The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum.
K Tanimoto, N R Cooper, J S Johnson, J H Vaughan
There are many examples of two penicillins acting synergistically, usually by one competitively inhibiting beta-lactamase, thus protecting the other from inactivation. There are few reports on penicillins antagonizing each other. Eight strains of three genera (Proteus, Escherichia, Pseudomonas) isolated at Boston City Hospital or Institut Pasteur, Paris, showed antagonism of carbenicillin or ampicillin by cephaloridine, cloxacillin, or 6-aminopenicillanic acid. Broth dilution tests showed that with seven of the eight strains the minimum inhibiting concentration (MIC) of the more active antibiotic was increased by 800-6,400% by low concentrations (often one-tenth the MIC) of the antagonist, whereas higher concentrations of "antagonist" were not as antagonistic, This suggested that two or more receptor sites of action for penicillins were present; the antagonist thus blocks the antibacterial action at the more sensitive site but acts additively with the antagonized antibiotic at the less sensitive site. The possibility that the mechanism of antagonism was induction of inactivating enzymes (beta-lactamase, penicillin acylase) was studied in two strains(one Escherichia coli and one Proteus rettgeri), and two antagonists were studied in detail. With E. coli cephaloridine was a poorer inducer of beta-lactamase than were the antagonized antibiotic and 6-aminopenicillanic acid. From these organisms, the good inducers of a beta-lactamase that acted on benzylpenicillin did not induce enzymes that inactivated carbenicillin. Thus, the mechanism of antagonism was not due to beta-lactamase induction.
J F Acar, L D Sabath, P A Ruch
We have used a continuous intravenous infusion of glucose (6 mg/kg/min), insulin (80 mU/min), epinephrine (6 mug/min), and propranolol (0.08 mg/min) to directly assess insulin resistance in 14 untreated adult onset diabetics with a mean (plus or minus SE) fasting plasma glucose level of 217 plus or minus 17 mg/100 ml. During the infusion endogenous insulin secretion is inhibited and steady-state plasma glucose and insulin levels are achieved after 90 min. Since similar steady-state levels of plasma insulin are achieved in all subjects, the plasma glucose concentration observed during the steady-state period is a measure of an individual's insulin resistance. Under these conditions, the mean (plus or minus SE) steady-state plasma glucose level of the 14 diabetic patients was 350 plus or minus 16 mg/100 ml, while that of 12 normal subjects was 121 plus or minus 4 mg/100 ml. Additional studies were performed in which control subjects and patients with diabetes had their fasting plasma glucose levels acutely raised or lowered to comparable levels before receiving the basic infusion mixture of glucose, insulin, epinephrine, and propranolol. The results of these studies indicated that differences in initial plasma glucose levels could not account for the different glucose responses of the two groups to the basic infusion. Finally, the mean (plus or minus SE) steady-state plasma glucose level of 104 plus or minus 17 mg/100 ml observed during the same basic infusion in five patients with fasting hyperglycemia (mean plus or minus SE, 142 plus or minus 12 mg/100 ml) secondary to chronic pancreatitis suggested that neither chronic hyperglycemia nor hypoinsulinemia per se necessarily lead to insulin resistance. These results demonstrate that marked insulin resistance exists in adult onset diabetics with fasting hyperglycemia. Since previous studies have documented the presence of insulin resistance in patients with chemical diabetes, the possibility exists that insulin resistance may be characteristic of adult onset diabetes mellitus.
H Ginsberg, G Kimmerling, J M Olefsky, G M Reaven
By use of a recently described method, which estimates the rate of gastric acid secretion by measuring the rate of sodium bicarbonate infusion needed to keep intragastric pH constant, gastric acid secretion rates and changes in serum gastrin were measured in five normal subjects while gastric pH was kept at 5.5, 4.0, 3.0, or 2.5. Preliminary experiments revealed that the method did not accurately measure acid secretion at a pH lower than 2.5. Stimulation of acid secretion was produced by gastric instillation of a solution of amino acids and cornstarch. The secretion rate with the amino acid meal was highest at pH 5.5 and was 60% of that produced by a steak meal at the same pH. As the pH of the amino acid meal was decreased, there was a stepwise reduction in acid secretion so that at pH 2.5 the rate was only half as great as at pH 5.5. The amino acid meal produced increases in serum gastrin that were also less marked than those produced by a steak meal. With amino acid stimulation, serum gastrin responses were similar at pH 5.5, 4.0, and 3.0, but no increase in gastrin could be measured when the meal was maintained at pH 2.5. A group of six patients with duodenal ulcers was compared with seven normal subjects at pH 5.5 and 2.5. Ulcer patients released more gastrin and secreted more acid at each time period at both pH values. More important, the degree of inhibition at pH 2.5 was significantly less in ulcer patients. For example, during the 2nd h after stimulation acid secretion was inhibited by only 30% in ulcer patients compared with 70% in normal subjects. These findings suggest a defect in autoregulation of gastrin release and gastric acid secretion at low pH in ulcer patients which may play a role in pathogenesis of this disease.
J H Walsh, C T Richardson, J S Fordtran
Family members from four generations were found to have polycythemia and increased whole blood O2 affinity (P50; 11 mm Hg; normal, 27 mm Hg). No abnormal hemoglobin bands were seen after electrophoresis on starch gel at pH 8.6 or agar gel at pH 6.0. Analysis of the oxygenated hemolysate by isoelectric focusing on polyacrylamide gel revealed two closely spaced bands. When deoxygenated hemolysate was analyzed in oxygen-free gels, the two components were more widely separated. About 40% of the patient's hemoglobin focused at a more acid pH than hemoglobin A, indicating a hemoglobin variant with impaired Bohr effect. Chromatography of globin in 8 M urea revealed two beta-chain peaks, the first of which was eluted at a lower buffer molarity than normal beta chain. Fingerprints of tryptic digests of the aminoethylated chains were done on silica gel thin-layer plates. Tp 14 from the abnormal beta chain had slower electrophoretic mobility and a greater Rf value. Amino acid analyses of this peptide gave values identical with those of betaTp 14, except that it contained one proline residue and no histidine. Since the one His in betaTp 14 is in position 143, hemoglobin Syracuse in alpha2beta2-143 His leads to Pro. Native Hb Syracuse could be separated from hemoglobin A on a carboxymethylcellulose column. The inclusion of 0.1 mM EDTA in the preparative buffers proved very useful in reducing the formation of methemoglobin. Oxygen equilibria of purified hemoglobin Syracuse showed high oxygen affinity (P50 value 12% that of hemoglobin A) and lack of cooperativity between subunits (Hill's n equals 1.1). The alkaline Bohr effect was about half that of hemoglobin A. The proline substitution at betaH21 disrupts the helical configuration and probably prevents the formation of salt bonds that are important in stabilizing the deoxy structure and contribute to the alkaline Bohr effect. Since beta143 His is a binding site for 2,3-diphosphoglycerate (2,3-DPG), it is not suprising that hemoglobin Syracuse had markedly impaired reactivity with 2,3-DPG. Hemoglobin Syracuse auto-oxidized more slowly than hemoglobin A, probably reflecting a slower rate of dissociation of oxygen from fully liganded hemoglobin.
M Jensen, F A Oski, D G Nathan, H F Bunn
The first step in the degradation of the steroid side chain during biosynthesis of bile acids from cholesterol in man was studied in microsomal and mitochondrial fraction of homogenate of livers from 14 patients. The microsomal fraction was found to catalyze an efficient 25-hydroxylation of 5,8-cholestane-3a,7a,12atriol. A small extent of 23-, 24-, and 26-hydroxylation of the same substrate was observed. 53-Cholestane-3a,7adiol was hydroxylated in the 25-position only to a very small extent. The mitochondrial fraction was found to catalyze 26-hydroxylation of cholesterol, 5-cholestene-3P,7a-diol, 5P-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one, and 5,0-cholestane-3a,7a,12a-triol. Addition of Mg++ stimulated the 26-hydroxylation of cholesterol but had no effect or an inhibitory effect on 26-hydroxylation of the other substrates, indicating a heterogeneity of the mitochondrial 26-hydroxylating system. The level of 26-hydroxylase activity towards different substrates varied considerably with different mitochondrial preparations. The roles of the microsomal and mitochondrial 26- hydroxylations as well as the microsomal 25-hydroxylation in biosynthesis of bile acids in man are discussed. The results indicate that microsomal 26-hydroxylation is less important than mitochondrial 26-hydroxylation under normal conditions. The possibility that microsomal 25-hydroxylation is important cannot be ruled out.
I Björkhem, J Gustafsson, G Johansson, B Persson
18 patients with osteogenic sarcoma were followed by serial measurements in vitro of tumor-specific cell-mediated cytotoxicity and of "active" and total rosette-forming T-cells. 13 of these patients have had or are currently receiving injections of osteogenic sarcoma-specific dialyzable transfer factor derived from healthy donors. In three patients with very small lesions, cytotoxicity was high before amputation and decreased within 2 mo after removal of tumor. Cytotoxicity was low at time of diagnosis in all patients with large tumor masses. The cytotoxicity of the patients' lymphocytes increased after administration of tumor-specific transfer factor in all patients so treated. Patients receiving nonspecific transfer factor showed evidence of declining cell-mediated cytotoxicity. Tumor-specific transfer factor may produce an increase in cell-mediated cytotoxicity to the tumor in patients with osteogenic sarcoma. This possibility is suggested by the pain and edema that occurred in the area of the tumor in patients who had metastatic disease when therapy was started and by lymphocytic infiltrates in the tumor, as well as by the increase in cell-mediated cytotoxicity and the increase in percentage of active rosette-forming cells from subnormal to normal. Serial measurements of cell-mediated cytotoxicity are helpful in monitoring the efficacy of transfer factor and other modes of therapy in these patients, and these measurements are the best available criteria for selection of donors of tumor-specific transfer factor.
A S Levin, V S Byers, H H Fudenberg, J Wybran, A J Hackett, J O Johnston, L E Spitler
Patients with osteogenic sarcoma (and related tumors), hypernephroma, and breast carcinoma, and their household contacts were tested for tumor-specific cell-mediated immunity against these tumors with the use of a short-term chromium-51 release assay. This assay, reproducible over many months and well-correlated with the clinical course of the patients, was used to demonstrate that household contacts of patients with osteogenic sarcoma and breast carcinoma have specific immunity against the tumor type with which they have been in contact. In both types of tumors, the range of cytotoxicity values produced by lymphocytes from the household contacts was significantly higher than that of the normal population. The incidence of immunity was much higher in household contacts of patients with breast carcinoma than in those of patients with osteogenic sarcoma. Immunity was found with equal frequency in men and women, as well as in genetically and nongenetically related household contacts (guardians, adopted children, spouses). Immunity against hypernephroma was not demonstrated in either patients with hypernephroma or their household contacts.
V S Byers, A S Levin, A J Hackett, H H Fudenberg
All 744 patients admitted to a Respiratory-Surgical Intensive Care Unit (RSICU) were included in a prospective study of the effects of a polymyxin (2.5 mg/kg body wt/day in six divided doses) or a placebo aerosol sprayed into the posterior pharynx and tracheal tube (if present), during 11 alternating 2-mo treatment cycles. The incidence of upper airway colonization in the RSICU with Pseudomonas aeruginosa was 1.6% during the polymyxin treatment cycles (total 374 patients) and 9.7% during the placebo cycles (370 patients) (X2 equals 23.2, P less than 0.01). 3 patients in the RSICU acquired Pseudomonas pneumonia, as defined by independent "blinded" assessors, during the polymyxin cycles while 17 acquired a Pseudomonas pneumonia during the placebo cycles (X2 equals 10.2, P less than 0.01). The overall mortality was similar in both placebo and polymyxin-treated groups (12.2 vs. 12.0%). Systemic antibiotic usage was similar in the different cycles; 49% of patients in the placebo and 53% in the polymyxin-treated groups received systemic antibiotics while in the RSICU.
J M Klick, G C du Moulin, J Hedley-Whyte, D Teres, L S Bushnell, D S Feingold
Colony-stimulating activity (CSA) is essential for in vitro differentiation of bone marrow cells into colonies of granulocytes and mononuclear cells. While blood monocytes and macrophages are a major source of CSA, recent studies have indicated that CSA may be produced by lymphocytes responding to immunologic stimulation. Lymphocytes, purified from spleens and thymuses of mice by glass wool columns, were incubated in CMRL-1066 medium with fetal calf serum in vitro. Lymphocytes from the thumus and spleen released CSA when cultured in vitro, with peak levels of CSA observed after 7 days of incubation. Stimulation of cultures with phytohemagglutinin, concanavalin A, or pokeweed mitogen resulted in a 2-5-fold increase in CSA release, with peak levels of CSA released after 4 days of incubation. Thymus-dependent lymphocytes were responsible for the release of CSA from unstimulated and mitogen-stimulated cultures, since the incubation of these cultures with rabbit anti-mouse T cell sera abolished their ability to release CSA. Anti-mouse B cell sera had no effect on the ability of lymphocyte cultures to release CSA. These studies suggest that thymocytes and thymus-derived lymphocytes can release CSA in vitro and may be responsible for the increase in CSA observed in certain immunologic reactions.
F W Ruscetti, P A Chervenick
It is well established that a number of organic anions are excreted by the liver into bile in association with a marked increase in bile flow. Previous studies have shown that iodipamide (3,3'-(adipoyl-diimino)bis[2,4,6-triiodobenzoic acid]), the radiographic contrast material used for intravenous cholangiography, is a potent choleretic. Experiments were performed in unanesthetized dogs to determine if the increased bile flow produced by iodipamide is canalicular or ductular in origin, to quantitate the choleresis associated with iodipamide and taurocholate excretion, and to correlate these findings with the results of in vitro studies in which the osmotic activities of iodipamide and taurocholate in both isotonic saline and bile were determined. The plasma erythritol clearance increase linearly with the excretion of iodipamide, indicating that iodipamide stimulates canalicular bile flow. The choleretic potency of iodipamide (22 ml/mmol) is approximately 3 times that of taurocholate (7.8 ml/mmol), yet the osmotic activity of iodipamide in bile (1.5 mosmol/mmol) is only twice as great as that of taurocholate in bile (0.8 mosmol/mmol). It therefore appears that, per unit of effective osmotic solute secreted, iodipamide carries more water into the bile canaliculi than does taurocholate.
G K Feld, P M Loeb, R N Berk, H O Wheeler
The purpose of the present series of experiments was to measure and compare the effects of an anticholinergic drug (isopropamide) and an antagonist of the histamine H2 receptor (metiamide) on food-stimulated acid secretion. Patients with duodenal ulcers were stimulated by a steak meal, and acid secretion was measured by in vivo intragastric titration. The largest dose of isopropamide that can be taken clinically without producing intolerable side effects (maximum tolerated dose) suppressed food-stimulated acid secretion by 35%. By contrast, metiamide in a 400-mg dose produced no side effects and almost completely abolished food-stimulated acid secretion. A dose-response curve revealed that a 50-mg dose of metiamide was required to suppress food-stimulated acid secretion by 50%. Further studies showed that metiamide and isopropamide are additive in suppressing food-stimulated acid secretion, and that metiamide has no effect on serum gastrin concentration or on gastric emptying.
C T Richardson, B A Bailey, J H Walsh, J S Fordtran
The presence of collagen in lung is fundamental in normal lung structure and function. Methods have been developed to examine human fetal and adult lung collagen with respect to its composition and synthesis. The second trimester fetal lung has a large number of cells per unit lung mass (36.6 plus or minus 2.7 mug DNA/mg dry wt) and relatively small amounts of collagen (17.0 plus or minus 5.3 mug collagen/mg dry wt). The number of cells per unit lung mass in the adult lung (11.1 plus or minus 3.4 mug DNA/mg dry wt) is 30% of the number of cells in the fetal lung, but the adult has 11 times more collagen (196 plus or minus 25 mug collagen/mg dry wt). The composition of fetal lung collagen can be partially characterized by extraction with salt at neutral pH, acetic acid, or guanidine. The extracted chains, representing 10% of the total lung collagen, chromatograph as alpha1 and alpha2 chains, each with a mol wt of 100,000 and an animo acid composition characteristic for collagen but not specific for lung. Short-term explant cultures of fetal and adult lung synthesize alpha chains which can be isolated by ion-exchange chromatography. These chains, representing 30-40% of the total collagen synthesized by the explants, coelectrophorese with extracted collagen chains on acrylamide gels: they are destroyed by clostridial collagenase and they have a mol wt of 100,000. Although the composition of the collagen synthesized by these explants can be only partially characterized, the rate of synthesis of both collagen and noncollagen protein can be quantitated. In fetal lung, 4.0 plus or minus 1.2% of the amino acids incorporated into protein per hour are incorporated into collagen. In normal adult lung, this percentage (4.2 plus or minus 0.9%) is remarkably similar. These values are almost identical to the relative rate of collagen synthesis in rabbit lung in the same age range. This technology should be applicable to answer specific questions regarding collagen synthesis and degradation in human lung disease.
K Bradley, S McConnell-Breul, R G Crystal
Spontaneous and chemically induced mutants with reduced ability to produce cholera enterotoxin (choleragen) as an extracellular protein were isolated from Vibrio cholerae strains 569B Inaba, a classical cholera vibrio, and 3083-2 Ogawa, an El Tor vibrio. By qualitative and quantitative immunological assay in vitro such mutants could be separated into different classes characterized either by production of no detectable choleragen (tox minus), or of small quantities of extracellular choleragen, or of large quantities of cell-associated choleragen but little extracellular choleragen. Analysis of proteins in concentrated culture supernates by electrophoresis in polyacrylamide gels showed that cultures from tox minus strains lacked proteins with electrophoretic mobilities corresponding with choleragen or the spontaneously formed toxoid (choleragenoid). Infant rabbits infected with the tox minus strains remained asymptomatic or developed milder symptoms than rabbits infected with the tox+ parental strains. When symptoms of cholera developed after inoculation with tox minus mutants, detectable numbers of tox+ revertants could be isolated from the intestines of the infected animals. Two tox minus strains, designated M13 and M27, caused no sumptoms and showed no evidence of reversion to tox+ during single passage in infant rabbits, and mutant M13 also remained avirulent and stably tox minus during six cycles of serial passage in infant rabbits. Strains M13 and M27 were also noncholeragenic in acult rabbit ileal loops. Quantitative cultures of the intestines from infected infant rabbits demonstrated that the avirulent mutant M13 can multiply in vivo and can persist in the intestinal tract for at least 48 h.
R K Holmes, M L Vasil, R A Finkelstein
Since oxygen-free polymorphonuclear neutrophils (PMN) cannot kill Staphylococcus aureus normally, the usual mechanisms for PMN bactericidal activity probably involve hydrogen peroxide or superoxide. Catalase can destroy hydrogen peroxide, and superoxide dismutase breaks down superoxide. Experiments were performed to study the influence of these enzymes (which are found in staphylococci) on virulence for mice or on leukocyte-bacterial interaction. 15 staphylococcal strains were injected i.p. into mice to quantitate virulence. There was good correlation between staphylococcal catalase activity and mouse lethality (r equals 0.88) but no correlation between staphylococcal superoxide dismutase activity and mouse lethality (r equals 0.14). Exogenous catalase (10,000 U/ml) increased the virulence of low-catalase staphylococci, but exogenous superoxide dismutase (200 mug/ml) did not alter the virulence of staphyloccal strains. C14=labeled high-catalase or low-catalase staphylococci were ingested equally well by PMN, with or without the addition of exogenous catalase. A high-catalase staphylococcal strain was killed relatively poorly by PMN, and addition of exogenous catalase (but not superoxide dismutase) decreased the ability of PMN to kill a low-catalase strain. Iodination of bacterial proteins by PMN is related to hydrogen peroxide, and a high-catalase staphylococcal strain was iodinated only 63% as much as a low-catalase strain. Addition of exogenous catalase decreased iodination of the low-catalase strain by 23%. These findings suggest that staphylococcal catalase protects intraphagocytic microbes by destroying hydrogen peroxide produced by the phagocyte. Thus, catalase may be a significant staphylococcal virulence factor.
G L Mandell
Globin chain synthesis was examined in erythroid cells of increasing maturity, fractionated from the whole bone marrow of beta-thalassemia heterozygotes by a density gradient centrifugation procedure. In experiments using total cell "globin," a gradient of alpha/beta chain ratios was observed, increasing with erythroid cell maturation from unity in the basophilic cells up to 2.0 in reticulocytes. Gel filtration of the lysates from these marrow fractions revealed the presence of free alpha chains even in the most immature cells, the amount of which increased with erythroid cell age; the total alpha/beta ratio derived from gel filtration experiments showed a gradient similar to that observed in the total globin experiments. However, the alpha/beta ratio of the hemoglobin fraction obtained by gel filtration remained constant throughout maturation at an average of 0.65. This latter finding is incompatible with balanced synthesis at any stage of red cell development and excludes the possibility that total beta chain production is higher in the early cells than in the later cells or that alpha chain production in the early cells is reduced to the level of beta chain synthesis. Furthermore, in a Hb S/beta-thalassemia marrow examined, the beta A/beta S ratio remained constant throughout maturation while the alpha/non-alpha ratio showed an increase like that observed in the simple beta-thalassemia heterozygotes. This argues strongly against increased synthesis from either the thalassemic or nonthalassemic beta chain gene being responsible for the balanced synthesis in the immature cells. These findings lead us to suggest that, in beta-thalassemia heterozygotes, a large alpha chain pool is present throughout erythroid cell maturation and that the observed increase in alpha/beta ratios is a function of the ability of those cells to degrade the excess alpha chains.
W G Wood, G Stamatoyannopoulos
Urine specimens from patients with multiple myeloma and Bence Jones proteinuria frequently contain low molecular weight proteins which correspond either to the amino-terminal, variant half (VL) or to the carboxyl-terminal, constant half (CL) of the Bence Jones protein. Analyses of urine specimens from such patients who had received high doses of corticosteroids as part of their treatment regimen revealed that concomitantly with a decrease in Bence Jones protein excretion was the appearance of a low molecular weight protein related to the Bence Jones protein but not identical to the VL or to the CL. Analyses of daily urine specimens obtained from one such patient over an extended time period revealed that a reproducible chain of events occurred during a treatment regimen which included oral administration of 75 mg of prednisone daily for 7 consecutive days. The amount of Bence Jones protein excreted decreased progressively, and by the 5th day was usually less than 10% of the pretreatment value. The urine specimen obtained on the 6th day of treatment was virtually devoid of Bence Jones protein but contained a newly appearing protein whose electrophoretic mobility was distinct from that of the Bence Jones protein or its VL or CL. Cessation of corticosteroid therapy resulted in a prompt disappearance of the new protein and in a progressive increase in the amount of Bence Jones protein excreted. The new protein was isolated from the urine of this patient and was purified for comparative studies with Bence Jones protein and with the VL and CL prepared by specific enzymatic cleavage of the Bence Jones protein. These studies revealed that the new protein was most related antigenically to the CL, but could be distinguished immunochemically from the CL. This new protein, a component found in vivo related to the constant half of the light polypeptide chain, was designated CL, and was structurally 25 amino acid residues longer than the CL, that is, the amino-terminus of the enzymatically prepared CL was at position 117 whereas that of the transitory new Bence Jones-related protein was at position 92 of the light polypeptide chain. Biosynthetic studies were performed with plasma cells derived from the bone marrow of this patient at a time when both the CL and the Bence Jones protein were being excreted; both proteins were identified in extracellular culture fluid by immunochemical techniques. Whether the CL is of synthetic or catabolic origin is presently not known; however, the detection of the CL and the absence of any detectable protein related to the VL in the extracellular culture fluid might imply a synthetic origin of the CL and suggest a corticosteroid-induced alteration in light chain synthesis.
A Solomon, C L McLaughlin, J D Capra
Interaction of D (the activated form of D) and B, factors of the properdin pathway, with C3b (the major cleavage fragment of C3) generates a convertase, C3B, which cleaves C3 and initiates the terminal complement sequence C5-C9. A functionally analogous more stable C3 convertase, CoVFB, ir formed by substituting cobra venom factor (CoVF) for C3b. Mixtures of highly purified CoVF, B, and D were chemotactic for human neutrophil polymorphonuclear leukocytes as assessed in Boyden chambers either by microscopic enumeration of migrating cells or by counting of 51Cr-labeled cells. Control mixtures containing CoVF, B, and D, reacted in the absence of Mg++, were hemolytically inactive and devoid of chemotactic activity. Over a range of doses, the chemotactic activity of mixtures yielding CoVFB correlated with their hemolytic activity. Pretreatment of neutrophils with mixtures containing CoVFB rendered them unresponsive to subsequent chemotactic stimulation by kallikrein of C5a, indicating cross-deactivation to other chemotactic factors. Similar neutrophil deactivation occurred after exposure to a mixture of C3b, B, and D in which C3B was formed; with short incubation times and high cell concentration C3B also exhibited some chemotactic activity. The chemotactic activity of C3B and CoVFB is an example of a biologic function arising from interactions among factors of the properdin pathway per se, as distinguished from the capacity of this pathway to activate C3 and the terminal complement sequence.
S Ruddy, K F Austen, E J Goetzl
This study has explored the nature of the molecular events which occur when C1 inactivator, a human plasma inhibitor of the complement, kinin-forming, coagulation, and fibrinolytic enzyme systems, interacts with C1s, plasmin, and trypsin. Purified inhibitor preparations demonstrated two bands, when examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The molecular weights of the major and minor bands were 105,000 and 96,000 daltons, respectively. The minor component appeared to be immunologically and functionally identical to the main C1 inactivator component. Loss of C1s and plasmin functional activity was associated with the formation of a 1:1 molar complex between the inhibitor and each enzyme. These complexes were stable in the presence of SDS and urea. The light chain of both these enzymes provided the binding site for C1 inactivator. Complex formation and enzyme inhibition occurred only with native and not with an inhibitor preparation denatured by acid treatment, thereby demonstrating the importance of conformational factors in the enzyme-inhibitor reaction. Although peptide bond cleavage of the C1 inactivator molecule by C1s was not documented, plasmin was found to degrade the inhibitor with the production of several characteristic derivatives. At least one of these products retained the ability to complex with C1s and plasmin. Trypsin, which failed to form a complex with C1 inactivator, degraded the inhibitor in a limited and sequential manner with the production of nonfunctional derivatives one of which appeared structurally similar to a plasmin-induced product. These studies therefore, provide new information concerning the molecular interactions between C1 inactivator and several of the proteases which it inhibits.
P C Harpel, N R Cooper
The function and several of the structural features of the C1 inactivator protein isolated from the plasma of a mother and daughter with the variant form of hereditary angioneurotic edema have been examined. These abnormal inhibitors shared immunologic identity with the normal C1 inactivator protein; however, they were inactive in inhibiting the functional activity of C1s. Analysis of the abnormal inhibitors by sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis suggested that each consisted of a single polypeptide chain, the mobility of which was slower than that of the normal C1 inactivator. The apparent molecular weight of the patients' inhibitors was 109,000 daltons as contrasted to 105,000 daltons, that of the normal C1 inactivator. The abnormal inhibitors failed to form a complex with C1s or plasmin as analyzed by SDS-acrylamide gels. The large proteolytic derivatives resulting from the plasmin- and trypsin-induced degradation of the abnormal inhibitors were approximately 3,000 daltons heavier than the corresponding products derived from normal C1 inactivator. Thus, the structural abnormality identified appeared to be a property of the core molecule. Treatment of the inhibitors with neuraminidase failed to demonstrate a difference between the normal and patient-derived C1 inactivator molecule. Neither were major differences found between the amino acid composition of the defective and normal inhibitors; however, the acidic amino acids tended to be higher in the patients' inhibitors, and the phenylalanine content lower. Thus, these studies have identified both structural and functional abnormalities in the C1 inactivator protein isolated from two related patients with hereditary angioneurotic edema. Examination of the interaction between endopeptidases and the inhibitors has further delineated the abnormal structural features.
P C Harpel, T E Hugli, N R Cooper
The effect of increased peritubule capillary oncotic pressure on sodium reabsorption by the proximal tubule of the dog was investistigated after extracellular volume expansion (ECVE) with Ringer's solution or during continued hydropenia. Control measurements were made after ECVE or during hydropenia and again during renal arterial infusion with hyperoncotic albumin solution. Absolute reabsorption by the proximal tubule was calculated from fractional reabsorption and single nephron filtration rates as determined by micropuncture. Direct measurements of efferent arteriole protein were used to determine efferent arteriolar oncotic pressure. Albumin infused into the renal artery after ECVE significantly increased efferent oncotic pressure by 17.6 plus or minus 5.3 mm Hg. Fractional and absolute reabsorption by the proximal tubule increased from 20 plus or minus 6 to 37 plus or minus 5% and from 22 plus or minus 6 to 36 plus or minus 7 nl/min, respectively. During hydropenia, the albumin infusion significantly increased efferent oncotic pressure by 15.0 plus or minus 4.4 mm Hg. However, in contrast to the effect seen during ECVE, neither fractional nor absolute reabsorption was changed, delta equals 0.3 plus or minus 1.5% and 3 plus or minus 5 nl/min, respectively. Single nephron filtration rates were not significantly different between the groups and were unchanged by the albumin infusion. Peritubule capillary hydrostatic pressures, measured with a null-servo device, were not changed by the albumin infusion in either group. Renal interstitial hydrostatic pressure, measured from chronically implanted polyethylene capsules, was decreased significantly from 7.2 plus or minus 0.9 to 3.4 plus or minus 0.6 mm Hg in the hydropenic group and from 0.6 plus or minus 0.6 to 4.8 plus or minus 0.7 mm Hg in the Ringer's expanded group. In the hydropenic group, the increase in efferent oncotic pressure was nearly compensated for by changes in interstitial forces so that the calculated net force for capillary uptake was almost unchanged, 17.8 mm Hg before vs. 21.4 mm Hg during the albumin infusion. The increased efferent oncotic pressure in the Ringer's expanded group was not compensated, so that the calculated net force for uptake was increased, 11.9 mm Hg before to 22.2 mm Hg during the albumin infusion. Thus, while the increase in efferent oncotic pressure during albumin infusion was not significantly different between the groups, absolute and fractional reabsorptions were increased only in the animals in which the extracellular volume was expanded. The results suggest that ECVE alters the effect of increased peritubule oncotic pressure on sodium reabsorption by the proximal tubule.
C E Ott, J A Haas, J L Cuche, F G Knox
Administration of 25 mg/kg uranyl nitrate (UN) to rats leads to a brief period of polyuria followed by progressive oliguria with death at 5 days. Factors that determine glomerular filtration rate (GFR) were examined in control Munich-Wistar rats (n equals 16) and 2 h after either 15 mg/kg (n equals 8) or 25 mg/kg (n equals 7) of UN (i.v.) utilizing direct measurements of hydrostatic and oncotic pressures and plasma flow. Total kidney GFR was reduced to 47% of control in the low dose group and to 21% in the high dose group. The simultaneous nephron filtration rate (sngfr) was 28.6 plus or minus 0.8 nl/min/g kidney wt in control, 29.1 plus or minus 1.0 in the low dose group, and 18.1 plus or minus 1.2 (P less than 0.001) in the higher dose group. This disparity in UN effect upon GFR and sngfr was due to tubular back-diffusion of solute through damaged epithelia beyond the early proximal tubule as demonstrated by microinjection of inulin and mannitol in the proximal tubule. Inulin "leak" persisted at 6 h after UN when tubular pressure had returned to normal. Comparison of sngfr measured in early vs. late proximal tubule revealed no difference after high dose UN, suggesting no significant leak of inulin from the early proximal tubule, and that the decreased sngfr was due to primary reductions in ultrafiltration. Nephron plasma flow was equal to control at both doses of UN. Also directly measured hydrostatic pressure gradient across the glomerular capillary was not changed. The effective filtration pressure achieved equilibrium in control of animals but became significantly positive at the efferent end of the capillary at both doses of UN and increased. Total glomerular permeability (LpA) was progressively reduced from control (0.089 plus or minus 0.005 nl/s/g kidney wt/mm Hg) at low dose UN (0.047 plus or minus 0.013) and high dose 0.024 plus or minus 0.003 nl/s/g kidney wt/mm Hg). Therefore UN decreases GFR by two mechanisms: (1) tubular damage leading to back-diffusion of solutes and (b) a primary reduction in sngfr due to reduced LpA.
R C Blantz
Platelets from individuals with familial hypercholesterolemia show increased sensitivity to the aggregating atents, epinephrine and ADP. Since the mechanism of this abnormal sensitivity is unknown, we examined, in vitro, the influence of the plasma lipid environment on the function of platelets. The composition of plasma lipids was altered by the addition of sonicated cholesterol-dipalmitoyl lecithin liposomes which were "cholesterol normal" (cholesterol-phospholipid mole ratio [C/P] equals 1.0, "cholesterol rich" (C/P eauals 2.2), or "cholesterol poor" (C/P equals 0). Cholesterol-normal liposomes had no influence on platelet lipids or platelet function. In contrast, after incubation for 5 h at 37 degrees C with cholesterol-rich liposomes, normal platelets acquired 39.2% excess cholesterol with no change in phospholipids or protein. The percent increase in platelet membrane cholesterol was three-fold that of the granule fraction. The acquisition of cholesterol by platelets was associated with a 35-fold increase in sensitivity to epinephrine-induced aggregation (P less than 0.001) and 15-fold increase to ADP aggregation (P less than 0.001), as determined both by aggregometry and by [13C]serotonin release. Response to thrombin or collagen was unchanged. Platelets incubated with cholesterol-poor liposomes underwent a selective loss of 21.4% cholesterol and this was associated with an 18-fold reduction in their sensitivity to epinephrine. These studies demonstrate that the cholesterol content of platelets is dependent on the lipid composition of the milier. Cholesterol acquired by platelets may exert its effect on platelet function by a modification of the platelet membrane.
S J Shattil, R Anaya-Galindo, J Bennett, R W Colman, R A Cooper
We have examined the mechanism of TCA-soluble orthophosphate (Pi) transfer across the membrane of mature human erythrocytes in normal subjects and in patients with X-linked hypophosphatemia (X-LH). The studies were carried out largely at pH 7.4 and 37 degrees C, in partial stimulation of conditions in vivo. (a) At physiological concentrations (1-2 mM) Pi enters the intact normal erythrocyte down its chemical gradient and under no conditions could we identify a steady-state trans-membrane gradient for Pi greater than 0.6. Calculations of the phosphate anion distribution ratio using the Nernst equation yield theoretical values that closely approximate observed values. (b) Glycolytic inhibitors have little effect on total entry of 32Pi inti erythrocytes but they do affect the intracellular distribution of Pi. In the presence of iodoacetamide, label accumulates almost exclusively in the orthophosphate pool and less than 1% enters the organic phosphate pool. (c) Specific activity measurements in unblocked cells indicate that Pi anion equilibrates first with its intracellular Pi pool. These initial findings imply that neither group translocation, nor energy coupling, influence Pi permeation into the human erythrocytes. (d) The relationship between 32P entry and extracellular Pi concentration is parabolic in the presence of chloride, and linear in the presence of sulfate. The kinetics of concentration dependent entrance cannot be examined and saturability of Pi entry cannot be identified under these conditions. (e) The competitive inhibitor arsenate partially inhibits the initial rate and steady-state flux of orthophosphate in erythrocytes treated with iodoacetamide to inhibit glycolysis. However, a significant portion of Pi transport escapes arsenate inhibition. (f) Activation energies for Pi entry, in nonglycolizing erythrocytes are much higher than those required by simple diffusion in an aqueous system. (g) Neither the inward or outward movement of Pi is modulated by trans-phosphate. These latter findings suggest that transport of phosphate across the human erythrocyte is compatible with slow facilitated diffusion with symmetry for influex and efflux. The transmembrane chemical distribution ratio, and the equilibrium flux of Pi were not different from normal in the X-LH erythrocyte. Nor did the extracellular Pi concentration, arsenate, or temperature affect Pi entry differently in the two types of cells. We dedjce that different gene products serve the diffusional type of Pi transport in the erythrocyte membrane and the saturable component of transepithelial absorption in the gut and kidney. Only the latter is affected by the X-LH mutation. The former is apparently present not only in erythrocytes but also in epithelial tissue, where it can serve the absorption of pharmacologic amounts of Pi in the therapeutic repair of the depleted phosphate pools in X-LH.
H S Tenenhouse, C R Scriver
Na-K-ATPase activity was measured with an ultramicromethod in single portions of the proximal and distal convolution and of the thick ascending limb of Henle from adrenalectomized rats and after treatment with 5 mug aldosterone per 100 g body wt. The activity in all tubular structures returned to normal within 1 h after injection. This rapid activation of Na-K-ATPase induced by hormone was completely prevented by actinomycin D and cycloheximide. It appears that this aldosterone effect on Na-K-ATPase requires an intact protein synthetic process.
U Schmidt, J Schmid, H Schmid, U C Dubach
Adenosine deaminase activity resides in various characteristic isozymes in red blood cells (RBC-ADA) and other tissues. Absence of RBC-ADA has been reported in a proportion of patients with autosomally inherited severe combined immunodeficiency (SCID). We have previously reported that the tissue isozymes of ADA are also deficient in children with SCID and RBC-ADA deficiency, although these isozymes differ from RBC-ADA in molecular weight, accessible SH groups, and electrophoretic mobility. The deficiency of all types of ADA in SCID implies that a catalytic unit of ADA in each isozyme is coded by the same structural gene. The relationship of RBC-ADA and the different tissue ADA isozymes is the subject of this paper. Incubation of RBC-ADA with ADA-deficient liver, kidney, and fibroblast extracts resulted in the appearance of new isozymes of ADA. These newly generated isozymes had the physicochemical and electrophoretic characteristics of the tissue-specific isozymes obtained from normal tissues. The electrophoretic mobility of the isozyme generated appeared to depend upon the tissue utilized and corresponded to the electrophoretic mobilities of the ADA isozymes found naturally in each of the different tissues. Additionally, the genetically determined polymorphism exhibited by RBC-ADA could be detected in the isozyme generated. Incubation with normal kidney also caused conversion of the RBC isozyme to the kidney form. These findings further support the concept that the catalytic activity of each of the several forms of the ADA enzyme resides in a single molecule coded at the same genetic locus as is defective in one form of SCID. The tissue-specific isozymes, which differ in electrophoretic mobility and molecular weight, are generated by interaction of the RBC catalytic unit with tissue-specific factors present in the different tissues of normal humans and patients.
In a patient with lifelong increased susceptibility to infection and multiple abnormalities in complement-mediated functions, the infusion of normal plasma had been seen to produce a prolonged partial correction of serum abnormalities. It was subsequently shown that the patient was genetically deficient in the C3b inactivator and that immunochemical depletion of C3b inactivator from normal serum resulted in abnormalities similar to those found in the patient's serum, including alternative pathway C3 activation. Highly purified C3b inactivator was obtained from the euglobulin fraction of normal human serum, sterilized by filtration, and infused intravenously. Partial or complete correction of almost all the known serum abnormalities was obtained. C3b almost disappeared from the serum within 4-5 h, as did Factor C activity. Native C3, C5, and serum hemolytic activity rose to normal or near-normal levels over 4 days and were sustained for another week. Factor B, properdin, opsonic activity, and bactericidal activity reached a level at least two-five times that found before the infusion within 24 h and fell over the next 5 days. These observations prove the primary role of C3b inactivator deficiency in the patient's disease and demonstrate clearly the curcial role in vivo of C3b inactivator in modulating alternative pathway activity.
J B Ziegler, C A Alper, R S Rosen, P J Lachmann, L Sherington