Issue published January 1, 1981

P hagocytic vesicles from rabbit lung macrophages produced superoxide in the presence of NADH or NADPH. At 37°C, these vesicles generated 51±7.8 nmol O₂⁻/min per mg protein in the presence of 0.5 mM NADPH. The apparent K_m for NADPH and NADH (66 and 266 μM, respectively), the pH optimum for the reaction (6.9), and the cyanide insensitivity were similar to properties of plasma membrane-rich fractions of stimulated polymorphonuclear leukocytes studied by others. The activity of the phagocytic vesicles was trypsin sensitive. The specific superoxide-generating activity of macrophage phagocytic vesicles isolated from cells incubated up to 90 min with phagocytic particles remained constant.Calcium in micromolar concentrations inhibited the NADPH-dependent O₂⁻-generating activity of phagocytic vesicles. In a physiological ionic medium (100 mM KCl, 2.5 mM MgCl₂, 30 mM imidazole-HCl, pH 6.9), a maximal inhibition of O₂⁻ generation by phagocytic vesicles of 80% was observed at 40 μM free Ca²⁺. The half maximum inhibitory effect was at 0.7 μM Ca²⁺. Variations of the calcium concentration resulted in rapid and reversible alterations in O₂⁻-forming activity. Preincubation of phagocytic vesicles in the presence of EGTA rendered their O₂⁻ generation rate in the presence of NADPH insensitive to alterations in the free calcium concentration. This desensitization by low EGTA concentrations (≤100 μM) was reversible by the addition of excess calcium, but desensitization by high EGTA concentrations (>1 mM) was not reversible by the addition of calcium either in the presence or absence of purified rabbit lung macrophage or bovine brain calmodulins. Furthermore, trifluoperazine, a drug that inhibits calmodulin-stimulated reactions, did not alter the activity or the calcium sensitivity of the superoxide-generating system of sensitive phagocytic vesicles.Peripheral plasma membrane vesicles (podosomes) prepared by gentle sonication of macrophages possessed on O₂⁻-generating system with similar properties to those of phagocytic vesicles.We conclude that the activated O₂⁻-generating system of rabbit lung macrophages has its initial localization in the plasmalemma and undergoes subsequent internalization into phagocytic vesicles, where it can function for prolonged periods of time. Calcium at concentrations likely to exist in macrophage cytoplasm exerts a regulatory effect on the activated system.

H uman growth hormone (hGH) is known to be a potent stimulator of somatomedin secretion in vivo. The induction of somatomedin by growth hormone has been difficult to study in vitro, however, because no organ containing a high concentration of somatomedin has been identified. Because fetal mouse explants have been shown to produce somatomedin in vitro, we have undertaken studies to determine whether postnatal human fibroblast monolayers also produce somatomedin, and if so, whether its production is regulated by other hormones. Quiescent human fibroblasts were exposed to serum-free minimum essential medium, and the medium was assayed for somatomedin concentration using a specific radioimmunoassay for somatomedin-C. A progressive rise in immunoreactive somatomedin to 0.08 U/ml per 10⁵ cells per 24 h was observed over 72 h of incubation. This was an underestimation of the actual concentration of immunoreactive somatomedin since the amount measured following acid treatment was at least fourfold higher than in the untreated medium. Growth hormone stimulated immunoreactive somatomedin production in a dose-dependent manner: 5 ng hGH/ml = 0.1 U/ml per 10⁵ cells; 50 ng hGH/ml = 0.25 U/ml per 10⁵ cells. Platelet-derived growth factor and fibroblast growth factor were also stimulatory, but epidermal growth factor, thyroxine, or cortisol had no effect. Media that had been exposed to human fibroblasts stimulated DNA synthesis in BALB/c 3T3 fibroblasts (a cell type that does not produce somatomedin). Medium-derived immuno-reactive somatomedin eluted from Sephacryl S-200 in two major peaks (150,000 and 8,000 mol wt). The higher molecular weight peak is similar to the one observed when whole serum was used. These studies provide a model system for studying the humoral and nonhumoral factors that control the biosynthesis of somatomedin by human tissues. Since immunoreactive somatomedin production may be a rate-limiting factor for fibroblast growth, the delineation of the hormonal control of somatomedin production should lead to a better understanding of the mechanisms controlling human fibroblast growth.

T o determine the effect of activation of the reticuloendothelial system on the localization of immune complexes in the kidney, a model of passive serum sickness nephritis in the mouse was used, with activation of the reticuloendothelial system with Corynebacterium parvum. Groups of mice, control and C. parvum-treated animals, were injected with BSA-125I-anti-BSA complexes containing 3 mg 125I-anti-BSA. Blood was obtained at 5 min, at 3 h, and at 12 h, when the animals were killed. Blood concentrations of BSA-125I-anti-BSA complexes were reduced in C. parvum-treated animals compared with controls. This appeared to be mediated by two effects, increased uptake of complexes in the liver and spleen, and enhanced degradation of immune complexes as measured by TCA-soluble radioactivity. In vitro studies using cultures of peritoneal macrophages also showed enhanced uptake of immune complexes. The amount of immune complexes deposited in the glomeruli of C. parvum-treated animals was reduced as determined by quantitation of radiolabeled material bound to isolated gomeruli and by immunofluorescence techniques. The results of the study emphasize the role of the reticuloendothelial system in the modulation of immune complex localization in the kidney and suggest a potential use of stimulants of the reticuloendothelial system in the therapy of immune complex nephritis.

T herapeutic doses of corticosteroids frequently induce eosinopenia; however, the mechanism(s) involved remain obscure. To investigate this question, we studied the effects of corticosteroids on eosinophil adherence and migration. Eosinophils from normal donors were prepared by dextran sedimentation and Hypaque gradient centrifugation to 45-96% purity. Adherence was measured by filtration of whole blood and isolated eosinophils through nylon wool columns. Before prednisone administration, adherence was 83.8±3.2% for eosinophils in heparinized blood and 82.1±3.2% for isolated eosinophils. 4 h after oral prednisone administration whole blood eosinophil adherence was reduced to 53.9±10.7%; at 24 and 48 h adherence was normal. In contrast, isolated eosinophils showed no decrease in adherence 4, 24, or 48 h after corticosteroid administration. Similarly, in vitro addition of hydrocortisone to isolated eosinophils at 0.01 and 2.0 mg/ml did not reduce adherence. Eosinophil migration was tested in modified Boyden chambers by “lower-surface” and “leading-front” methods, using zymosan-activated serum and buffered saline to assess chemotactic and random migration, respectively. In vitro incubation of eosinophils with hydrocortisone or methylprednisolone produced a dose-dependent inhibition of chemotaxis. Using lower-surface methods the minimal concentration effecting substantial inhibition was 0.01 mg/ml for both drugs. At 2.0 mg/ml hydrocortisone and methylprednisolone inhibited eosinophil chemotaxis 82.6±4.4% and 85.0±3.5%, respectively. Using leading-front chemotaxis techniques significant inhibition was detected at 0.001 mg/ml hydrocortisone. Eosinophils incubated and washed free of corticosteroids responded normally to chemoattractants, indicating that the inhibitory effect of these drugs was reversible. Hydrocortisone at 2 mg/ml inhibited random eosinophil migration, although this effect was not apparent at lower concentrations. Corticosteroids did not act as chemotactic factor inactivators and were not toxic as measured by trypan blue exclusion. Eosinophils obtained from donors who had received 40 mg of prednisone orally for four days showed normal chemotactic responses, probably reflecting the fact that the cells were washed free of plasma before testing. In contrast, incubation of eosinophils in plasma from donors who had received a 300-mg bolus of hydrocortisone induced 46.1±4.5% more inhibition of chemotaxis than did incubation in normal plasma. These results indicate that: (a) eosinophil adherence is transiently reduced following in vivo corticosteroid administration, (b) eosinophil chemotaxis is inhibited by both in vitro and in vivo administration of corticosteroids, and (c) the chemotaxis inhibiting effect is nontoxic, cell-directed, dose-dependent and reversible. Inhibition of eosinophil adherence and chemotaxis may in part explain how corticosteroids produce eosinopenia and decrease the local accumulation of eosinophils.

N itrofurantoin, a commonly used urinary tract antiseptic, has been associated with idiosyncratic pulmonary and hepatic damage. We have used human lymphocytes in vitro to explore the biochemical basis of susceptibility to nitrofurantoin toxicity. The drug itself did not damage the cells as assessed by trypan blue dye exclusion. In the presence of a mouse hepatic microsomal drug-activating system, however, nitrofurantoin metabolites produced dose dependent toxicity to the lymphocytes. Inhibition of the enzyme epoxide hydrolase did not enhance toxicity; the metabolite thus does not appear to be a furan epoxide. Binding of reactive metabolites to cell macromolecules may lead directly to cell death, or in vivo, by acting as haptens to secondary immunologic responses. The metabolite caused a dose-dependent depletion of lymphocyte glutathione content. Cells from a patient with glutathione synthetase deficiency showed markedly enhanced nitrofurantoin toxicity. The findings suggest that glutathione plays a major role in protecting cells from nitrofurantoin-induced damage, and that studies of lymphocyte toxicity and glutathione metabolism in patients experiencing idiosyncratic reactions to nitrofurantoin may lead to elucidation of the biochemical and genetic basis of drug susceptibility.

A patient with acquired agammaglobulinemia had an antihelper T cell factor that was identified as an immunoglobulin of the IgG class. The factor specifically bound to the TH2- T cell subset and, in the presence of complement, abolished the helper effect of normal T cells. The antihelper T cell antibody preceded by several years the appearance of suppressor TH2+Ia+ T cells, at which time the clinical course rapidly deteriorated. Plasmapheresis resulted in lymphocytosis and reappearance of a functionally intact helper T cell population. It did not affect the suppressor cells. Conversely, total thymectomy resulted in a temporary disappearance of the TH2+Ia+ suppressor cells, but did not decrease the levels of the autoantibody to helper T cells. Neither of these treatments reversed the state of agammaglobulinemia.

T o elucidate the metabolic abnormality of musclar dystrophy, 27 kinds of enzyme activity in various organs of control and dystrophic mice were examined. The organs examined included muscle, bone, heart, testis, uterus, spleen, thymus, submaxillary gland, stomach, pancreas, liver, kidney, brain, and lung. The activities of 14 different aminopeptidases, 5 endopeptidases, 4 glycosidases, phosphatase, esterase, and ribonuclease were measured. Most of the enzyme activities were significantly elevated in muscles and bones of dystrophic mice. These organs were similar in their patterns of enzyme abnormality. Among the 14 kinds of aminopeptidase activity studied, the degree of increased activity was greater for the aminopeptidases (AP):Ala-AP, Leu-AP, Met-AP, Phe-AP, Trp-AP, Gly-Pro-Leu-AP. In addition to aminopeptidases, there were significant increases in activities of chymotrypsinlike enzyme, cathepsin C, cathepsin D, several glycosidases and neutral ribonuclease in the muscles of dystrophic mice. Similarly increased enzyme activity was also observed in organs other than muscle and bone. Furthermore, protein content in most organs was higher in dystrophic mice than in those of control mice. These abnormalities were seen in both males and females. The present results suggest that there are extensive abnormalities in the protein metabolism in dystrophic mice. It seems therefore that the therapeutic approach to muscular dystrophy should be studies not only from the well-known abnormality of intramuscular endopeptidases, but from other aspects as well.

T he chemotactic migration of leukocytes is preceded by an alteration in the cells' shape from round to a characteristic polar configuration. We have developed an assay that shows that human monocytes, when exposed to chemoattractant in suspension, assume this polarized shape. The three types of chemo-attractants studied, a chemotactic lymphokine, complement-activated serum, and the N-formylated oligopeptides, all induced polarization in a time, temperature, and dose-dependent fashion. Nonchemotactic agents such as mitogens or phorbol myristate acetate did not induce polarization. At 37°C, polarization was rapid (<1 min) and was inhibitable by cytochalasin B, sodium azide, or low temperature. A series of N-formylated oligopeptides were studied and their activity in inducing polarization correlated closely (r > 0.99) with their chemotactic activity. Of the entire population of circulating monocytes there is a subpopulation of cells that is capable of polarizing in response to chemotactic stimuli. The maximum percentage of monocytes which polarized to any chemotactic factor was ∼60%. Furthermore, the combination of several chemotactic factors could not increase the percentage of polarized monocytes above the maximum obtained with an optimal dose of any single chemoattractant. The data also demonstrate that high doses of a chemoattractant can induce a state of cross-desensitization in monocytes that blocks the response of the cells to other types of chemotactic factors. These results support the concept that the monocytes that do respond to chemotactic stimuli are capable of responding to any of several attractants.

W hen supernates from the established human breast cancer cell line MCF-7 were applied to fetal rat long bones that had been labeled with 45Ca and devitalized to remove endogenous bone cells, mineral was released from the bones. The release of bone mineral by MCF-7 supernates was associated with increased basal release of hydrolytic enzyme activity by the tumor cells. The basal release of lysosomal enzymes and collagenolytic activity by MCF-7 cells with approximately twice that of mouse 3T3 cells, which did not cause mineral release by the fetal rat bones. Release of hydrolytic enzymes and bone mineral-releasing activity was increased by colchicine and vinblastine, drugs that inhibit microtubule assembly, but not affected by lumicolchicine. Time-course experiments performed on MCF-7 cells with or without colchicine showed that release of cathepsin D and collagenolytic activity was associated more closely with release of bone mineral and degradation of bone matrix than was the release of N-acetylglucosaminidase. The release of previously incorporated [3H]proline from the bones exposed to MCF-7 cell cultures was more closely associated with release of collagenolytic activity by MCF-7 cells than with release of cathepsin D or N-acetylglucosaminidase. These data suggest that breast cancer-mediated bone resorption in vitro is positively correlated with release of hydrolytic enzymes by the tumor cells, and release of these enzymes is enhanced by disassembly of microtubules.

T he possible occurrence of immune complexes (IC) in serum and in cerebrospinal fluid (CSF) has been studied in 36 patients with African trypanosomiasis (Trypanosoma brucei gambiense). In serum, very high levels of IC were detectable by the ¹²⁵I-C1q-binding and by the conglutinin-binding assays with positive results in 94 and 87%, respectively, of untreated patients. Circulating IC were found in both early and late stages of the disease, without significant quantitative differences; their size was 15-25S. There was a significant negative correlation between C3 values and C1qBA. Our studies suggest that circulating IC occurring during trypanosomiasis may be the expression of a polyclonal B cell activation. Indeed, there was a significant correlation (P < 0.001) between the levels of circulating IC and either the levels of IgM (mean value 12.5±7.2 mg/ml) or with the levels of rheumatoid factor-like antiimmunoglobulin antibodies that were detected by solid phase radioimmunoassay in 74% of the patients.IC were detected in 31 of 35 CSF samples, with a marked elevation in patients with definite involvement of the central nervous system as compared with earlier stages of sleeping sickness. The occurrence of IC in CSF was not related to an impairment of the blood-brain barrier as shown by analysis of CSF/serum albumin ratios. The level of IC in CSF did not correlate with the serum level and, therefore, circulating IC do not appear to cross efficiently an unimpaired blood-brain barrier. The analysis of IgG, IgM, and albumin concentrations in serum and CSF demonstrates a marked intracerebral immunoglobulin synthesis in patients with manifestations of meningoencephalitis. There was a correlation between CSF-C1q binding assay and this local IgG synthesis.These data are consistent with a local formation of IC in CSF in patients with active meningoencephalitis. The results obtained in eight patients followed during therapy suggest that the presence of IC in CSF may be an indicator of a continuing central nervous system disease and that the quantitation of CSF-IC may be useful for monitoring patient care.

P ulmonary sarcoidosis is a disorder in which local granuloma formation is perpetuated by activated lung T lymphocytes. The present study suggests that lung T lymphocytes may also play a critical role in modulating local production of antibodies in this disorder. In untreated patients with pulmonary sarcoidosis, the numbers of IgG- and IgM-secreting cells per 10³ lung lymphocytes are markedly increased compared with those in normal individuals (P < 0.001 and P < 0.01, respectively); the numbers of IgA-secreting cells in lavage fluid of these patients are not increased (P > 0.2). In contrast to lungs, the numbers of IgG-, IgM-, and IgA-secreting cells in blood of patients with this disorder are similar to those in normal individuals (P > 0.2, each comparison). In patients with pulmonary sarcoidosis, there is a direct correlation between the percentage of bronchoalveolar cells that are T lymphocytes and the percentage of bronchoalveolar cells that secrete IgG (r = 0.79; P < 0.001); in normal individuals there is no such relationship (P > 0.2). When purified sarcoid lung T cells from patients with high proportions of T lymphocytes in their lavage fluid were co-cultured with blood mononuclear cells from normal individuals (without added antigens or mitogens), the B lymphocytes in these normal mononuclear cell suspensions were induced to differentiate into immunoglobulin-secreting cells (P < 0.01). In contrast, blood T lymphocytes from these same patients and lung T lymphocytes from sarcoidosis patients with low proportions of T lymphocytes in their lavage fluid did not stimulate normal B cells to produce immunoglobulin (P > 0.2, all comparisons). These findings suggest that in pulmonary sarcoidosis (a) the lung is an important site of immunoglobulin production; (b) activated lung T lymphocytes play an important role in modulating this local production of antibody, and thus are likely to modulate the polyclonal hyperglobulinemia observed in these individuals.

H uman as well as murine granulocytes have been shown to kill the larval stages of helminth parasites; the mechanism of this cell-mediated cytotoxicity is, however, poorly understood. The present study was designed to assess the role of peroxidative processes in killing of schistosomula of Schistosoma mansoni by human granulocytes in vitro. The rate of H₂O₂ production by human neutrophils, eosinophils, and basophils was measured upon incubation with schistosomula alone or in the presence of specific antibody or complement. Opsonized parasites (antibody and/or complement) increased the rate of H₂O₂ production by neutrophils, eosinophils, and basophils by respective percentages of 500, 500, and 371. The rate of H₂O₂ release was directly related to the number of granulocytes and to the proportion of cells attached to the surface of the schistosomula. Increased hydrogen peroxide release occurred by 10 min of incubation and was demonstrable up to 16 h after addition of leukocytes to schistosomula. The primary source of this oxygen product was found to be the granulocytes adherent to the schistosomula and not those that remained unattached. Hydrogen peroxide production by neutrophils and eosinophils was quantitatively similar (schistosomula coated with antibody plus complement stimulated 5 × 10⁶ neutrophils and eosinophils to release H₂O₂ at respective rates of 0.35 and 0.40 nmol/min). Granulocyte-mediated parasite killing correlated with rate of H₂O₂ generation; both processes were inhibited by catalase. To define further the role of oxidative metabolites, neutrophils and eosinophils of two subjects with chronic granulomatous disease were used; marked reduction of granulocyte-mediated parasite mortality was observed.Peroxidase was required for H₂O₂-mediated killing. Addition of the peroxidase inhibitors azide (1 mM), cyanide (1 mM), or aminotriazole (1 cM) to neutrophilschistosomula mixtures significantly reduced parasite cytotoxicity (P < 0.01); similar reduction was observed when eosinophils were used (P < 0.01). Fixation of halide (iodide) to trichloroacetic acid-precipitable protein (2.4-6.0 nmol/h per 10⁷ neutrophils) was demonstrated in the presence of granulocytes, opsonins, and parasites; this process was completely inhibited by 1 mM azide.These data indicate that contact between the surfaces of human granulocytes and schistosomula results in release of cellular hydrogen peroxide and iodination. The generation of H₂O₂ and its interaction with peroxidase appear to be crucial in effecting in vitro granulocyte-mediated parasite cytotoxicity.

B rush border membrane vesicles were isolated from rabbit renal cortex by Mg⁺⁺-precipitation and differential centrifugation. ³⁶Cl⁻ and [³H]glucose uptakes were simultaneously determined by a rapid filtration technique. Lysis of the vesicles with distilled water abolished 90-95% of the radioactivity on the filters, suggesting that nearly all of the ³⁶Cl⁻ and [³H]glucose counts represented uptake into an osmotically reactive intravesicular space. Inwardly directed K⁺ gradients plus valinomycin stimulated ³⁶Cl⁻ uptake, demonstrating a conductive pathway for chloride uptake into brush-border membrane vesicles. ³⁶Cl⁻ uptake could also be stimulated by inwardly directed proton gradients (pH_outside < pH_inside). This effect was seen in the absence of sodium, as well as in the presence of valinomycin when the vesicles had equal K⁺ concentrations inside and out. An “overshoot” phenomenon was observed when external ³⁶Cl⁻ was 2 mM and the external pH was lowered from 7.5 to 6.0 or to 4.5. The effect of the proton gradient was presumed to be different from the conductive mechanism because (a) the stimulation of ³⁶Cl⁻ uptake by inwardly directed K⁺ diffusion potentials was additive to the proton gradient effect, and (b) competition studies revealed statistically significant effects of thiocyanate on the conductive pathway, but not on the proton-driven pathway.HCl cotransport or anion exchange are electrically neutral mechanisms which could couple ³⁶Cl⁻ uptake to inwardly-directed proton gradients in a brush border membrane vesicle. If both electrically neutral and conductive path ways for chloride transport are present in the luminal membrane of the proximal tubule, then the mechanism as well as the direction of net chloride transport will be influenced by the nature of the accompanying cation transport process.

T he extent of inhibition of monovalent cation active transport in Purkinje fibers and myocardium in response to toxic and inotropic doses of digitalis were studied in the dog to elucidate the factors underlying the different relative sensitivities of these tissues to the toxic arrhythmogenic effects of digitalis. Monovalent cation transport inhibition was assessed by measuring uptake of the K⁺ analog Rb⁺ in samples of myocardium and Purkinje fibers after in vitro ouabain exposure and after acute or chronic administration of digoxin in vivo. The active uptake of Rb+ was determined as the difference between total uptake and uptake in the presence of 1.0 mM ouabain. Mean active uptake of Rb⁺ by Purkinje fibers from control hearts was 1.62±0.11 (SEM) nmol/mg wet wt per 15 min, significantly greater than the value of 0.49±0.05 for myocardium (P < 0.001). Concentration-effect curves for inhibition of monovalent cation active transport by in vitro exposure to graded concentrations of ouabain showed that the concentration for half-maximal inhibition of monovalent cation transport, IC₅₀, for Purkinje fiber transport was 0.4 μM, significantly less than the value of 1.4 μM for myocardial samples. Dogs were given toxic doses of digoxin (0.3 mg/kg i.v.). At onset of sustained ventricular tachycardia, they were killed and monovalent cation transport measured in myocardial and Purkinje fiber samples. Active Rb⁺ uptake was inhibited by 44% in myocardial samples, whereas a significantly greater inhibition of 76% was noted in Purkinje fibers (P < 0.01). Similar data were obtained for both transmural myocardial biopsy samples and subendocardial trabecular samples obtained from regions adjacent to Purkinje fibers. Another group of dogs received a nontoxic dose of 0.02 mg/kg i.v. daily for 6 d. Myocardium showed a 17% reduction in Rb⁺ active uptake compared to control animals receiving no drug, whereas Purkinje fiber transport was reduced in these dogs to a significantly greater extent averaging 44% (P < 0.001). Thus, both toxic and inotropic (non-toxic) doses of digoxin inhibited monovalent cation transport in Purkinje fibers to a greater extent than in myocardium. This difference in apparent sensitivity of monovalent cation transport to digoxin may contribute to the previously reported tendency of digitalis toxic arrhythmias to arise in Purkinje fibers.

W e have studied the relative concentrations of the human immunoreactive (IR) peptides γ-lipotropin (hγLPH, [1-58]hβLPH), β-lipotropin (hβLPH), and β-endorphin (hβEND, [61-91]hβLPH) using gel exclusion chromatography together with a specific radio-immunoassay (RIA) for hγLPH and a RIA that (because hβEND is the COOH-terminus of the hβLPH molecule) measures both hβEND and hβLPH on an equimolar basis. In normal subjects, basal plasma IR-hγLPH was often undetectable (<12.5 fmol/ml), but ranged up to 21 fmol/ml, and IR-hβEND/hβLPH was 10.8±0.7 fmol/ml; previous studies by others suggest that most of the IR-hβEND/hβLPH was probably hβLPH. Both IR-hγLPH and IR-hβEND/hβLPH were significantly elevated (P < 0.001) in patients undergoing chronic hemodialysis (101.5±12.7 and 23.8±2.0 fmol/ml, respectively). Their IR-hγLPH coeluted with standard hγLPH as a single peak, and IR-hβEND/hβLPH coeluted with hβLPH; no distinct peak of IR-hβEND was observed. In patients with ACTH/LPH hypersecretion due to Addison's disease, Nelson's syndrome, or ectopic ACTH syndrome, IR-hγLPH and IR-hβEND/hβLPH were both elevated, and IR-hβEND/hβLPH eluted as two peaks, one coeluting with hβLPH and the other with hβEND. The molar concentrations of all three peptides were significantly correlated with one another. The lower concentrations of endogenous IR-hβEND observed may be due in part to its apparent shorter plasma half-life, as estimated in an Addison's patient given a cortisol infusion. The biologic significance of these three peptides in circulating blood is still unknown. The increased levels of hβLPH and hγLPH in plasma of patients with chronic renal failure suggest that the kidney may be an important organ for their metabolism.

A monoclonal antibody (anti-B1) specific for a unique B cell surface differentiation antigen was used to characterize the malignant cells from patients with leukemias or lymphomas. All tumor cells from patients with lymphomas or chronic lymphocytic leukemias, bearing either monoclonal kappa lambda light chain, expressed the B1 antigen. In contrast, tumor cells from T cell leukemias and lymphomas or acute myeloblastic leukemia were unreactive. Approximately 50% of acute lymphoblastic leukemias (ALL) of non-T origin and 50% of chronic myelocytic leukemia in blast crisis were also anti-B1 reactive. moreover, 21 of 28 patients with the common ALL antigen (CALLA) positive form of ALL were anti-B1 positive, whereas 0 of 13 patients with CALLA negative ALL were reactive. These observations demonstrate that an antigen present on normal B cells is expressed on the vast majority of B cell lymphomas and on approximately 75% of CALLA positive ALL, suggesting that these tumors may share a common B cell lineage.

A sensitive and precise competitive-displacement double-antibody radioimmunoassay was developed for the human plasma enzyme lecithin-cholesterol acyltransferase (LCAT; Ec 2.3 1.43). The ability of plasma from various animal species to displace labeled human LCAT from goat anti-human LCAT could be ranked in the following order: man and sheep > nonhuman primates > cat or dog > pig > rabbit or guinea pig > mouse > rat. Normolipidemic subjects had levels of LCAT of 6.14 +/- 0.98 micrograms/ml (mean +/- SD, n = 66). Subjects with dysbeta-lipoproteinemia had the highest plasma LCAT levels (7.88 +/- 0.39 micrograms/ml, n = 7, P < 0.05), followed by hypercholesterolemic subjects (7.00 +/- 1.30, n = 41) and hypertriglyceridemic subjects (6.96 +/- 1.3, n = 10). LCAT-deficient subjects had the lowest enzyme levels (0.89, 0.83, and 0.05 micrograms/ml, respectively, and two subjects with no detectable enzyme). Males had lower LCAT levels (6.42 +/- 1.05 micrograms/ml, n = 90, for all subjects; 5.99 +/- 1.03, n = 44, for normolipidemics) than females (7.01 +/- 1.14, n = 34, for all subjects P < 0.01; 6.44 +/- 0.79, n = 22, for normolipidemics, P < 0.01). LCAT levels correlated significantly with total cholesterol (males, r = 0.384, P < 0.001; females, r = 0.519, P < 0.002); and total triglyceride (only in females, r = 0.512, P < 0.002). LCAT levels in females correlated inversely with HDL cholesterol (r = 0.341, P < 0.05) and apoprotein D (r = 0.443, P < 0.02), but no such relationship existed in males.

I n this study we present evidence that in human erythrocytes NADH-cytochrome b5 reductase (methemoglobin reductase) is not only soluble but also tightly bound to the membrane. The membrane methemoglobin reductase-like activity is unmasked by Triton X-100 treatment, and represents about half of the total activity in the erythrocytes. Like the amphiphilic microsomal-bound cytochrome b5 reductase, the erythrocyte membrane-bound enzyme is solubilized by cathepsin D. Because this treatment is effective on unsealed ghosts but not on resealed (inside-in) ghosts, it is concluded that the enzyme is strongly bound to the inner face of the membrane. The erythrocyte membrane enzyme is antigenically similar to the soluble enzyme. The two forms of enzyme are specified by the same gene, in that both were found defective in six patients with recessive congenital methemoglobinemia. We suggest that the cytochrome b5 reductase of the erythrocyte membrane is the primary gene product. A posttranslational partial proteolysis probably gives rise to the soluble form of the enzyme, which serves as a methemoglobin reductase.

W e determine the effects of alfalfa top saponins on cholesterol and bile acid balance in eight cynomolgus macaques (Macaca fascicularis). The monkeys ate semipurified food containing cholesterol with or without added saponins. The saponins decreased cholesterolemia without changing the levels of high density lipoprotein-cholesterol; hence, they reduced the total cholesterol/high density lipoprotein-cholesterol ratio. Furthermore, they decreased intestinal absorption of cholesterol, increased fecal excretion of endogenous and exogenous neutral steroids and bile acids, and decreased the percent distribution of fecal deoxycholic and lithocholic acids. The fecal excretion of fat was also slightly increased, but steatorrhea did not occur. We saw no signs of toxicity in the monkeys after 6 or 8 wk of saponin ingestion. The data suggest that alfalfa top saponins may be of use in the treatment of patients with hypercholesterolemia, but long-term studies on possible toxicity are needed before this therapy can be recommended for humans.

R ecent work indicates that subpopulations of human fecal bacteria, averaging ∼1% of the total viable fecal flora, degrade the oligosaccharide side chains of hog gastric mucin, which structurally resembles human epithelial mucins. Here we report studies to determine whether degradation of mucin oligosaccharides is related to glycosidase production by bacteria growing in anaerobic fecal cultures. Triplicate cultures containing hog gastric mucin were inoculated with serially diluted feces from each of seven healthy subjects. When the stationary growth phase was attained, mucin oligosaccharide degradation and both cell-bound and extracellular activities of four glycosidases were measured in each culture. Cell-bound β-d-galactosidase, β-N-acetylglucosaminidase, and sialidase were present in bacteria growing at all levels of fecal inocula, including 10⁻¹¹ g. In contrast, extracellular activities were present in every culture inoculated with 10⁻⁴−10⁻⁷ g feces, but were diminished or absent in cultures inoculated with 10⁻⁸−10⁻¹¹ g feces. Bacterial autolysis was an unlikely cause of extracellular glycosidase activity, since p-nitrophenyl-α-l-fucosidase remained cell bound in cultures at every level of fecal inoculum. Degradation of mucin oligosaccharides was associated with extracellular, but not with cell-bound β-d-galactosidase, β-N-acetylglucosaminidase, and sialidase. Among the seven subjects, the estimated most probable numbers (MPN) of fecal bacteria producing extracellular β-d-galactosidase, β-N-acetylglucosaminidase, and sialidase ranged from 10⁶−10¹⁰/g dry fecal wt, were comparable to the MPN of mucin-degrading bacteria, and were significantly smaller than the MPN of total fecal bacteria.We interpret these findings as evidence for the existence of bacterial subpopulations in the normal fecal flora that produce extracellular glycosidases, and that these subpopulations have a major role in degrading the complex oligosaccharides of mucin in the gut lumen.

H uman peripheral blood monocytes attached to Candida albicans hyphae in the absence of serum and damaged the hyphae without completely ingesting them. Attachment and damage was not augmented by the addition of serum. Damage to hyphae was quantitated by a previously developed metabolic assay that measured leukocyte-induced reduction in uptake of [¹⁴C]cytosine by the hyphae. Use of cells from patients with hereditary disorders of leukocyte function, chronic granulomatous disease, and myeloperoxidase deficiency indicated that myeloperoxidase-independent and nonoxidative mechanisms could sometimes damage hyphae where oxidative mechanisms were impaired. Damage to hyphae by normal monocytes was inhibited by concentrations of sodium azide and sodium cyanide that primarily affect myeloperoxidase activity, as well as by halide-free conditions, catalase, and putative antagonists of hypochlorous acid or singlet oxygen. Iodination of hyphae, a myeloperoxidase and hydrogen peroxide-dependent process of monocytes, was similarly inhibited by sodium azide, sodium cyanide, and catalase. Under anaerobic conditions, damage to hyphae was reduced by 64.0-68.4%. In contrast, inhibitors of potential nonoxidative antifungal mechanisms, iron salts to saturate iron chelators, and polyanionic amino acid polymers to neutralize cationic proteins did not block damage to hyphae by monocytes. Preparations rich in lysosomal granules from fractionated normal monocytes also did not damage hyphae. Overall, it appeared that oxidative mechanisms were most important for damage to hyphae by normal monocytes.Electron microscopy confirmed that Candida hyphae were damaged and probably killed by monocytes, but monocytes appeared to sustain significant damage in the process. In the absence of serum, monocyte cell membranes became closely approximated to Candida cell walls. It appeared that some Candida could escape this partial engulfment, as they were seen floating free with vesicular trilaminar membrane remnants covering hyphal surfaces. In general, monocytes appeared to be damaged by interactions with Candida hyphae more than neutrophils had been in previous studies.

W e investigated the ability of malaria-infected and normal mice to clear particulate immune complexes consisting of autologous erythrocytes sensitized with either IgG or complement.Normal mice rapidly clear autologous erythrocytes optimally sensitized with IgG and the liver plays a major role in their sequestration. Clearance of optimally sensitized erythrocytes is complement-dependent because cobra venom factor-treated, normal mice failed to clear these cells rapidly, unless they had been pre-treated with fresh mouse serum.In the initial phase of Plasmodium berghei infection, clearance of the optimally sensitized erythrocytes was markedly increased over that observed in normal mice. 2 wk after infection, however, clearance was minimal. This defect was most likely the consequence of the hypocomplementemia observed at this stage of infection since sensitized erythrocytes were removed rapidly from the blood if they had been coated with C3 in vitro before injection into malarial mice.The functional activity of the complement receptors of phagocytic cells was assayed in malarial mice by the injection of autologous erythrocytes coated with C3 and C4 in the absence of antibody. The complement-coated erythrocytes were rapidly removed from the blood, accumulated in the liver, and then gradually returned to the circulation. Similar patterns were observed in normal animals, but the degree of clearance was considerably higher in the malarial mice late in infection. It appears, therefore, that complement receptors remain functional throughout the infection.Erythrocytes suboptimally sensitized with IgG were cleared minimally by normal mice. This clearance was not complement-dependent and was mediated mainly by the spleen. During malaria, clearance of these particles was initially enhanced but later it was abolished.The association of hypocomplementemia with a major splenic defect in clearance late in malaria infection may explain the accumulation of immune complexes in pathological sites observed in this disease.

T o determine whether hydralazine, a systemic vasodilator, exerted a similar effect on the pulmonary circulation, we studied the circulatory changes in dogs during three interventions: (a) the control state during room air ventilation; (b) during continuous hypoxic ventilation with 10% oxygen, and maintaining continuous hypoxic ventilation; and (c) after 1 mg/kg hydralazine intravenously.Ventilation with 10% oxygen caused the mean pulmonary artery pressure to increase from 10±1.2 to 23±2.4 mm Hg (P < 0.01) and the pulmonary arteriolar resistance to increase from 1.51±0.19 to 5.87±1.10 U (P < 0.01). Hydralazine significantly lowered the pulmonary artery pressure (23.0±2.4 to 14.3±1.5 mm Hg, P < 0.01) and the pulmonary arteriolar resistance (5.87±1.10 to 2.87±0.52 U, P < 0.01). Femoral artery pressure, pulmonary artery wedge pressure, heart rate, and cardiac output remained unchanged throughout.To ascertain the contribution of the prostaglandin system to the pulmonary vasodilator effects of hydralazine, we pretreated a group of dogs with the prostaglandin synthetase inhibitor, indomethacin, 5 mg/kg s.c., twice daily for 2 d. These animals then underwent identical studies.The pretreated dogs had comparable base-line and hypoxia hemodynamic data. However, hydralazine had no effect on pulmonary artery pressure (23.3±1.6 vs. 21.7±2.3 mm Hg, NS) or pulmonary arteriolar resistance (8.03±1.09 vs. 7.14±1.42, NS) during continuous hypoxic ventilation in the indomethacin-pretreated group. Pretreatment with indomethacin did not, however, block the pulmonary vasodilator effects of intravenous prostacyclin (PGI₂). Pretreatment with meclofenamate, a cyclo-oxygenase inhibitor structurally unrelated to indomethacin, also blocked the effects of hydralazine during hypoxic ventilation. These data suggest that hydralazine exerts a pulmonary vasodilatory effect during hypoxia-induced pulmonary vasoconstriction, and that this vasodilator effect may be mediated by prostaglandins.

C hronic hypoxic lung diseases are associated with abnormal blood pressure regulation. Because the lung is the principal site of angiotensin conversion and because hypoxia decreases converting enzyme activity, we examined whether angiotensin converting enzyme activity was impaired in lung disease. 12 dogs received a 6 wk course of aerosolized and intratracheal papain that produced moderate panlobular emphysema. These dogs and 24 control dogs were anesthetized and sampling catheters were placed under fluoroscopic control. Angiotensin conversion was measured by a blood pressure response bioassay. Pulmonary converting enzyme activity was also assessed by infusing bradykinin (BK) and using radioimmunoassay to measure the instantaneous clearance of BK and the concentration of BK in the pulmonary artery which first produced spillover of BK into left atrial blood. Angiotensin conversion was reduced in the emphysematous dogs to 81.1% (13.2 SD) from 92% (6 SD) in the control dogs (P < 0.01). Instantaneous clearance of BK in the emphysematous dogs was only slightly reduced (93%), despite reduction in their Pao₂ to 75 mm Hg, indicating that the greatest proportion of the perfused vascular bed was exposed to alveolar Po₂ of >90 mm Hg. However, the barrier to BK passage provided by the lung, and measured by the spillover level, was reduced ¼ to ½ that observed in control animals. That the defect was promptly corrected by supplemental oxygen indicates that regional pulmonary vascular converting enzyme activity had been impaired by regional alveolar hypoxia, which permitted some peptide to pass through the lungs unmetabolized. Determination of peptide metabolism in the lungs may provide a useful measure of regional alveolar hypoxia and may lead to new ways of assessing lung injury.

T he activity of prolylcarboxypeptidase (PCP), or angiotensinase C, was measured in lung tissues, leukocytes, and cultured human cells using Cbz-Pro-[14C]Ala as a substrate. A lysosomal fraction of homogenized rat or human lung contained most of the PCP activity in that tissue. Polymorphonuclear neutrophils, macrophages, and lymphocytes isolated from human blood had PCP activity. Fibroblasts cultured from human tissues had the highest activity (0.56-1.15 mumol/h per 10(6) cells), more than endothelial cells cultured from human pulmonary arteries. PCP of cultured human fibroblasts was similar to the human renal enzyme because it was resistant to moderate heating and was not inhibited by p-chloromercuriphenyl sulfonic acid. These properties and the substrate specificity distinguish PCP from cathepsin A, which is also in fibroblasts. Antibody to human renal PCP reacted with fibroblast PCP in immunofluorescence, indicating common antigenic determinants. Hydrocortisone changed PCP activity in fibroblasts in parallel with changes in beta-glucuronidase activity and cell-protein concentration; the activity was depressed at low concentration of the hormone. PCP activity was also found in synovial fluid from arthritic joints and in fibroblasts from the synovium. That PCP is found in both inflammatory exudates and in cells that appear at sites of inflammation indicates that, in addition to inactivating angiotensins, this enzyme may have a role in inflammation.

H uman breast cyst fluids were shown to contain low concentrations of IgA (15-78 micrograms/ml) and IgG (33-145 micrograms/ml). The IgA:IgG ratios in individual breast cyst fluids ranged from 1:0.6 to 1:4. These levels are considerably higher than their ratio in serum (1:7). IgA from 33% of the 40 fluids examined, and IgG from 10% of the fluids, reacted with the murine mammary tumor virus (MuMTV). The reactivity was detected by an enzyme-linked immunosorbent assay that measures antibody binding to both the envelope glycoprotein and core protein of the virus. In a second series of experiments. IgA from 28% of 40 breast cyst fluids reacted only with MuMTV while IgA from 30% of the fluids was reactive with both MuMTV and the Rauscher murine leukemia virus. Antigen reactive with antiserum to the 28,000-dalton MuMTV core protein (p28), was also identified in a 165,000-g pellet fraction from breast cyst fluids. In individual fluids, the extent of IgA binding to MuMTV was positively correlated (P less than or equal to 0.01) with the binding of anti-p28 antibody to the pellet of the breast cyst fluid. Fractions with the buoyant density of retroviruses (1.16-1.18 g/ml) or their cores (1.21-1.25 g/ml) were isolated from breast cyst fluids. These fractions contained a DNA polymerase capable of utilizing the reverse transcriptase-specific template, dG12-18 x poly rCm. In addition, they reacted with antiserum to MuMTV p 28 but not with antiserum to the 30,000-dalton Rauscher murine leukemia virus core protein.

T he ability of heparin glycosaminoglycan to prevent formation of the properdin-stabilized amplification C3 convertase is independent of antithrombin binding activity and requires substitution of the amino sugar and a degree of oxygen (O)-sulfation which could be on the uronic acid or the amino sugar. Preparations of heparin glycosaminoglycan isolated by different techniques from different species (rat, human, and porcine) exhibited an equivalent capacity to inhibit generation of the amplification C3 convertase. Hyaluronic acid, which is devoid of O-sulfation, had no inhibitory activity; chondroitin 4-sulfate of rat and whale origins, chondroitin 6-sulfate of rat and shark origins, and dermatan sulfate from porcine skin are O-sulfated on the galactosamine and had minimal activity. Porcine heparin glycosaminoglycan, isolated on the basis of affinity for antithrombin III, had no greater anticomplementary activity than porcine glycosaminoglycan, which failed to bind antithrombin III and had essentially no anticoagulant activity. Nitrogen (N)-desulfation of porcine heparin reduced anticomplementary activity to the level of the other sulfated mucopolysaccharides, and both N-resulfation and N-acetylation restored the original activities, thereby indicating a requirement for N-substitution, but not N-sulfation. N-resulfation of N-desulfated and O-desulfated heparin did not restore any activity, thus indicating that O-sulfation and N-substitution represent independent, critical structural requirements for the anticomplementary activity of heparin glycosaminoglycan. Inasmuch as N-desulfated-N-acetylated heparin had no anticoagulant activity, the nature of the N-substitution completely distinguishes the plasma-protein effector pathway that is inhibited.

T o determine whether renal prostaglandins participate in the regulation of renal blood flow during acute reduction of cardiac output, cardiac venous return was decreased in 17 anesthetized dogs by inflating a balloon placed in the thoracic inferior vena cava. This maneuver decreased cardiac output from 3.69±0.09 liters/min (mean±SEM) to 2.15±0.19 liters/min (P < 0.01) and the mean arterial blood pressure from 132±4 to 111±5 mm Hg (P < 0.01) and increased total peripheral vascular resistance from 37.6±2.5 to 57.9±4.8 arbitrary resistance units (RU) (P < 0.01). In marked contrast, only slight and insignificant decreases in the renal blood flow from 224±16 to 203±19 ml/min and renal vascular resistance from 0.66±0.06 to 0.61±0.05 arbitrary resistance units (ru) were observed during inflation of the balloon. Concomitant with these hemodynamic changes, plasma renin activity and plasma norepinephrine concentration increased significantly in both the arterial and renal venous bloods. Plasma concentration of prostaglandin E₂ in renal venous blood increased from 34±6 to 129±24 pg/ml (P < 0.01). The subsequent administration of indomethacin or meclofenamate had no significant effect on mean arterial pressure, cardiac output, and total peripheral vascular resistance, but reduced renal blood flow from 203±19 to 156±21 ml/min (P < 0.01) and increased renal vascular resistance from 0.61±0.05 to 1.05±0.21 ru (P < 0.01). Simultaneously, the plasma concentration of prostaglandin E₂ in renal venous blood fell from 129±24 to 19±3 pg/ml (P < 0.01). Administration of indomethacin to five dogs without prior obstruction of the inferior vena cava had no effect upon renal blood flow or renal vascular resistance. The results indicate that acute reduction of cardiac output enhances renal renin secretion and the activity of the renal adrenergic nerves as well as renal prostaglandin synthesis without significantly changing renal blood flow or renal vascular resistance. Inhibition of prostaglandin synthesis during acute reduction of cardiac output results in an increased renal vascular resistance and reduced renal blood flow. Accordingly, that data provide evidence that renal prostaglandins counteract in the kidney the vasoconstrictor mechanisms activated during acute reduction of cardiac output.

A lveolar hypoxia causes pulmonary vasoconstriction; we investigated whether hypoxia could also impair pulmonary vasodilation. We found in the isolated perfused rat lung a delay in vasodilation following agonist-induced vasoconstriction. The delay was not due to erythrocyte or plasma factors, or to alterations in base-line lung perfusion pressure. Pretreating lungs with arachidonic acid abolished hypoxic vasoconstriction, but did not influence the hypoxia-induced impairment of vasodilation after angiotensin II, bradykinin, or serotonin pressor responses. Progressive slowing of vasodilation followed angiotensin II-induced constriction as the lung oxygen tension fell progressively below 60 Torr. KCl, which is not metabolized by the lung, caused vasoconstriction; the subsequent vasodilation time was delayed during hypoxia. However, catecholamine depletion in the lungs abolished this hypoxic vasodilation delay after KCl-induced vasoconstriction. In lungs from high altitude rats, the hypoxia-induced vasodilation impairment after an angiotensin II pressor response was markedly less than it was in lungs from low altitude rats. We conclude from these studies that (a) hypoxia impairs vasodilation of rat lung vessels following constriction induced by angiotensin II, serotonin, bradykinin, or KCl, (b) hypoxia slows vasodilation following KCl-induced vasoconstriction probably by altering lung handling of norepinephrine, (c) the effect of hypoxia on vasodilation is not dependent on its constricting effect on lung vessels, (d) high altitude acclimation moderates the effect of acute hypoxia on vasodilation, and (e) the hypoxic impairment of vasodilation is possibly the result of an altered rate of dissociation of agonists from their membrane receptors on the vascular smooth muscle.

N ormal and antibiotic-pretreated staphylococci were incubated with human neutrophils to determine the interactions between cells and antimicrobials in the killing of the organisms. Staphylococcus aureus 502A pretreated during log-phase growth with subinhibitory (¼ minimum inhibiting concentration) (MIC) concentrations of penicillin G were more susceptible to killing by normal neutrophils than untreated bacteria (intracellular survival 0.17±0.04 vs. 1.5±0.38%, mean±SEM, respectively, at 35 min in 14 experiments; P < 0.01 by t test). Furthermore, this enhanced susceptibility to killing was observed even when phagosome formation was inhibited by cytochalasin B (65.6±4.6% pencillintreated vs. 30.5±4.5% untreated killed at 30 min in 14 experiments, P < 0.001). Pretreatment of S. aureus with vancomycin similarly enhanced susceptibility to killing by cytochalasin B-treated polymorphonuclear leukocytes (PMN), whereas pretreatment with gentamicin did not.The enchancement of killing by pretreatment with cell wall-active antibiotics was present in a dose-response fashion to 1/16th the MIC. It required specific antimicrobial activity; i.e., penicillin activity was inhibited by penicillinase or by incubation with bacteria at 4°C. It also required active cellular metabolism and intact neutrophils. For antibiotic-pretreated bacteria to be killed by normal and cytochalasin B-treated cells, phagocytosis or binding to the cells was essential via a serum opsonindependent mechanism. In experiments with the cytochalasin B-treated cells, all bound penicillin-treated bacteria were killed vs. only a fraction (70%) of the bound untreated bacteria. Penicillin in 10 times the MIC had no direct effects on PMN phagocytic, metabolic, or microbicidal functions against a nonsusceptible organism, Candida albicans.The results indicate a cooperative effect between cell wall-active antibiotics at low concentrations and human PMN in the killing of staphylococci. The model establishes conditions for the study of the mechanisms involved in the cooperation of these bactericidal systems.

T he inherited structural polymorphism in the fourth component of complement was studied in the family of a child with homozygous deficiency of this protein. It was shown that a number of family members, including the child's parents, carried a C4 haplotype, C4A*QO C4B*QO, that produced no detectable protein at either the Chido (C4B) or Rodgers (C4A) locus. The family contained individuals with one, two, three, or four expressed C4 genes, and the mean serum C4 levels in such individuals roughly reflected the number of structural genes.

W e studied the effects of continuous negative external chest pressure (CNECP) produced by a cuirass appliance on lung water and protein transport in sheep with chronic lung lymphatic fistulas. We compared data obtained during periods of mechanical ventilation (base line) to period of CNECP, using identical ventilatory support. Three groups were studied: six sheep were studied before and after application of CNECP for 1 h (control) and again after induction of a pulmonary vascular permeability defect (PVPD) by infusing live Pseudomonas bacteria (group I); another six sheep were studied under control conditions before and after prolonged application of CNECP for over 4 h (group II); 10 sheep were studied 24 h after a Pseudomonas infusion (PVPD), before and after 4 h of CNECP (group III).Compared to base-line data, CNECP produced significant increases in functional residual capacity and decreases in pulmonary blood volume, pulmonary artery pressure, and left atrial pressure in all groups. Lung lymph flow (Q_L) was unchanged during the 1st h of CNECP and therefore was not significantly different from base line in group I, but after prolonged CNECP a steady-state decrease in Q_L as well as a decrease in lung albumin transport was found in groups II and III (P < 0.05 by paired t test). We postulate that decreased pleural pressure during CNECP produces decreased hydrostatic filtration pressures, thereby decreasing Q_L. Reduction of both convective forces as well as microvascular surface area available for solute exchange account for decreases in lung albumin transport. Histologic examination and gravimetric studies on four sheep failed to demonstrate increase in lung water accumulation after 4 h of CNECP. We conclude that CNECP mechanically reversed the PVPD in our group III sheep. These physiologic effects of CNECP may be of benefit in the management of patients with adult respiratory distress syndrome.

P lasma 25-hydroxyvitamin D and 24, 25-dihydroxy-vitamin D [24,25-(OH)₂D] concentrations were measured in normal and chronically dialyzed anephric humans and pigs. Measurement of the 24, 25-(OH)₂D was preceded by three purification steps involving one Sephadex LH-20 column and two high-pressure liquid chromatographic columns. The final high-pressure liquid chromatography step involved resolution of 25-hydroxy-vitamin D₃-26,23 lactone and 25,26-dihydroxy-vitamin D₂ from 24,25-dihydroxyvitamin D₂ and 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂D₃]. The total 25-hydroxyvitamin D [25-hydroxyvitamin D₂ plus 25-hydroxyvitamin D₃ (25-OHD₃)] was 31.7±3.6 ng/ml in the plasma of eight anephric human subjects and 40.1±3.7 ng/ml in five normal human subjects. Six of the eight anephric patients had undetectable (<0.2 ng/ml) 24,25-(OH)₂D concentrations. Two of the eight patients had very low (0.51 and 0.41 ng/ml), but detectable, 24,25-dihydroxyvitamin D₂. The normal human volunteers had plasma 24,25-(OH)₂D concentrations of 2.8±0.7 ng/ml. Chronically dialyzed anephric and normal pigs were given intramuscular injections of massive amounts (5 × 10⁶ IU) of vitamin D₃ immediately after surgery (day 0) and again on day 7. In anephric pigs, plasma 25-OHD₃ progressively rose from 12±4 ng/ml on day 0 to 705±62 ng/ml on day 10. The 25-OHD₃ concentrations in normal pigs rose from 8±2 ng/ml on day 0 to 439±64 ng/ml on day 10. Plasma 25-OHD₃ was higher in anephrics throughout the experiment, and concentrations were significantly higher (P < 0.05) on days 9 and 10. Plasma 24,25-(OH)₂D₃ concentrations declined progressively in anephric pigs from 3.6±0.6 ng/ml on day 0 to 3.2±0.7 ng/ml on day 2. During days 4-10, plasma 24,25-(OH)₂D₃ was not apparent until plasma 25-OHD₃ was >400 ng/ml. In control pigs, plasma 24,25-(OH)₂D₃ was elevated from 4.3±0.6 ng/ml on day 0 to 178±2.7 ng/ml on day 3. Plasma 24,25-(OH)₂D₃ was significantly higher (P < 0.05) in controls on days 1-8. At the end of the experiment (day 10), 24,25-(OH)₂D₃ concentrations were similar and not significantly different in both groups (87.0±18.4 ng/ml in anephric and 110.3±32.1 ng/ml in normal pigs). The identity of the 24,25-(OH)₂D₃ isolated from anephric pig plasma was confirmed by mass spectroscopy. Our data suggest that anephric humans receiving normal dietary levels of vitamin D₃ have little or no ability to produce 24,25-(OH)₂D. However, we have shown that pigs produce 24,25-(OH)₂D₃ when plasma 25-OHD₃ is extremely high (>400 ng/ml).

P roteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin.

T he purpose of the present study was to evaluate the significance of immunogenetic factors on the survival of pancreatic allografts in beagle dogs. Donors and recipients were leukocyte antigen (DLA)-typed and mixed lymphocyte culture (MLC)-tested. Recipients were made diabetic by total pancreatectomy and immediately implanted intraperitoneally with a vascularized, free-draining (duct unligated) pancreatic segmental (FDPS) allograft. Two groups of dogs were studied. In group I consisting of donor-recipient littermates, recipients were immunosuppressed with prednisone and azathioprine (n = 16 dogs), or not immunosuppressed (n = 4). In group II, recipients were made specifically unresponsive by total body radiation, autologous marrow implantation, and kidney transplantation from DLA-MLC identical donors, 1 yr before FDPS transplantation from the corresponding original kidney donors.Survival of the FDPS grafts in group I was inversely related to pretransplant MLC reactivity, irrespective of DLA genotyped match between donor and recipient. Thus, immunosuppressed high MLC reactors (n = 8) rejected FDPS grafts between 7 and 14 d, whereas immunosuppressed low MLC reactors (n = 8) accepted grafts for 25 to 260+ days, and nonimmunosuppressed low MLC reactors (n = 4) accepted grafts for 9-55 d. Rejection (hyperglycemia) of FDPS grafts was sudden, permanent, and unpredictable despite weekly intravenous glucose tolerance tests with measurements of glucose disappearance rates and serum insulin responses. Nevertheless, serial in vitro cell-mediated lymphocytotoxicity (CML) assays revealed increases in CML before graft rejection in low MLC reactors, and decreases in both CML and MLC responses before graft rejection in high MLC reactors. FDPS graft survival was indefinite (>6 mo) in group II dogs, despite low-grade MLC reactivity (2:4 dogs) and CML responses (4:4 dogs). Biopsies of FDPS grafts at 6 mo in normoglycemic dogs showed disappearance of exocrine tissue and coalescence of islets in both groups I and II, but with less fibrosis in group I (immunosuppressed).These results indicate that (a) pancreatic islets in vascularized grafts (FDPS) may survive indefinitely in the presence of a good tissue match best predicted by MLC testing, (b) tissue specific histocompatibility factors appear to be common enough between kidney and pancreas to allow for long-term survival of both organs transplanted from the same donor, at least in appropriate recipients (group II), and (c) immunosuppression is associated with less fibrosis in FDPS allografts.

I n previous studies subjects with familial polyposis, the autosomal dominant disease leading to colon cancer, excreted higher levels of fecal cholesterol than normal subjects, with decreased conversion to degradation products. Findings suggested fecal cholesterol degradation as a marker of hereditary predisposition to colon cancer. Current measurements now have shown that affected individuals and asymptomatic progeny in a second population group with inherited predisposition to colon cancer are low converters of fecal cholesterol. The latter group consisted of highly colon cancer prone families without polyposis, in which patterns of inheritance similar to the autosomal dominant pattern of familial polyposis were observed. 24-h stool collections were obtained from 72 subjects who consumed mixed western diets. Mean percent degradation of fecal cholesterol to coprostanol, coprostanone, cholestanol, and cholestanone revealed significant decreases in fecal cholesterol conversion in affected and asymptomatic subjects in colon cancer prone families without polyposis (P < 0.001) compared to controls. This is in addition to those with familial polyposis (P < 0.001), and extends this marker of colon cancer susceptibility to a second population group with hereditary predisposition to colonic neoplasia.

L ymphocytes obtained from the blood of normal individuals and six patients with newly diagnosed multiple myeloma were separated into T and non-T cell populations by rosette-formation with sheep erythrocytes, and were then assayed for the presence of surface membrane Fc receptors. When compared with normal individuals, four patients with IgG myeloma had a three- to fourfold increase in T cells with IgG receptors (T gamma cells) and two patients with IgA myeloma had a two- to threefold increase in T cells with IgA receptors (T alpha cells). Patients with IgG or IgA myeloma had normal numbers of non-T lymphocytes with surface receptors for IgG and IgA, respectively. The finding that human myeloma is accompanied by elevated numbers of T cells with Fc receptors for the heavy chain class of the myeloma protein: (1) may account for the apparent "monoclonal" lymphocyte population in patients with myeloma; (b) extends to humans similar observations made in mice with secretory plasmacytomas; and (c) is of interest because T cells with Fc receptors are immunoregulatory lymphocytes.

W e have identified a clinically distinct subset of lupus erythematosus patients marked by the presence of a histologically proven, nonscarring variety of cutaneous LE (subacute cutaneous LE) in which there is a very high frequency of the human leukocyte antigens (HLA) B8 and DR3. Differences in the configuration of their skin lesions allowed a separation of the patients into two clinical subgroups; annular and papulosquamous. HLA-B8 was increased in the annular subgroup (81%, corrected P (Pc) < 0.007) and combined group (65%, Pc < 0.004). HLA-DR3 was present in all 11 of the annular patients (10%, Pc < 0.00008). In addition, HLA-DR3 was present in increased frequencies in the papulosquamous subgroup (60%, Pc < 0.04) and combined group (77%, Pc < 0.00008). Thus, HLA-DR3 positive individuals have a relative risk of 10.8 for developing subacute cutaneous LE of either type and an even greater relative risk (67.1) for the annular variety. The HLA phenotype A1, B8, DR3 was also found more commonly in the annular (73%, P < 0.00008) and combined patient groups (46%, P < 0.004). These HLA associations, which are stronger than ever before reported for any form of LE, did not result from the concurrent presence of subclinical Sjögren's syndrome. Thus, subacute cutaneous LE can now be added to the growing list of HLA-B8, DR3-associated diseases that have autoimmune features.