Issue published September 1, 1981

H uman blood mononuclear cells (BMC) in short-term culture secrete one or more factors that induce degradation of matrix proteoglycan and collagen in cartilage explants in organ culture. Induction of matrix degradation took place both in nasal septum and articular cartilage explants in the presence of the mononuclear cell supernates. Cartilage degradation in this system was absolutely dependent on the presence of live chondrocytes. Matrix depletion did not occur in dead cartilage explants cultured with active supernates. Supernates obtained from unstimulated BMC showed variable cartilage matrix degrading activity (MDA). BMC stimulated with phytohemagglutinin (PHA) showed increased MDA, which in one dilution experiment was found to be five times higher than that in the unstimulated control supernate. Concanavalin A and pokeweed mitogen were also shown to stimulate release of MDA. Time experiments showed that most of the degrading activity was released by the mononuclear cells during the first day of culture. The cellular origin of MDA was investigated with the aid of partially purified BMC subpopulations. Removal of adherent cells resulted in a decrease of MDA release. Purified T lymphocytes failed to show enhanced MDA release in spite of their ability to mount a virtually intact proliferative response to PHA. Purified adherent cells also failed to show enhanced PHA-dependent MDA release. Nevertheless, restoration of PHA-dependent MDA release took place in reconstituted cell populations containing both T lymphocytes and monocytes. These experiments suggest that MDA may be released by adherent mononuclear cells, presumably monocytes, and that the PHA-dependent increase in MDA release may be mediated by T lymphocytes. Partial characterization of MDA by gel chromatography showed one active fraction corresponding to an apparent molecular weight ranging from 12,000 to 20,000. The fraction was also shown to degrade cartilage matrix only in the presence of live chondrocytes. These results demonstrate that factors released by human BMC mediate degradation of matrix proteoglycan and collagen in intact cartilage explants through chondrocyte activation. This pathogenic mechanism may play a role in in vivo cartilage destruction in chronic inflammatory joint diseases.

I ntravenous histamine causes high amplitude repetitive phasic contractions of the in vivo cat pylorus but has little effect on the antrum and duodenum. The genesis of this phasic response was studied using a pinned perfused catheter with openings at the pylorus, antrum, and duodenum. 2-Pyridylethylamine, an H1 agonist, produced phasic contractions similar to histamine whereas dimaprit, an H2 agonist, did not. Conversely, histamine-induced excitation is competitively antagonized by the H1 inhibitor diphenhydramine but not by the H2 inhibitor cimetidine. Thus histamine excitation is mediated through H1 receptor stimulation. Tetrodotoxin caused inhibition of the histamine response indicating that pyloric excitation is partly mediated through a neural pathway. To identify the nature of this pathway adrenergic and cholinergic blockers were used. Atropine, hexamethonium, and propranolol had no effect on the histamine response. Phentolamine and reserpine increased the magnitude of the histamine response. Conversely, phenylephrine blocked the histamine response. We conclude: histamine induces high phasic contractions in the pylorus; this effect is mediated through neural (nonadrenergic noncholinergic) and myogenic H1 receptors; alpha adrenergic stimulation inhibits the histamine response and alpha adrenergic blockade and catecholamine depletion increase it.

W e have demonstrated that human plasma contains a heparin-dependent inhibitor of thrombin that is distinguishable from antithrombin III (AT III). When a 1:50 dilution of plasma was incubated with greater than or equal to 0.01 U/ml heparin and 1 U/ml 125I-thrombin, the labeled thrombin B-chains became incorporated into two complexes of Mr-96,000 and Mr-85,000 that were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Neither complex was detectable at heparin concentrations less than 0.01 U/ml. When a limiting amount of 125I-thrombin was present, the proportion of radioactivity incorporated into each of the two complexes varied with the heparin concentration. Thus, the Mr-85,000 complex predominated at 0.01-5 U/ml heparin, whereas the Mr-96,000 complex predominated at 5-100 U/ml heparin. The Mr-85,000 complex reacted with antibodies to human AT III and comigrated with the purified thrombin-AT III complex. The Mr-96,000 complex did not react with antibodies to AT III or to alpha 1-antitrypsin, and it was detected in normal quantities after incubating 125I-thrombin with plasma immunodepleted of AT III, alpha 2-antiplasmin, alpha 2-macroglobulin, C1 inactivator, alpha 1-antichymotrypsin, or inter-alpha-trypsin inhibitor. The protein that combines with thrombin to form the Mr-96,000 complex was estimated to be present at a minimum concentration of 90 +/- 26 micrograms/ml (mean +/- SD) in identical to any of the known plasma protease inhibitors and that at relatively high heparin concentrations in vitro it reacts with thrombin more rapidly than does AT III.

H ereditary pyropoikilocytosis (HPP) is a hemolytic anemia characterized by microspherocytosis, poikilocytosis, and an unusual thermal sensitivity of erythrocytes. We have investigated the contribution of abnormal membrane skeletal assembly to these abnormal HPP erythrocyte properties. Skeletons prepared from fresh HPP ghosts with Triton X-100 were considerably more fragile than skeletons from control erythrocytes. Spectrin, the major skeleton component, extracted at 0 degrees C from normal erythrocytes, was present primarily as tetramers and high molecular weight complexes. In contrast, spectrin extracted from HPP erythrocytes under identical conditions contained a significant amount of dimers with a concomitant decrease of tetramers. Furthermore, spectrin dimers from HPP erythrocytes differed from normal spectrin dimers in their failure to reassociate into tetramers both in solution and in the membrane. Presumptive HPP carriers (asymptomatic mothers of the two patients) exhibited a mild but reproducible increase of spectrin dimers in 0 degrees C extracts and a defective reassociation of spectrin dimers of tetramers both in solution and in the membrane. We conclude that in HPP, self-association of spectrin dimers into tetramers is defective, which accounts for the instability of membrane skeletons.

L inked DNA polymorphisms can be used to study the evolution of structural gene mutations. Both the beta S-(beta 6Glu leads to Val) and beta C-(beta 6Glu leads to Lys) genes are common in West Africa. We have analyzed their linkage to a polymorphic Hpa 1 site appearing 3' to the beta-globin gene locus in selected populations from Wes Africa. A large reservoir of beta A-genes linked to 13-kilobase Hpa 1 fragments with a frequency of 17-18% has been identified. In addition, the beta S- and beta C-genes in Togo are found to be tightly linked to the 13-kilobase Hpa 1 fragment, whereas 72% of the beta S-genes in the Ivory Coast reside on the 7.6-kilobase Hpa 1 fragment. These studies are consistent with the selection and expansion of two different chromosomes bearing beta S-genes in at least two physically close, but ethnically separate regions of West Africa, with subsequent diffusion to North, Equatorial, and East Africa.

A utoantibodies in the serum from a patient with connective tissue disease have been used to define a high molecule weight acidic nuclear protein antigen. The antigen tentatively termed Ku, after the first two letters of patient's name, has distinct physicochemical properties and immunological specificities that distinguish it from previously reported antigens. The Ku antigen has an apparent 300,000 mol wt as determined by gel filtration and sucrose density gradient ultracentrifugation techniques. The antigen is destroyed by trypsin, mild heating, and pH variations greater than 10 and less than 5. Treatment with ribonuclease or deoxyribonuclease did not affect the antigenic reactivity. The Ku antigen was demonstrated in the soluble extracts of human, calf, and rabbit, but not of rat tissues. Purified antibody localized the Ku antigen within the nuclei of human liver where a "reticular" pattern of immunofluorescence was seen. Of 330 patients with various connective tissue diseases, 9 had precipitating antibodies to the Ku antigen. Preliminary results of clinical analysis indicated that antibody to the Ku antigen might become a useful marker for a group of patients with clinical characteristics of both polymyositis and scleroderma with a good prognosis.

M onocytes, macrophages, and neutrophils will demonstrate several important cellular functions in response to synthetic formylated oligopeptides. N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (fNLPNTL) was a potent chemoattractant for human blood monocytes; a 1.0-nM concentration induced a maximal chemotactic response. Binding of 125I-labeled fNLPNTL to the monocyte formyl peptide receptor was rapid, specific, and saturable at 4, 24, or 37 degrees C. At 4 degrees C, monocytes from several different donors demonstrated between 10,000 and 18,000 receptors/cell with a dissociation constant (Kd) of 1.7-2.7 nM. The association of the 125I peptide with the cells was irreversible at the elevated temperatures and exceeded the amount of surface receptor by approximately four-fold, suggesting receptor-mediated peptide endocytosis. Processing of rhodamine-labeled fNLPNTL by monocytes was observed directly by video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization of the peptide. Monocytes incubated with fNLPNTL displayed a temperature dependent loss of surface binding capacity (receptor down-regulation). This decrease was due to a decrease in surface receptor number rather than to a decrease in receptor affinity. A dose-response curve for peptide-induced receptor down-regulation correlated with a dose-response curve for 125I-labeled fNLPNTL uptake, suggesting that each uptake event led to the loss of one surface receptor. Surface receptor replenishment following down-regulation was rapid and not dependent on new protein synthesis, but was inversely related to both the time and peptide concentration used to induce down-regulation. An exact correlation between receptor down-regulation and functional deactivation of the chemotactic response could not be demonstrated.

A patient with xerocytosis was found to have swimming-induced intravascular hemolysis and shortening of erythrocyte life-span. In a microviscometer, xerocytes were more susceptible than normal erythrocytes to hemolysis by shear stress. Fractionation of normal and abnormal cells on discontinuous Stractan density gradients revealed that increasingly dehydrated cells were increasingly more shear sensitive. This sensitivity was partially corrected by rehydrating xerocytic erythrocytes by means of the cation-ionophore nystatin in a high potassium buffer. Conversely, normal erythrocytes were rendered shear sensitive by dehydrating them with nystatin in a low potassium buffer. This effect of dehydration was entirely reversible if normal cells were dehydrated for less than 4 h but was only partially reversed after more prolonged dehydration. It is likely that dehydration of erythrocytes results in shear sensitivity primarily because of concentration of cell contents and reduced cellular deformability. With prolonged dehydration, secondary membrane changes may potentiate the primary effect. This increased shear sensitivity of dehydrated cells may explain atraumatic exercise-induced hemolysis in xerocytosis as cardiac output is shifted to vessels of exercising muscles with small diameters and high shear rates.

E nterococci are resistant to penicillin killing in vivo and in vitro. Because some bacteria resistant to penicillin killing have reduced autolytic activity, we examined the lysis of clinical enterococcal isolates suspended in buffer (spontaneous lysis), and compared it with their susceptibility to antibiotic-induced lysis and killing. We found significant correlations between spontaneous and antibiotic-induced lysis, using five antibiotics that inhibit cell wall synthesis (penicillin, cephalothin, bacitracin, cycloserine, and vancomycin). Among isolates, strains more rapidly lysed by one antibiotic were more rapidly lysed by the other antibiotics, and more susceptible to spontaneous lysis. In studies involving a single strain grown in different media, spontaneous lysis also correlated closely with antibiotic-induced lysis. These results are consistent with a common mechanism for spontaneous and antibiotic-induced lysis, such as the autolytic enzyme system. Human serum was one of the least permissive media tested for enterococcal growth and antibiotic-induced lysis and killing. We suggest that the inhibitory effect of human serum on growth and the activation of the enterococcal autolytic enzyme system may be a critical factor in the resistance of enterococcal endocarditis to treatment with penicillin alone.

C umulative evidence indicates that there is an increased accumulation of calcium in dystrophic muscle and that this may have a pathophysiological role in the progression of the dystrophic process. The accumulation may be related to a defect of the plasma membrane. Because parathyroid hormone (PTH) stimulates calcium influx into the cytosol, the chronic effects of surgical ablation of the parathyroid glands on muscle Ca, Mg, protein synthesis, and histology, as well as plasma creatine phosphokinase (CPK), Ca, and Mg, were studied in normal and dystrophic (BIO 14.6) hamsters. Thyroparathyroidectomized (TPTX) hamsters receiving replacement doses of l-thyroxine were killed at age 90 d, 55 d after TPTX. In intact dystrophic hamsters, the Ca content in the heart was 20 times higher than in normal animals and was reduced by half in TPTX dystrophic hamsters. Similar results were observed in diaphragm and rectus femoris. No abnormalities in Mg content were observed in intact or TPTX dystrophic hamsters. Ether-extractable fat of the heart and diaphragm was reduced in dystrophic hamsters and was not modified by TPTX. Protein synthesis was enhanced in the diaphragm of dystrophic hamsters but was not changed by TPTX. The concentration of CPK in plasma was elevated in dystrophic hamsters and fell significantly after TPTX. In the latter animals, microscopic examination of the heart showed lesser signs of dystrophy, particularly in the degree of fibrosis.To determine the degree of dystrophy at the age when TPTX was performed, identical analyses were made in 35-d-old hamsters. Definitive histological signs of dystrophy were observed, and although the Ca content in heart, diaphragm, and rectus femoris was elevated, the values were lower than in 90-d-old intact and TPTX dystrophic hamsters. This indicates that chronic TPTX in dystrophic hamsters reduces, but does not arrest, the dystrophic process.In normal hamsters, a 50% reduction in plasma Ca concentration was observed 6 h after TPTX; 55 d after TPTX, however, plasma Ca was within normal limits in both normal and dystrophic hamsters. No parathyroid tissue was observed in serial sections of the trachea and adjacent tissues in TPTX animals. This suggests that in chronically TPTX hamsters fed a standard laboratory diet, plasma Ca can be maintained by mechanisms independent of parathyroid function.The data indicate that in dystrophic hamsters TPTX causes a marked reduction in: (a) muscle Ca accumulation, (b) levels of plasma CPK and, (c) intensity of histological changes in the heart. These changes were independent of the levels of plasma Ca and were not observed in normal hamsters. We conclude that PTH accentuates the dystrophic process, probably by enhancing the already increased Ca flux into muscle (apparently caused by defective sarcolemma). We postulate that normal secretion of PTH may have a deleterious effect in congenital or acquired conditions associated with altered plasma membranes.

D ietary phosphorus restriction (PR) prevents uremia in rats with nephrotoxic serum nephritis (NSN). One possible mechanism by which PR could be protective would be through the suppression of parathyroid hormone. To evaluate this possibility two separate protocols were designed. In the first rats were thyroparathyroidectomized (TPTX) before (n = 11) or 5 wk after (n = 7) NSN induction and compared to sham-operated parathyroid intact rats with NSN (n = 12). At the end of the 23-wk study, intact rats were azotemic, plasma creatinine 3.80±0.81 mg/100 ml vs. 0.65±0.07 for TPTX rats (P < 0.001). During the study 75% of intact rats died of uremia in contrast to none of the TPTX rats (P < 0.001). Renal histological damage was greatly diminished and calcification prevented in TPTX rats. The proteinuria of the heterologous phase was unaffected, but the protein excretion and hypertriglyceridemia (HTG) of the autologous phase were markedly decreased in the TPTX rats. The degree of HTG and proteinuria had a high positive correlation (P < 0.001). Late TPTX also produced significant decreases in proteinuria and HTG regardless of the degree of azotemia, and prevented azotemia if the plasma creatinine at the time of TPTX was ≤0.85 mg/100 ml.In additional studies selective parathyroidectomy (PTX) was performed. The adequacy of this procedure was documented by showing a similar fall in plasma Ca and urinary cyclic AMP in PTX animals as found in TPTX animals. However, selective PTX had no effect on proteinuria, histologic damage, or functional deterioration. These studies further showed that early, histologic damage and functional deterioration preceeded renal parenchymal calcification. Because animals were pair fed and both groups were given 1,25-dihydroxycholecalciferol to normalize serum Ca and P levels these studies exclude alterations in plasma Ca and P levels, dietary intake, urinary P excretion, and vitamin D administration in promoting the protective effect of TPTX on renal function.We conclude that TPTX is equally effective in preventing functional deterioration and more effective in reducing proteinuria in NSN than PR. The mechanism of this protective effect remains to be elucidated, since it does not primarily involve either the elimination of parathyroid hormone or the prevention of renal parenchymal calcification.

T he lymphocyte transformation responses to purified preparations of two extracellular products of group A streptococci (blastogen A and nuclease B), to phytohemagglutinin, and to Candida albicans antigen were measured in tonsillar and peripheral blood lymphocytes from patients with rheumatic heart disease (RHD) and suitably matched nonrheumatic (control) subjects.The mean phytohemagglutinin dose responses of tonsillar and peripheral lymphocytes from RHD patients were essentially indistinguishable from those of controls. In contrast, the responses of tonsillar and peripheral blood lymphocytes to the two extracellular products of group A streptococci were significantly lower in RHD patients than in nonrheumatic control subjects. Candida antigen produced very little stimulation of lymphocytes in any of the subjects.The geometric means of antibody levels against streptolysin O, nuclease B, and nicotinamide adenine dinucleotidase showed no consistent differences between the control group and the group of RHD subjects. Group A streptococci were isolated from the tonsils of ∼25% of both groups of subjects.The RHD patients clearly had a depressed cellular immune response to the two purified streptococcal extracellular antigens. The equal frequency in recovery of group A streptococci from tonsils and the absence of consistent difference in titers of humoral antibodies to streptococcal extracellular antigens, particularly nuclease B, suggest that this differential response is not due to a lower level of stimulation by repeated exposure to group A streptococcal products.

I ncubation of a 0.5% suspension of washed, normal mouse erythrocytes with ferriprotoporphyrin IX (FP) at 37 degrees C and pH 7.4 caused potassium loss, swelling, increased susceptibility to hypotonic lysis, and finally hemolysis. Hemolysis was not inhibited by incubation in the dark, malonyldialdehyde was not produced, and various free radical scavengers had no effect on the hemolysis. Only the sulfhydryl compounds, cysteine, dithiothreitol, and mercaptoethanol protected erythrocytes from FP. Potassium loss reached 90% within 30 min of exposure to 5 microM FP. This amount of FP caused greater than 50% hemolysis within 2.5 h. Sucrose (0.1 M) completely prevented hemolysis but had no effect on potassium loss. Likewise, reducing the temperature from 37 to 25 degrees C greatly retarded hemolysis but had no effect on potassium loss. These observations indicate that FP impairs the erythrocyte's ability to maintain cation gradients and induces hemolysis by a colloid-osmotic mechanism.

E xperiments were made to investigate the effect of four anesthetic drugs that are commonly used in surgical practice on the postoperative growth of mouse tumors in syngeneic recipients. These experiments revealed that some of the anesthetics when applied for surgical excision of the local tumor, strongly accelerated postoperative progression of spontaneous lung metastases produced by the 3LL Lewis lung carcinoma and by the B16 melanoma. Some of the drugs caused the appearance of metastases in organs, such as the liver, in which spontaneous metastases are not usually produced by these tumors. A T10 sarcoma clone that does not produce detectable metastases in immune intact mice even following intravenous injection, did produce metastases when injected into animals treated with pentothal sodium.

M acrophages were shown by the use of glomerular cell culture and morphologic techniques to be present in large numbers within the glomeruli of rabbits with acute serum sickness (AcSS) and in a passive model of the autologous phase of antiglomerular basement membrane (GBM) antibody-induced glomerulonephritis (PAGBMN). To determine the part played by these cells in the glomerular injury, animals were treated with a sheep anti-rabbit macrophage serum (AMS) or normal sheep serum (NSS). NSS administration had no effect on the development of either model of glomerulonephritis. The use of AMS reduced the number of circulating monocytes and prevented the accumulation of macrophages within glomeruli in both models (AcSS/NSS, mean 126/glomerulus, range 40-251; AcSS/AMS, mean 8, range 1-44; PAGBMN/NSS, mean 52, range 27-69; PAGBMN/AMS, mean 5, range 2-7). The AMS-treated rabbits had only minor histologic lesion and profound reduction in proteinuria (AcSS/NSS, mean 516 mg/24 h, range 200-991; AcSS/AMS, mean 41, range 3-161; PAGBMN/NSS, mean 335, range 55-975; PAGBMN/AMS, mean 10, range 2-24). Similar studies in the heterologous phase of glomerular injury induced by the same anti-GBM antibody revealed no effect of the AMS on this polymorphonuclear leukocyte-related phase of injury, demonstrating the selectivity of the antisera. Complement depletion, with cobra venom factor, did not affect the development of glomerulonephritis nor the accumulation of macrophages in either model. Inhibition of macrophage accumulation can largely prevent these forms of experimental glomerulonephritis, thereby implicating macrophages as mediators of glomerular injury and consequent proteinuria.

A series of monoclonal antibodies to T cell surface antigens were used to characterize peripheral lymphoid populations from patients with a variety of immunodeficiency diseases. Several disorders of T cell differentiation were observed to occur in severe combined immunodeficiency. One subtype of severe combined immunodeficiency was associated with failure to develop lymphocytes that express any thymus specific antigens, another with failure to differentiate beyond the early prothymocyte-thymocyte (T9+, T10+) stage, while a third subtype was associated with failure to differentiate beyond a late thymocyte (T3+, T4+, T5+/T8+, T10+) stage. In contrast, patients with thymic aplasia (DiGeorge syndrome) had a diminished but detectable population of mature T cells. Imbalances in immunoregulatory T cells with a relative excess of suppressor cells were found in 9 of 17 patients with spontaneously occurring acquired agammaglobulinemia. In one of the latter individuals, there was an activated suppressor T cell population expressing Ia antigens (T+/T8+, Ia+). Another had no inducer T4+ cells. Patients with X-linked agammaglobulinemia frequently had an abnormal ratio of inducer to suppressor cells as well as an absence of circulating surface immunoglobulin-bearing cells. No such abnormalities were noted in normals or individuals with selective immunoglobulin (Ig)A deficiency. Taken together, these findings support the notion that several immunodeficiency states may occur as a consequence of defective T cell maturation or imbalances in immunoregulatory T lymphocyte subpopulations.

T he effect of epinephrine on basal and insulin-stimulated glucose uptake in perfused hindlimbs of fed rats was studied. Insulin increased glucose uptake in a dose-dependent manner from a basal value of 1.5±0.3 up to a maximum value of 5.3±0.9 μmol/min per 100 g with 6 nM (1 m U/ml). Epinephrine at 10 nM and 0.1 μM also increased glucose uptake to 2.6±0.1 and 3.1±0.1 μmol/min per 100 g, respectively. These same concentrations of epinephrine, however, suppressed the insulin-stimulated glucose uptake to 3.2±0.3 μmol/min per 100 g. Both the stimulatory and inhibitory effects of epinephrine on glucose uptake were completely reversed by propranolol, but were not significantly altered by phentolamine.Uptake of 3-O-methylglucose and 2-deoxyglucose into thigh muscles of the perfused hindlimbs was stimulated fivefold by insulin, but was unaffected by epinephrine. Epinephrine also did not inhibit the stimulation of uptake by insulin. Epinephrine decreased the phosphorylation of 2-deoxyglucose, however, and caused the intracellular accumulation of free glucose. These last two effects were more prominent in the presence of insulin. Whereas epinephrine caused large rises in glucose-6-P and fructose-6-P, insulin did not alter the concentration of these metabolites either in the absence or presence of epinephrine.These data indicate that: (a) epinephrine has a stimulatory effect on glucose uptake by perfused rat hindlimbs that does not appear to be exerted on skeletal muscle; (b) epinephrine does not affect hexose transport in skeletal muscle; (c) epinephrine inhibits insulin-stimulated glucose uptake in skeletal muscle by inhibiting glucose phosphorylation. It is hypothesized that the inhibition of glucose phosphorylation is due to the stimulation of glycogenolysis, which leads to the accumulation of hexose phosphates, which inhibit hexokinase.

H uman neutrophils stimulated with phorbol myristate acetate were able to destroy suspensions or monolayers of cultured human endothelial cells. Neutrophil-mediated cytotoxicity was related to phorbol myristate acetate concentration, time of incubation and neutrophil number. Cytolysis was prevented by the addition of catalase, while superoxide dismutase had no effect on cytotoxicity. The addition of the heme-enzyme inhibitors, azide or cyanide, markedly stimulated neutrophil-mediated damage while exogenous myeloperoxidase failed to stimulate cytolysis. Neutrophils isolated from patients with chronic granulomatous disease did not destroy the endothelial cell targets while myeloperoxidase-deficient neutrophils successfully mediated cytotoxicity. Endothelial cell damage mediated by the myeloperoxidase deficient cells was also inhibited by catalase but not superoxide dismutase. The addition of purified myeloperoxidase to the deficient cells did not stimulate cytotoxicity. Glucose-glucose oxidase, an enzyme system capable of generating hydrogen peroxide, could replace the neutrophil as the cytotoxic mediator. The addition of myeloperoxidase at low concentrations of glucose oxidase did not increase cytolysis, but at the higher concentrations of glucose oxidase it stimulated cytotoxicity. The destruction of endothelial cells by the glucose oxidase-myeloperoxidase system was inhibited by the addition of hypochlorous acid scavengers. In contrast, neutrophil-mediated cytolysis was not effectively inhibited by the hypochlorous acid scavengers. Based on these observations, we propose that human neutrophils can destroy cultured human endothelial cells by generating cytotoxic quantities of hydrogen peroxide.

P revious studies have demonstrated that hyperosmolar NaCl and mannitol stimulate immunoreactive prostaglandin E (iPGE) production by slices of inner medulla (IM), whereas urea inhibits this process. In the present study, the roles of Ca2+ and calmodulin in the control of PGE synthesis in IM and the basis for the differential actions of solutes were examined. A23187 increased [14C]arachidonate (AA) release and iPGE accumulation in the presence but not in the absence of media Ca2+ whereas stimulation by hypertonic NaCl or mannitol was well expressed with Ca2+ or in Ca2+-free buffer containing 2 mM EGTA. Hypertonic urea and trifluoperazine (TFP), an inhibitor of actions of the Ca2+-CaM complex, suppressed increases in [14C]AA release and iPGE induced by A23187, NaCl, or mannitol. By contrast, increases in iPGE in response to exogenous AA were not altered by urea or TFP. Ca2+ (25-100 microM) increased acyl hydrolase (AH) activity in EGTA washed (4 degrees C) 100,000 g particulate fractions of IM threefold, thereby restoring AH activity to the higher basal values of particulate fractions not washed with EGTA. This action of Ca2+ was blocked by hypertonic urea of TFP, whereas AH activity was not influenced by NaCl or mannitol in the presence or absence of Ca2+. In contrast to their effects on AH activity, hypertonic urea and TFP did not alter conversion of AA to PGE2, PGF2 alpha, or PGD2 by IM microsomal fractions. Ca2+-induced increases in particulate AH were blunted after partial depletion of endogenous CaM-like activity. Ca2+ action was restored by addition of purified exogenous CaM, but not by addition of other small acidic proteins, including troponin C. The findings support a role for CaM in the regulation of PGE synthesis in the IM at the level of Ca2+-responsive AH activity. They further imply that urea suppresses PGE synthesis in IM through inhibition of AH and a reduction in the availability of endogenous AA for conversion to PGE.

W e have recently described an assay system for human peripheral blood megakaryocyte colony-forming unit cells (CFU-M) using an anti-platelet glycoprotein antiserum probe to define megakaryocyte colonies grown in vitro. This system was applied to study the nature and regulation of human bone marrow CFU-M. In the absence of a specific megakaryocyte growth-promoting factor, 12.4 +/- 3.0 (means +/- SEM) megakaryocyte colonies were cloned per 5 X 10(5) cells cultured. Colonies were present after 6 d of incubation reaching peak numbers between days 10 and 14 and slowly decreasing thereafter. Erythropoietin in concentrations of up to 4 U/ml failed to augment colony numbers. Also failing to enhance megakaryocyte colony plating efficiency were media containing burst-promoting activity and colony-stimulating activity. A medium conditioned by human embryonic kidney cells, which has been previously demonstrated to contain thrombopoietin, also had no effect on megakaryocyte colony numbers. In contrast, sera from three patients with severe aplastic anemia produced significant enhancement of CFU-M-derived colony formation in vitro. Both the number of megakaryocyte colonies present and the number of megakaryocytes per colony were increased in proportion to the final concentration of aplastic anemia serum. In the presence of 10% aplastic anemia serum, cultured megakaryocyte colony numbers were linear with respect to the number of bone marrow mononuclear cells plated suggesting a clonal origin of each of the colonies. This in vitro assay for bone marrow CFU-M is a reliable means by which to study the regulation of human megakaryocytopoiesis. Initial data suggest that megakaryocyte production is stimulated by a factor detectable in aplastic anemia serum that may be distinct from other known hematopoietic stem cell regulators.

T urnover and clearance of lung surfactant phospholipids were studied with particular reference to myoinositol-induced perturbation in the acidic phospholipids. Administration of myoinositol decreased [³H]palmitate and [³²P]phosphate incorporation into phosphatidylglycerol by 80-90% in whole lung, and by 94-99% in lamellar bodies and in alveolar lavage. The increased incorporation of radioactive isotopes into phosphatidylinositol following myoinositol, was inverse to the decrease in phosphatidyl-glycerol incorporation. Myoinositol treatment affected neither content nor labeling of phosphatidylcholine or disaturated phosphatidylcholine as studied within 50 h of administration. Phosphatidylglycerol was pulse labeled by intravenous [³²P]phosphate and [³H]palmitate, followed by myoinositol. The biological half-lives of phosphatidylglycerol in the microsomal fraction, lamellar bodies, and alveolar lavage were 1.6, 4.6, 5.4 h (with ³H), and 2.8, 6.5, 7.0 h (with ³²P), respectively.³²P-labeled lung surfactant tracer was applied to the airways in saline suspension and the clearance of phospholipid radioactivity was measured in alveolar lavage, alveolar macrophages, lamellar bodies and lung homogenates. The clearance rates of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol as studied in whole lung over 6 h were 3.4-5.8% h. Only a small amount of phospholipid radioactivity was recovered in the alveolar macrophage fraction (including bis-[monoacylglycerol]phosphate). Phospholipid radioactivity in alveolar lavage fell to 40-70% of the maximum during the 1st h, and to 5-18% over the next 6 h. During 2 h after the application of phospholipids, the radioactivity in the lamellar body fraction increased, and the specific radioactivities approached those in alveolar lavage. The association of phosphatidylglycerol with lamellar bodies was unaffected by myoinositol.Phosphatidylinositol entered more slowly than did phosphatidylglycerol from microsomes to the alveolar lavage fraction, and from alveolar lavage to lamellar bodies. These differences may be of importance regarding the poor performance of phosphatidylinositol-containing surfactant at birth. Further investigations are needed to clarify the possible role for the postulated bidirectional surfactant flux between the lamellar body and alveolar lavage fractions in maintaining the activity of surfactant.

T o investigate the role of non-ACTH pituitary peptides on steroidogenesis, we studied the effects of synthetic β-lipotropin, β-melanotropin, and β-endorphin on aldosterone and corticosterone stimulation using rat adrenal collagenase-dispersed capsular and decapsular cells. β-lipotropin induced a significant aldosterone stimulation in a dose-dependent fashion (10 nM-1 μM). β-endorphin, which is the carboxyterminal fragment of β-lipotropin, did not stimulate aldosterone production at the doses used (3 nM-6 μM). β-melanotropin, which is the middle fragment of β-lipotropin, showed comparable effects on aldosterone stimulation. β-lipotropin and β-melanotropin did not affect corticosterone production in decapsular cells. Although ACTH^1-24 caused a significant increase in cyclic AMP production in capsular cells in a dose-dependent fashion (1 nM-1 μM), β-lipotropin and β-melanotropin did not induce an increase in cyclic AMP production at the doses used (1 nM-1 μM). The β-melanotropin analogue (glycine[Gly]¹⁰-β-melanotropin) inhibited aldosterone production induced by β-lipotropin or β-melanotropin, but did not inhibit aldosterone production induced by ACTH^1-24 or angiotensin II. Corticotropin-inhibiting peptide (ACTH^7-38) inhibited not only ACTH^1-24 action but also β-lipotropin or β-melanotropin action; however it did not affect angiotensin II-induced aldosterone production. (saralasin [Sar]¹; alanine [Ala]⁸)-Angiotensin II inhibited the actions of β-lipotropin and β-melanotropin as well as angiotensin II. These results indicate that (a) β-lipotropin and β-melanotropin cause a significant stimulation of aldosterone production in capsular cells, (b) β-lipotropin and β-melanotropin have a preferential effect on zona glomerulosa cells, (c) β-melanotropin contains the active peptide core necessary for aldosterone stimulation, (d) the effects of these peptides on aldosterone production may be independent of cyclic AMP, and (e) the receptors for β-lipotropin or β-melanotropin may be different from those for ACTH or angiotensin II.

I n experimental models of glomerular and nonglomerular renal disease, single nephron filtration rate and proximal tubular reabsorption of fluid decrease or increase in parallel in the same nephron. To assess whether intrinsic adaptations in proximal tubular function, i.e., changes that are independent of the peritubular or humoral milieu, contribute to this phenomenon, segments of rabbit late superficial proximal convoluted tubules (PCT) were studied by in vitro perfusion. PCT were obtained from normal kidneys, from remnant kidneys, and from kidneys embolized with microspheres. Single nephron filtration rates are increased in the remnant and decreased in the embolized kidneys. Whereas the embolized-kidney rabbits were nonazotemic (the contralateral kidney was left in situ), the remnant-kidney animals were uremic. In order to study a nonazotemic model of increased single nephron filtration rate, PCT were also obtained from uninephrectomized rabbits.Significant compensatory hypertrophy occurred in the PCT of the remnant kidney. Net fluid reabsorption (Jv) per unit length was increased by ∼60%; Jv per unit luminal surface area was the same as in the normal PCT. Transepithelial potential difference (PD) was significantly greater than normal. This was associated with a reversal of the normal permselective properties (P_Cl > P_Na) of the late superficial PCT so that P_Na exceeded P_Cl. The changes could not be ascribed to some undetermined effect of the uremic state in vivo, since increases in tubule size, Jv per unit length, and PD also occurred in PCT from nonazotemic uninephrectomized rabbits.In contrast, Jv, per unit length or per unit luminal surface area, was decreased by ∼50% in PCT from embolized kidneys and PD was also reduced. In these tubules, the normal permselective properties were also reversed. Tubule size, however, was not significantly different from normal.The increases or decreases in Jv that occurred in the different disease models were not dependent on tubular fluid flow rate or the uremic milieu in vitro.These studies indicate that intrinsic proximal tubular function is modified by the disease state in vivo and that the “memory” of this adaptation is expressed in the in vitro situation. The changes in Jv observed in vitro parallel the increases or decreases in single nephron filtration rates that occur in vivo. Compensatory hypertrophy, with an attendant increase in luminal surface area, could explain the increased Jv per millimeter in the remnant kidneys, but the adaptation observed in the embolized kidneys cannot be ascribed to changes in tubule size.

W e examined the role of metabolic CO2 production in the hyperpnea of muscular exercise by comparing the response of alveolar ventilation to moderate levels of exercise with the response to venous infusion of CO2 at rest. Studies were performed in four awake sheep that were trained to run on a treadmill. The sheep had been cannulated for veno-venous extracorporeal perfusion so that CO2 could be infused into the peripheral venous blood through membrane lungs in the perfusion circuit. The sheep breathed room air through an endo-tracheal tube inserted through a tracheostomy, and samples of expired gas were collected for measurement of the rates of CO2 production and O2 consumption. All measurements were made in the steady state. In each of the four sheep, the relationship between alveolar ventilation and the rate of CO2 production could be described by a single linear function (r greater than 0.99; P less than 0.001), regardless of whether CO2 production was increased by exercise, venous CO2 infusion, or combinations of both procedures. This relationship applied for values of CO2 production up to 350% of control. In contrast, no unique relationship was found between the rate of alveolar ventilation and either the rate of O2 consumption, cardiac output, or mixed venous blood gas pressures. The findings indicate that the hyperpnea of mild to moderate steady-state exercise can be attributed to the associated increase in the rate of CO2 production. Therefore, there is no need to invoke obligatory nonmetabolic stimuli to account for the ventilatory response to steady-state exercise.

P revious studies have shown that bile salt concentrations in human blood taken from the placenta at birth of term infants are in the range found in adults. A 125I-radioimmunoassay procedure and capillary gas liquid chromatography-mass spectrometry have been used in this investigation to measure serum bile salt concentrations in premature and normal term infants. It was found that the serum bile salt concentration in samples taken at birth in premature infants were also similar to that of adults. In the week after birth the serum bile salt concentration rose four- to sevenfold in each of the infant groups. The increase was independent of gestational age and the "health" of the child. A similar increase was observed in term infants. Thus, hypercholemia is physiologic in newborn infants. In conjunction with other abnormalities of the enterohepatic circulation of bile salts there are profound implications in the newborn for the metabolism and excretion of those endogenous and exogenous substances that are dependent on the secretion of bile salt by the liver. In addition, speculations concerning the role of parenteral nutrition in the induction of cholestasis in premature infants should be made with caution.

T he status of suppressor T cells (Ts) was assessed in seven children with the hyper IgE syndrome (recurrent staphylococcal infections, eczematous skin rash, and elevated serum IgE) to determine whether a deficiency in Ts is associated with increased IgE synthesis. When circulating T cells and their subsets were enumerated with the aid of monoclonal antibodies that identify T cells (T3), helper/inducer T cells (T4), and suppressor/cytotoxic T cells (T8), there was a selective deficiency of T3+ cells (51.7±11.2% vs. 66±5% for normal controls) and of T8+ cells (7.5±4.4% vs. 22±4% for normal controls) but not of T4+ cells (36.5±7.5% vs. 37±3% for normal controls).Suppressor T cell function was assessed by examining the ability of mononuclear cells incubated for 48 h with concanavalin A to suppress the proliferation of fresh autologous mononuclear cells in response to the mitogens phytohemagglutinin and pokeweed mitogen. All seven patients were severely deficient in concanavalin A-inducible suppressor cells.In vitro de novo synthesis of IgE in 6-d cultures of peripheral blood lymphocytes was measured in four patients by a solid-phase radioimmunoassay. Mononuclear cells from all four patients synthesized spontaneously increased quantities of IgE in vitro (4,950±3,760 pg/10⁶ cells vs. 250±215 pg/10⁶ cells for eight normal controls). IgE synthesis was suppressed by the addition of parental T cells to the culture. Elimination of the T8+ subset, but not of the T4+ subset, by complement-dependent lysis resulted in the loss of the capacity of parental T cells to suppress IgE synthesis.These results suggest that a deficiency of Ts underlies the elevated IgE levels observed in the hyper IgE syndrome.

H uman blood mononuclear cells exposed to concanavalin A or phytohemagglutinin secrete a soluble factor that arrests the growth of human synovial fibroblastic cells in culture. Once the growth-inhibitory effect is initiated it cannot be reversed by washing the fibroblastic cells, by refeeding with nonconditioned fresh serum-containing medium, by trypsinization, EDTA treatment, or a combination of these procedures. Media from nonstimulated mononuclear cells, fibroblastic cells, or the lectins themselves do not contain similar inhibitory activity that can be detected by the present culture systems. This lectin-dependent, growth-inhibitory activity does not have a cytotoxic effect on the fibroblasts but increases their adhesiveness to plastic or glass surfaces, and the cells tend to assume a less fibroblastic morphology. The growth-inhibitory activity is stable in the cold and is nondialyzable or ultrafilterable, but the activity is rapidly lost at temperature between 60 degrees and 70 degrees C and at pH 2.0. The growth-arrested cells secrete more glycosaminoglycan per cell in the medium and synthesize more cell surface glycosaminoglycan than the controls. However, the increased glycosaminoglycan synthesis cannot be explained as being entirely secondary to a cell density effect as it is also observed when adjustments are made for the differences in growth rates.

T he possible suppressive effects of 24,25-dihydroxycholecalciferol on secondary hyperparathyroidism and increased bone resorption were investigated in adult rats raised on a diet normal in calcium, phosphorus, and vitamin D, and subjected to acute bilateral nephrectomy. The animals had received subcutaneous radiocalcium 4 wk before the experiment. 5 h after nephrectomy an increase in serum total calcium, ⁴⁵Ca-specific activity, citrate, phosphorus, and magnesium concentrations were observed. Serum immunoreactive parathyroid hormone increased, while serum calcitonin decreased. The osteoclast count in the tibial metaphyses was augmented. The biochemical and histological changes observed were partly parathyroid hormone and calcitonin independent, as they also occurred in parathyroidectomized hypocalcemic rats. Pretreatment with 650 pmol of 24,25-dihydroxycholecalciferol 16 h before nephrectomy prevented bone calcium mobilization and diminished the rise in serum total calcium and citrate both in parathyroid-intact and in parathyroidectomized animals. In parathyroid-intact rats, serum immunoreactive parathyroid hormone and calcitonin remained normal in spite of the fall in serum-ionized calcium, and the number of osteoclasts did not increase. In parathyroidectomized rats, 24,25-dihydroxycholecalciferol did not prevent the postnephrectomy rise in the osteoclast count. This latter observation suggests that this metabolite exerts its effect on bone either by acting on cells other than osteoclasts, i.e., the osteocytes, or by inhibiting cell activity. At equimolar dosage 1,25-dihydroxycholecalciferol had a potent stimulatory effect on bone resorption. This effect of 1,25-dihydroxycholecalciferol was partly blocked by the simultaneous administration of 24,25-dihydroxycholecalciferol.The potential clinical significance of these observations remains to be determined.

T he effects of insulin-dependent diabetes mellitus on glucose transport activity and on the concentrations of glucose transport systems in the plasma and low density microsomal membranes in adipose cells isolated from streptozotocin-induced diabetic rats have been examined. Glucose transport activity was assessed by measuring 3-O-methylglucose transport and the concentration of glucose transport systems estimated by measuring specific D-glucose-inhibitable cytochalasin B-binding. Basal glucose transport activity decreases from 0.19 to 0.12 fmol/cell per min with the induction of diabetes, but remains constant per unit cellular surface area and is accompanied by a constant 6 pmol of glucose transport systems/mg of membrane protein in the plasma membrane fraction. Maximally insulin-stimulated glucose transport activity decreases from 3.16 to 1.05 fmol/cell per min and from 0.26 to 0.12 amol/micrometers 2 per min, and is accompanied by a decrease from 25 to 15 pmol of glucose transport systems/mg of plasma membrane protein. These diminished effects of insulin on glucose transport activity and the concentration of glucose transport systems in the plasma membrane fraction are paralleled by a 45% decrease in the basal number of glucose transport systems per milligram of membrane protein in the low density microsomal membrane fraction, the source of those glucose transport systems appearing in the plasma membrane in response to insulin. Thus, the "insulin resistant" glucose transport of the adipose cell in the streptozotocin-induced diabetic rat appears to be the consequence of a depletion of glucose transport systems in the intracellular pool.

T he influence of calcitonin (CT) on various stages of bone formation was investigated. A demineralized collagenous bone matrix-induced bone forming system in rats was used to temporally segregate chondrogenesis and osteogenesis. Administration of CT (15 Medical Research Council Units [MRCU]) daily) at the initiation of matrix-induced bone formation (BF) resulted in a 76% stimulation of BF as measured by 45Ca incorporation and alkaline phosphatase activity. This increase was due, in part, to a stimulation of cartilage and bone precursor cell proliferation monitored by the rate of [3H]thymidine incorporation and ornithine decarboxylase activity. Chondrogenesis on day 7 as measured by 35SO4 incorporation was increased by 52% with CT treatment. To rule out the possibility of a secondary response due to parathyroid hormone, similar studies were done in parathyroidectomized animals and CT stimulation of BF was still observed. However, when CT injections were started after cartilage formation (day 8) there was no stimulation of BF but a significant decrease in 45Ca incorporation was observed. These results indicate CT has two actions: (a) when CT is administered during the initial phases of bone formation, it increases BF due to a stimulation of proliferation of cartilage and bone precursor cells; and (b) when CT is administered after bone formation has been initiated, subsequent bone formation is suppressed.

F our male transsexual subjects were given a superactive luteinizing hormone-releasing hormone (LHRH) analogue, D-tryptophan-6-LHRH at daily doses of 100 micrograms for 3--6 mo. A decrease in beard growth, acne, and erectile potency was noted; the latter was documented objectively with the recordings of nocturnal penile tumescence episodes. Plasma testosterone and dihydrotestosterone levels fell to castrate values; basal prolactin and luteinizing hormone levels showed a small decline, whereas the acutely releasable luteinizing hormone was significantly suppressed. A rise of plasma testosterone from castrate to normal levels was demonstrable with the use of human chorionic gonadotropin. Discontinuation of treatment led to a normalization of erectile potency and plasma testosterone. The suppression of Leydig cell function by D-tryptophan-6-LHRH might have wide application in reproductive biology and in endocrine-dependent neoplasia (where it could replace surgical castration).