steady-state system. The binding induced by thrombin was specific because it was inhibited by a 100-fold molar excess of unlabeled F. VIIIVWF factor, by rabbit monospecific antibody against Factor VIII, and was not inhibited by an excess of fibrinogen or fibronectin. Binding induced by thrombin required metabolically active platelets, in contrast to a system with ristocetin that also prompted binding to glutaraldehyde-treated platelets. The thrombin effects on binding of 125I-F. VIIIVWF was not observed when platelets were washed with EDTA-containing buffers; EDTA and EGTA both inhibited thrombin-induced binding. Platelet membrane glycoproteins were required because enzymatic stripping od them from the platelet surface with chymotrypsin reduced binding 2.5-5.0-fold. Prostacyclin, in the concentration range of 1 to 50 nM, had two distinct effects on the receptor for F. VIIIWVF: (a) it prevented exposure of this receptor when added 10 min before thrombin, and (b) it reversed the binding of 125I-F. VIIIVWF to the platelet receptor when added 30 min after thrombin and the ligand, ie., when binding was at equilibrium. The dual effect of prostacyclin on the receptor for F. VIIIVWF was reproduced by dibutyryl cyclic AMP.

M edullasin, a new serine protease found in bone marrow cells, increased markedly human natural killer cell activity. Whereas the natural killer cell activity measured immediately after the treatment with medullasin remained almost on the same level as the control, an incubation at 37 degrees C for several hours increased markedly the natural killer cell activity of the lymphocytes treated with medullasin. Enhancement of the natural cytotoxicity was caused by the treatment with physiologic concentrations of the protease (5-20 micrograms/ml). Inhibitors of medullasin such as phenylmethylsulfonyl fluoride and elastatinal prevented the activation of natural cytotoxicity. Depletion of lymphocytes bearing Fc receptors for IgG abolished the enhancement of natural killer cell activity by medullasin. Interferon activity was not detected in the supernatant of lymphocyte cultures stimulated with medullasin. The medullasin enhanced further the natural killer cell activity of lymphocytes stimulated with interferon. Medullasin activity was detected neither in unstimulated nor stimulated (by concanavalin A or phytohemagglutinin) human lymphocytes. The protease was released easily from human mature granulocytes into culture medium. It is considered from these results that the level of human natural killer cell activity is regulated by medullasin released by mature granulocytes.

T his study investigated the defective natural killer (NK) cell activity in two patients with the Chediak-Higashi syndrome (CHS) using both a standard 51-chromium release microcytoxicity and a single cell-in-agarose assay against K562 and Molt-4 target cells. CHS patients were deficient in overall maximum NK capacity, but had normal percentages of potentially cytotoxic target bindng cells. the relative number of TBC that could kill bound targets (i.e., "active" NK cells) was significantly depressed in CHS patients when compared with normal controls. The diminished CHS active NK cells that were present, however, were capable of recycling and lysing multiple target cells during the assay period. In vitro interferon (INF) treatment of normal and CHS effector cells did not alter target cell binding, but did increase the maximum NK capacity, percentage of active NK cells and the maximum recycling capacity, as well as the rate of lysis. These studies indicate that the depression of NK activity in patients with CHS is secondary to a deficiency of active NK cells. The CHS active NK cells that are present, however, are capable of normal target lysis and recycling. Potentially cytotoxic pre-NK cells, which can bind but not kill target cells, can be activated by in vitro IFN to develop lytic activity. Thus, IFN treatment may be of potential benefit to the immune surveillance network of CHS patients by activating a population of pre-NK cells to express their cytotoxic potential.

T he concentration of multiplication stimulating activity (MSA), an insulinlike growth factor (IGF), is high in fetal rat serum. We now report that MSA is exclusively associated wth an albumin-size binding protein in fetal rat serum; the growth hormone-dependent, gamma globulin-size binding protein, which predominates in the older animal, is absent from fetal rat serum. When ¹²⁵I-MSA was incubated with fetal rat serum and then gel filtered on Sephadex G-200, specific radioactivity eluted in the void volume (peak I) and the albumin region (peak III); by contrast, specific radioactivity eluted mainly in the gamma globulin region (peak II) in adult rat serum. Pools of the Sephadex G-200 fractions were chromatographed on Sephadex G-50, in 1 M acetic acid, to separate the binding protein from IGF activity. Analysis of IGF activity by chick embryo fibroblast bioassay, competitive protein binding assay, and MSA by radioimmunoassay revealed that all the IGF activity and MSA in fetal rat serum resided in peak III. Measurement of MSA binding capacity of the stripped binding protein by Scatchard analysis demonstrated that the majority of binding capacity also was found in peak III in fetal rat serum; most of MSA binding capacity was in peak II in adult rat serum. In fetal rat sera, in addition to the peak III binding protein, which is the major carrier of endogenous MSA, there is a component in peak I capable of specifically binding ¹²⁵I-MSA. This component elutes as a single species from a Sepharose-6B column. As MSA associated with peak III gradually declined in early neonatal life, peak II-associated IGF activity measured by chick embryo fibroblast bioassay showed a rise of activity with a peak at 5 d of neonatal life, a nadir at 20 d, with an increase again to attain adult levels. These studies demonstrate that the MSA binding protein in the fetus is different from the growth hormone-dependent binding protein in adult life.

T wo siblings with m ild hemorrhagic symptoms had combined functional deficiencies of vitamin K-dependent clotting factors. Prothrombin (0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) were most severely affected. Antigenic amounts of affected coagulation factors were normal and normal generation of thrombin activity occurred in the patients' plasmas after treatment with nonophysiologic activators that do not require calcium for prothrombin activation. Hepatobilary disease, malabsorptive disorders, and plasma warfarin were not present. Both parents had normal levels of all coagulation factors. The patients' plasmas contained prothrombin that reacted both with antibody directed against des-gamma-carboxyprothrombin and native prothrombin. Crossed immunoelectrophoresis of patients' plasmas and studies of partially purified patient prothrombin suggested the presence of a relatively homogeneous species of dysfunctional prothrombin, distinct from the heterologous species found in the plasma of warfarin-treated persons. These studies are most consistent with a posttranslational defect in hepatic carboxylation of vitamin K-dependent factors. This kindred uniquely possesses an autosomal recessive disorder of vitamin K-dependent factor formation that causes production of an apparently homogeneous species of dysfunctional prothrombin; the functional deficiencies in clotting factors are totally corrected by oral or parenteral administration of vitamin K1.

W e have studied a man suspected of having primary cortisol resistance on the basis of high 24-h mean plasma cortisol levels (27.4 micrograms/dl) and no stigmata of Cushing's syndrome. His son had slightly elevated 24-h mean plasma cortisol levels (9.9 micrograms/dl; normal 7.52 micrograms/dl). Both had high plasma protein unbound cortisol and increased urinary free cortisol. Plasma ACTH concentration was high, and both were resistant to adrenal suppression by dexamethasone. The father appeared to have mineralocorticoid excess resulting in hypertension, hypokalemia, and metabolic alkalosis. This was found to be due to markedly elevated plasma levels of deoxycorticosterone and corticosterone. The son, who was normotensive, had mildly increased plasma corticosterone and normal deoxycorticosterone levels. To study the apparent end-organ resistance to cortisol, we examined the glucocorticoid receptor in the white cells and fibroblasts of these patients. In both tissues, using both whole cell and cytosol assays, the glucocorticoid receptor was found to have reduced affinity for dexamethasone. In the cytoxol assays, a reduced receptor number was found as well. We conclude that cortisol resistance is a rare familial syndrome owing to an abnormal glucocorticoid receptor with a decreased affinity for cortisol.

T he results of studies in a patient with congenital deficiency of Factor XI who developed an inhibitor are presented. The patient presented with a severe, apparently spontaneous bleed into the thigh, which progressed despite infusion of fresh frozen plasma, but which responded promptly to activated prothrombin complex. During therapy with plasma his clotting time and Factor XI level were unresponsive and a Factor XI inhibitor titer of 6,000 U/ml was attained. The inhibitor was isolated and found to be polyclonal immunoglobulin G (IgG), predominantly of subclass 4. The specificity of the antibodies for Factor XI was shown by the ability of isolated inhibitor bound to polyacrylamide beads to remove Factor XI selectively from normal plasma. The binding of ¹²⁵I-labeled factor XI to the inhibitor was studied and an affinity constant of 1.65 × 10¹⁰ liter/mol was found. Complexing of the antibodies with Factor XI was shown to block multiple activities of the clotting factor. Factor XI complexed with antibody did not bind to high molecular weight kininogen or undergo activation and cleavage by two-chain Factor XII. The complex of activated Factor XI with inhibitor prevented the cleavage and activation of Factor IX. Hence the inhibitor appears to act by binding to multiple sites on the Factor XI molecule and preventing its interaction with other molecules. Clinically these interactions of the inhibitor with Factor XI result in a state of severe Factor XI deficiency.The clinical circumstances of the case, with severe hemorrhage refractory to plasma infusion but readily responsive to an alternate clot-promoting agent, suggest that a defect of intrinsic system activation was critical, supporting the inference that Factor XI does participate in normal hemostasis. The clinical course of this patient, who has only had two documented hemorrhages in the presence of the inhibitor, is not as severe as that of patients with severe Factor VIII or IX deficiency. This suggests that physiologic activation of Factors XI and IX does not occur exclusively in series because deficiency of factors XII, XI, VIII, and IX should then have similar hemostatic consequences. We propose that independent mechanisms for bypass of Factors XII and XI are important in physiologic activation of coagulation.

T he effect of pulmonary blood flow on the exchange between the circulating was marginating pool of polymorphonuclear leukocytes (PMN) was examined in three sets of experiments. In the first we used the double indicator dilution technique with labeled PMN and erythrocytes (RBC) to calculate the percent extraction and percent recovery of PMN at different levels of cardiac output (CO). In the second group of experiments we took advantage of the wide range of blood flow in the lung to determine the effect of regional blood flow on regional PMN retention, and in the third set we measured total leukocyte (WBC) and PMN counts in simultaneous samples from the pulmonary artery and aorta over a wide range of cardiac output. The studies showed that 80-90% of the labeled PMN were removed in a single pass through the lung and that regional retention of labeled PMN and A-V differences for unlabeled PMN increased with decreasing blood flow. The data for regional retention of labeled PMN and the A-V differences observed for unlabeled cells both fit the equation Y = A + Be-cx (where A + B = 100), which showed that PMN accumulate in the lung as blood flow is reduced. We conclude that a dynamic equilibrium exists between the circulating and marginating pools of leukocytes in the lung and that blood flow primarily effects the reentry of cells into the circulating pool so that the marginating pool of PMN within the lung accumulates cells when blood flow is reduced below 7 ml/min per g.

T o determine the characteristics of and mechanisms causing the bradycardia during sleep apnea (SA), both patients with SA and normals were studied. Evaluation of six consecutive SA patients demonstrated that bradycardia occurred during 95% of all apneas (central, obstructive, and mixed) and became marked with increased apnea length (P less than 0.01) and increased oxyhemoglobin desaturation (P less than 0.01). Heart rate slowed 9.5 beats per minute (bpm) during apneas of 10-19 s in duration, 11.4 bpm during 20-39s apneas, and 16.6 bpm during 40-59-s apneas. Sleep stage had no effect unexplained by apnea length or degree of desaturation. Oxygen administration to four SA patients completely prevented the bradycardia although apneas lengthened (P less than 0.05) in three. Sleeping normal subjects did not develop bradycardia during hypoxic hyperpnea but, instead, HR increased with hypoxia in all sleep stages, although the increase in HR was not as great as that which occurred while awake. Breath holding in awake normals did not result in bradycardia during hyperoxia (SaO2 = 99%), but was consistently (P less than 0.01) associated with heart rate slowing during room air breath-holds (-6 bpm) at SaO2 = 93%, with more striking slowing (-20 bpm) during hypoxic breath-holds (P less than 0.01) at SaO2 = 78%. Breath holding during hyperoxic hypercapnia had no significant effect on rate. Breath holding in awake SA subjects demonstrated similar findings. We conclude that the bradycardia of SA is a consistent feature of apnea and results from the combined effect of cessation of breathing plus hypoxemia.

S omatostatin (SRIF) is localized in the hypothalamus, extrahypothalamic brain, and throughout the gastrointestinal tract. Release of gastrointestinal SRIF-like immunoreactivity (SRIF-LI) is under nutrient regulation but the effect of nutrients on neural SRIF-LI is unknown. The present studies examined the effects of glucose uptake and metabolism and hormones influencing glucose disposition on SRIF-LI release from medial basal hypothalamus (MBH) and cerebral cortex (Cx) incubated in Krebs-Ringer bicarbonate containing bacitracin. After a preincubation to achieve stable secretion, tissues were incubated for 20 min in 14 mM glucose (basal) and then, for 20 min in fresh medium with test materials. MBH SRIF-LI release was inversely related to medium glucose concentration with release in the absence of glucose (235±42 pg/MBH per 20 min) more than five times that in the presence of 25 mM glucose (46±4 pg/20 min). In the presence of 14 mM glucose MBH SRIF-LI release was stimulated above basal by agents interfering with glucose uptake including 3-O-methyl-d-glucose (42 mM; 70±5 vs. 42±3 pg/20 min, P < 0.05), phlorizin (50 mM; 351±63 vs. 29±2 pg/20 min, P < 0.001) or cytochalasin B (20 μM; 110±7 vs. 22±2 pg/20 min, P < 0.001). Inhibition of glucose metabolism by 2-deoxy-d-glucose resulted in dose-related stimulation of MBH SRIF-LI release (maximal at 28 mM; 201±28 pg/20 min vs. 32±4 pg/20 min, P < 0.001). Viability of MBH was unimpaired by incubation in the absence of glucose or following exposure to 2-deoxy-d-glucose as determined by retention of SRIF-LI responsiveness to stimulation by potassium (60 mM) or neurotensin (5 μM). In contrast, Cx SRIF-LI release was slightly inhibited by decreases in medium glucose and unaffected by inhibition of glucose uptake or metabolism. These results provide evidence for nutrient regulation of MBH but not Cx SRIF-LI release and may explain inhibition of growth hormone seen in the rat in response to hypoglycemia. Insulin (10 nM-1 μM) stimulated MBH but not Cx SRIF-LI release while glucagon was without effect. Our previous demonstration that MBH SRIF-LI release was stimulated by somatomedin-C, but not insulin at physiologic concentrations, is consistent with an action of insulin through the somatomedin-C receptor at the doses studied. Our studies indicate a regional specificity for the control of SRIF secretion within the brain and suggests the possibility of a role for hypothalamic SRIF in metabolic regulation.

I n 100 patients with various types of endocrine dysfunction, we measured bone mineral density (BMD) at the midradius (greater than 95% cortical bone) and distal radius (75% cortical and 25% trabecular bone) by single photon absorptiometry and at the lumbar spine (greater than 66% trabecular bone) using the new technique of dual photon absorptiometry. BMD in each endocrine disorder deviated in at least one site from the sex-specific age regression of 187 normal subjects. For patients with primary hyperparathyroidism, hypercortisolism, and hyperthyroidism this deviation was negative (suggesting bone loss), whereas for patients with secondary hyperparathyroidism due to chronic renal failure, acromegaly, and postsurgical hypoparathyroidism it was positive (suggesting bone gain). When all six states of endocrine dysfunction were compared concomitantly by multivariate analysis of variance, the profile of the changes in BMD differed significantly (P less than 0.001), indicating a nonuniform response of bone to the various hormonal alterations. When values for BMD at each of the three scanning sites were compared the midradius and distal radius did not differ significantly; either of the radius measurements, however, differed significantly (P less than 0.001) from the lumbar spine. Thus, the BMD of the axial skeleton cannot be reliably predicted from measurements made in the appendicular skeleton. We conclude that the effects of endocrine dysfunction on bone density are complex and are both disease and site specific.

T hese studies have been undertaken to evaluate the role of the brain noradrenergic and dopaminergic pathways in the regulation of the secretion of thyrotropin-releasing hormone (TRH) in the central nervous system (CNS) and pancreas of the neonatal rat. When CNS stores of norepinephrine (NE) were selectively reduced by the subcutaneous administration of the dopamine-β-hydroxylase inhibitor FLA-63, TRH concentrations were significantly reduced throughout the brain. However, when CNS stores of both NE and dopamine (DA) were depleted by the subcutaneous administration of the tyrosine hydroxylase inhibitor α-methyl-ρ-tyrosine (α-MT), TRH concentrations in the brain were not significantly altered.FLA-63 and α-MT did not significantly reduce pancreatic catecholamine concentrations, indicating that in the basal state, these agents predominantly deplete central catecholamine stores. Nevertheless, pancreatic TRH concentrations were markedly reduced by FLA-63, and this effect was significantly attenuated by the simultaneous intracerebroventricular (icv) administration of NE. In contrast to the effects of FLA-63, α-MT caused a significant increase in pancreatic TRH concentrations, and this effect was significantly lessened by icv DA. To determine whether the sympathetic nervous system might be one route by which these central effects are mediated, a chemical sympathectomy was induced with guanethidine. This treatment selectively reduced pancreatic concentrations of NE, and caused a marked increase in pancreatic TRH concentrations.From these observations, we conclude the following: (a) within the central nervous system, both NE and DA are involved in regulating brain TRH secretion or biosynthesis, and the direction of action of these two neurotransmitters appears to be opposite; (b) pancreatic TRH secretion or biosynthesis is also controlled by the brain noradrenergic and dopaminergic systems, and the net effects of each of these pathways appears to be opposite; (c) at least one route by which impulses from the brain may travel and modulate pancreatic TRH secretion or biosynthesis is by the sympathetic nervous system.

C ardiovascular actions of insulin were studied by intravenous infusions of insulin (4 and 8 mU/kg per min) in normal conscious dogs. This resulted in increases in cardiac output, heart rate, and left ventricular derivative of pressure with respect to time (dP/dt) and dP/dt/P, as blood glucose was reduced. The inotropic and chronotropic effects of insulin were not related to hypoglycemia, as they persisted even when blood glucose was restored to control values or when it was prevented from falling by a simultaneous infusion of glucose. These cardiac effects were accompanied by increases in plasma catecholamines, and were abolished by propranolol pretreatment. Both plasma epinephrine and norepinephrine increased during insulin hypoglycemia, but only norepinephrine increased during insulin infusion when euglycemia was maintained.Mean arterial blood pressure did not change significantly during insulin hypoglycemia, but rose if euglycemia was maintained, probably due to the selective increase in norepinephrine in the latter condition. A pressor response also occurred in propranolol-pretreated dogs during insulin hypoglycemia, but was abolished when the animals also had been pretreated with phentolamine, indicating that the vasoconstrictor action of insulin was mediated via alpha adrenergic receptors.Insulin infusion increased left ventricular work and myocardial blood flow in dogs with and without hypoglycemia. Myocardial blood flow, however, did not change significantly during insulin infusion in dogs pretreated with propranolol. As propranolol also diminished the inotropic response, it appears that the increase in myocardial blood flow caused by insulin in the normal dog is causally related to the increased myocardial metabolic demand.Insulin also produced vasomotor effects on other vascular beds. In skeletal muscle, blood flow was increased under all study conditions, except during insulin hypoglycemia after propranolol-pretreatment when unopposed alpha-mediated vasoconstriction was present. The persistent increase in flow during both alpha and beta adrenergic blockade suggests that insulin has a direct dilator effect on skeletal muscle vasculature. In the adrenal gland, flow was increased except during euglycemia, when no rise in plasma epinephrine was observed, suggesting coupling between adrenal flow and catecholamine release. In the splanchnic bed, flow was decreased during euglycemia, when plasma norepinephrine rose, and during beta adrenergic blockade with propranolol, when unopposed alpha-mediated vasoconstriction also predominated. A similar pattern was found in the kidney, except that renal blood flow also fell after combined alpha and beta adrenergic blockade. The results show that the vasomotor effects on regional flows are mediated both via adrenergic mechanisms, and in the case of skeletal muscle and kidney, via mechanisms unrelated to sympathetic stimulation.

T he cellular location and carbohydrate specificities of a glycoprotein recognition system on rat hepatic sinusoidal cells have been determined. Purified preparations of endothelial, Kupffer, and parenchymal cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. ¹²⁵I-labeled agalactoorosomucoid, an N-acetylglucosamine-terminated glycoprotein, was selectively taken up in vitro by endothelial cells. Uptake was shown to be protein dependent, calcium ion dependent, and saturable, and could be described by Michaelis-Menten kinetics (apparent K_m 0.29 μM; apparent maximum velocity 4.8 pmol/h per 5 × 10⁶ cells). Uptake was inhibited not only by N-acetylglucosamine, mannose, and mannan but also by glucose, fructose, and a glucose-albumin conjugate. Inhibition by glucose was competitive over a wide range of concentrations and was almost 100% at a glucose concentration of 56 mM. Fasting and the induction of diabetes mellitus prior to isolation of cells was associated with 60% reductions in the recovery of endothelial cells. Uptake by cells isolated from fasted rats was enhanced (apparent maximum velocity 14.3 pmol/h per 5 × 10⁶ cells without change in the apparent K_m). These observations suggest that fasting is associated with a marked increase in the mean number of glycoprotein receptors per endothelial cell isolated from normal rats. This effect of fasting could be due to upregulation of glycoprotein receptors on endothelial cells or to the selective isolation of a subpopulation of endothelial cells from fasted animals that bears more glycoprotein receptors per cell than does another subpopulation of these cells. In addition, in vivo studies of the fate of intravenously administered ¹²⁵I-agalactoorosomucoid indicated that its rate of disappearance from plasma, hepatic accumulation, and catabolism were slower in diabetic than in normal rats. The results suggest that modulation of a carbohydrate-mediated glycoprotein recognition system located on hepatic endothelial cells can be induced by glucose and glucose-conjugated proteins and by fasting and diabetes mellitus. The findings in this study suggest a mechanism for abnormal glycoprotein metabolism in diabetes mellitus.

T wo series of derivatives of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), including a dechlorinated analog of W-7 (W-5) and various aminoalkyl chain analogs of W-7 (A-3, A-4, A-5, I-240, A-6) were synthesized and their structure-activity relationships with calmodulin antagonistic actions and their potencies in inhibiting human platelet aggregation in vitro were investigated. Their binding affinities to calmodulin in the presence of 100 microM Ca2+ were dependent both on the chlorination of the naphthalene ring and on the length of aminoalkyl chain. The ability of these derivatives to inhibit Ca2+-dependent phosphorylation of 20,000-dalton myosin light chain from platelets correlated well with the magnitude of their binding affinity to calmodulin. W-7(10-100 microM) inhibited in a dose-dependent manner platelet aggregation induced by collagen (2 micrograms/ml), ADP (5 microM), epinephrine (1 microgram/ml), sodium arachidonate (0.83 mM), thrombin (0.125 U/ml), and A-23187 (10 microM). The IC50 value (concentration producing 50% inhibition of aggregation) of W-7 was lower in arachidonate- and collagen-induced aggregation than in ADP- or epinephrine-induced aggregation. A good correlation between the potency in inhibition of collagen-induced aggregation by W-7 and its derivatives and their affinities to calmodulin was obtained (r = 0.94). Thus, the inhibitory mechanism of these compounds may be due to their effect on Ca2+-calmodulin-dependent processes, such as 20,000-dalton myosin light chain phosphorylation. These data also support the hypothesis that the calmodulin-mediated system has an important role in platelet function.

t he stimulation of lymphocytes from rheumatoid arthritis patients with pokeweed mitogen produces a large number of plasma cells that express the dominant cross-reactive idiotype previously found on monoclonal IgM anti-gamma-globulins from patients with mixed cryoglobulinemia. Similar experiments with the cells of normal individuals show a much lower percentage of these cells with a lower intensity of staining with the fluorescent reagents utilized. Efforts to demonstrate rheumatoid factor in the same cells by fluorescent staining with aggregated gammaglobulin were entirely unsuccessful. This also proved to be the case for pokeweed mitogen-stimulated cells from the mixed cryoglobulinemic patients with large amounts of rheumatoid factor in the serum, despite high percentages of cells expressing the cross-reactive idiotype and also the individual idiotype. On the other hand, native plasma cells from synovial tissue of rheumatoid arthritis patients showed some cells with both the cross-reactive idiotype and aggregate staining. The exact reason for the failure to demonstrate rheumatoid factor by aggregate staining in pokeweed mitogen-stimulated cultures remains to be determined despite considerable effort to resolve the problem. The most likely possibility is that these plasma cells are relatively immature and have not accumulated polymeric IgM in their cytoplasm to the degree seen in synovial tissue plasma cells. The monomeric forms are readily recognized by the antiidiotypic antibodies and these reagents appear to be of particular value for cellular studies of this type.

A cetylation of platelet cyclooxygenase by oral aspirin is dose dependent and cumulative with repeated administration. However, no single dose of aspirin has been found to be completely selective of platelet thromboxane (TX) synthesis inhibition in man. We determined the dose dependence, cumulative nature and selectivity of aspirin effects on platelet TXB₂ and renal prostaglandin (PG) and prostacyclin (PGI₂) production. We measured, by radioimmunoassay, serum TXB₂ levels after whole blood clotting and urinary excretion of PGE₂, PGF_2α, and 6-keto-PGF_1α, before and after single or repeated oral aspirin doses given to 46 healthy subjects. Single doses of 6-100 mg aspirin resulted in a linear (r = 0.92, P < 0.01) inhibition of platelet TXB₂ production, ranging from 12 to 95% after 24 h. A daily dose of 0.45 mg/kg given for 7 d produced a cumulative and virtually complete inhibition of platelet TXB₂ production, without significantly reducing the urinary excretion of PGE₂, PGF_2α, and 6-keto-PGF_1α in both healthy men and women. The platelet inhibitory effect of this regimen was maintained unaltered throughout 1 mo of therapy, with no evidence of cumulative inhibition of renal PG-synthesis. Moreover, furosemide-induced renal PGI₂ synthesis and renin release were unaffected by chronic low-dose aspirin. Following cessation of aspirin therapy, platelet TXB₂ production returned toward control values at a similar rate as after a single higher dose.We conclude that in healthy subjects: (a) aspirin causes a dose-dependent inhibition of platelet TXA₂ production, with no obvious sex-related difference; (b) the inhibitory effect of daily low-dose aspirin is cumulative on platelet TXA₂ but not on renal PG-synthesis; (c) during chronic low-dose aspirin therapy, renal PGI₂-producing cells are readily activable by furosemide at a time of virtually complete suppression of platelet cyclooxygenase activity.

R ecessive dystrophic epidermolysis bullosa, a genodermatosis characterized by dermolytic blister formation in response to minor trauma, is characterized by an incresaed collagenase synthesis by skin fibroblasts in culture. Since preliminary studies of partially purified recessive dystrophic epidermolysis bullosa collagenase suggested that the protein itself was aberrant, efforts were made to purify this enzyme to homogeneity, so that detailed biochemical and immunologic comparisons could be made with normal human skin fibroblast collagenase. Recessive dystrophic epidermolysis bullosa skin fibroblasts obtained from a patient documented to have increased synthesis of the enzyme were grown in large scale tissue culture and both serum-free and serum-containing medium collected as a source of collagenase. The recessive dystrophic epidermolysis bullosa collagenase was purified to electrophoretic homogeneity using a combination of salt precipitation, ion-exchange, and gel-filtration chromatography. In contrast to the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase bound to carboxymethyl-cellulose at Ca²⁺ concentrations at least 10 times higher than those used with the normal enzyme. Additionally, this enzyme was significantly more labile to chromatographic manipulations, particularly when serum-free medium was used. However, rapid purification from serum-containing medium yielded a preparation enzymatically equivalent to normal human skin collagenase. Like the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase was secreted as a set of two closely related zymogens of ∼60,000 and ∼55,000 daltons that could be activated by trypsin to form enzymically active species of ∼50,000 and ∼45,000 daltons, respectively. Amino acid analysis suggested slight variations between the normal and recessive dystrophic epidermolysis bullosa collagenases. Cyanogen bromide digests demonstrated peptides unique to the enzyme from each source. The recessive dystrophic epidermolysis bullosa proenzyme was significantly more thermolabile at 60°C than the normal, a finding that correlated with an approximate fourfold decrease in the affinity of the mutant enzyme for Ca²⁺, a known activator and stabilizer of human skin collagenase. Aside from the altered affinity for this metal cofactor, kinetic analysis of the structurally altered recessive dystrophic epidermolysis bullosa collagenase revealed that its reaction rates and substrate specificity for human collagen types I-V were identical to those for the normal enzyme. Likewise, enzymes from both sources displayed identical energies of activation and deuterium isotope effects. Antisera were raised to the normal and putatively mutant procollagenases respectively, and, although they displayed a reaction of identity in double diffusion analysis, immunologic differences were present in enzyme inhibition and quantitative precipitation studies. These studies indicate that recessive dystrophic epidermolysis bullosa is characterized by the increased synthesis of an enzymically normal, but structurally aberrant, collagenase.

M orphologic and biochemical studies suggest that actin in human platelets polymerizes in response to various stimuli and that shortening of actin filaments can be regulated by calcium. We report that human platelets contain gelsolin, a protein of Mr 91,000 that binds reversibly to actin in the presence of calcium. Platelet gelsolin exhibits immunologic crossreactivity with rabbit macrophage gelsolin and shortens actin filaments as demonstrated by viscosity measurements and gel point determinations. Gelsolin is active in micromolar calcium concentrations and its effects upon actin filaments are reversible. Gelsolin may be a dynamic regulator of actin filament length in the human platelet.

W e studied the production of and response to interleukin-2 (IL-2) by peripheral blood T lymphocytes from 19 systemic lupus erythematosus (SLE) patients who received no treatment at the time they were studied. Eight had active disease and the rest were in remission. Results were compared with those obtained in 12 healthy subjects of similar age. T cells from SLE patients, whether activated with phytohemagglutinin or in autologous mixed lymphocyte reactions, were found to yield little IL-2, to have a low response to IL-2 from its own, or other sources, and to absorb IL-2 poorly, IL-2 produced by SLE cells, albeit scant, was absorbed normally by activated T cells from normal subjects. Our findings may contribute to the understanding of the immunoregulatory defect in SLE.

T he present study examined the effect of various unopsonized strains of influenza A virus on release of myeloperoxidase (MPO) and acid phosphatase in polymorphonuclear leukocytes (PMNL). These results were correlated with the effect that these same viruses had on bactericidal activity in PMNL. Several strains of virus inhibited the fusion of azurophil granules with phagosomes containing Staphylococcus aureus. These same strains inhibited the extracellular release of MPO from PMNL (39-59%) and caused depressed killing (42-77%). In contrast, one of the influenza viruses (X-47a) did not inhibit PMNL MPO release or killing. The data indicate a close relationship between the ability of influenza virus to ablate normal intracellular lysosome-phagosome fusion with subsequent depression of bactericidal functions of PMNL.