Chlamydia trachomatis, Ureaplasma urealyticum (T-mycoplasma), and Hemophilus vaginalis have previously been considered possible etiological agents in nongonococcal urethritis (NGU). In this study, current C. trachomatis infection was confirmed by culture and (or) micro-immunofluorescence serology in 26 of 69 men experiencing afirst episode of NGU, and 1 of 39 with no urethritis. Serum IgM immunofluorescent antibody to chlamydia was demonstrated in 16 of 20 men with chlamydia culture positive NGU, and 3 of 39 with chlamydia culture negative NG, and none of 34 with no urethritis. 9 of 10 culture positive men with less than or equal to 10 days symptoms developed immunofluorescent antibody seroconversion in paired sera. U. realyticum was isolated significantly more often and in significantly higher concentration from first voided urine from chlamydia-negative cases of NGU than from chlamydia-positive NGU. Ureaplasmacidal antibody titers increased fourfold in six men, four of whom had negative cultures for for unreaplasma. H. vaginalis was isolated from c9 of 33 men with no urethritis and 2 of 69 with NGU. C. trachomatis is susceptible, and U. urealyticum is resistant to sulfonamides. A 10-day course of sulfisoxazole therapy produced improvement in 13 of 13 chlamydia-positive, unreaplasma-negative, and only 14 of 29 chlamydia-negative, unreaplasma-positive NGU cases (P less than 0.002). Thus, culture, serology, and response to therapy support the etiologic role of chlamydia in NGU. Quantitative culture and response to therapy suggest U. unrealyticum may cause many cases of chlamydia-netative NGU.
W R Bowie, S P Wang, E R Alexander, J Floyd, P S Forsyth, H M Pollock, J S Lin, T M Buchanan, K K Holmes
Serum IgE concentrations were determined and IgE turnover studies were performed in control individuals as well as in patients with several disease states. Patients with common variable hypogammaglobulinemia, thymoma and hypogammaglobulinemia, ataxia telangiectasia, and selective IgA deficiency had significantly decreased mean serum IgE concentrations. In turnover studies, this was found to be due to decreased IgE synthesis. In spite of these depressed mean values, some patients with common variable hypogammaglobulinemia had normal serum IgE concentrations and synthetic rates. Patients with the Wiskott-Aldrich syndrome had a significantly elevated mean serum IgE concentration. In one of four patients studied with the turnover technique, a strikingly high IgE concentration was present and was associated with an elevated IgE synthetic rate. Three other patients had both normal serum IgE concentrations and synthetic rates. Patients with chronic lymphocytic leukemia had significantly decreased mean serum concentrations and synthetic rates for IgE. The depressed IgE synthesis was associated with a significantly prolonged IgE half-life. Patients with Hodgkin's disease had significantly increased serum IgE concentrations. One of three patients studied had a high serum IgE concentration and synthetic rate of IgE. The two other patients had normal serum IgE concentrations associated with normal synthetic rates. Finally patients with protein-losing enteropathy or familial hypercatabolic hypoproteinemia had normal IgE concentrations associated with normal IgE metabolic parameters. In these cases, the disorder in the catabolic rate was not severe enough to affect the total amount of circulating IgE because IgE normally has a very high fractional catabolic rate. In general, IgE levels in a variety of disease states were correlated with IgE synthetic rates and abnormalities in the catabolic rate of IgE in disease did not exert an important effect on IgE concentration.
A Iio, W Strober, S Broder, S H Polmar
The cuase for the intestinal hyperabsorptionof calcium (Ca) in various forms of hypercalciurias was explored by a careful measurement of plasma 1 alpha, 25-dihydroxycholecalciferol [1 alpha, 25-(OH)I D] and by an assessment of intestinal Ca absorption and of parathyroid function. In 18 cases of primary hyperparathyroidism (PHPT), the mean plasma concentration of 1 alpha, 25-(OH)2D was significantly increased (4.9 +/- 2.2 SD ng/dl vs. 3.4 +/- 0.9 ng/dl for the control group), and was significantly correlated with fractional Ca absorption (alpha) (r = 0.80, P less than 0.001). Plasma 1 alpha, 25-(OH)2D was also correlated with urinary Ca (P less than 0.05), but not with serum Ca or phosphorus (P), P clearance, urinary cyclic AMP, or serum immunoreactive parathyroid hormone. In 21 cases of absorptive hypercalciuria (AH), plasma 1 alpha, 25-(OH)2D was elevated in one-third of cases, and the mean value of 4.5 +/- 1.1 ng/dl was significantly higher than that of the control group (P less than 0.01). Since relative hypoparathyroidism may be present, the normal absolute value of plasma 1 alpha, 25-(OH)2D, found in two-thirds of cases of AH, may be considered to be inappropriately high. Moreover, in the majority of cases of AH, the data points relating plasma 1 alpha, 25-(OH)2D and alpha fell within 95% confidence limits of values found in non-AH groups (including PHPT). The results suggest that the intestinal hyperabsorption of Ca in PHPT aw AH may be vitamin D dependent. However, the disturbance in vitamin D metabolism may not be the sole cause for the high Ca absorption in AH, since in some patients with AH, the intestinal Ca absorption appears to be inapp
R A Kaplan, M R Haussler, L J Deftos, H Bone, C Y Pak
Cancer-free individuals from family agregates of seemingly hereditary colon carcinoma were studied to determine the nature of their cell-mediated immune capacities in miexed leukocyte culture. Members of families who demonstrated no evidence of a precancerous condition such as polyposis coli did demonstrate substantial cellular immunopathology. Of these, 44% showed a decreased responsiveness of their peripheral mononuclear cells to allogeneic stimuli, and in a number of these individuals this deficiency clearly manifested itself as an inappropriate suppression of potentially normal lymphocyte blastogenic capacities by an adherent population of mononuclear leukocytes. This in vitro defect of recognitive immunity appears to be the same type of defect that has already been described for individuals with established maligancies. The pattern of phenotypic expression of this immunopathology within these families is not inconsistent with an hereditary disorder. Individuals from families with a known hereditary somatic precancerous condition usually did not demonstrate this immunopathology. It is appropriate to speculate that the defect of recognitive immunity in the former families could be contributory to the genesis of the colon carcinoma.
N T Berlinger, C Lopez, M Lipkin, J E Vogel, R A Good
The effects of chronic phosphate depletion on renal tubular function were evaluated by micropuncture and free water clearance studies in the dog. Proximal tubular punctures demonstrated that chronic hypophosphatemia led to a reduction in ratio of tubular fluid to plasma inulin in late superficial tubular from 1.59+/-0.08 in control animals to 1.29+/-0.06 in phosphate-depleted dogs, with proportional inhibition of calcium and sodium reabsorption. The chronic decrease in proximal tubular fluid reabsorption was confirmed by the analysis of sustained water diuresis in conscious, phosphate-depleted dogs, before and after repletion of body PO4 stores, and in control animals. Urine flow rate/100 ml glomerular filtration rate (V/GFR) was significantly higher in PO4 DEPLETION THAN CONTROL (15.8+/-1.1 VS. 10.7+/-0.82). In addition, acetazolamide infusion did not increase V/GFR in phosphate-depleted dogs (15.8+/-1.1 vs. 17.16+/-0.9), supporting the conclusion that inhibition of proximal tubular fluid reabsorption was responsible for the elevated urine flow rate. PO4 repletion over 5 days reduced V/GFR to 9.2+/-0.7 despite no change in urine osmolality and no change in GFR, further suggesting a specific reversible alteration in proximal tubular reabsorption in phosphate depletion. Although hypercalciuria was a constant finding in phosphate depletion (fractional excretion of calcium of 2.04+/-0.4% vs. 0.47+/-0.13% in controls), the enhanced distal delivery of calcium was not a crucial factor; acute phosphate infusion reduced urinary calcium excretion to control values without affecting the reduced proximal tubular reabsorption in either intact or thyroparathyroidectomized phosphate-depleted dogs the change in distal nephron calcium reabsorption was independent of parathyroid hormone (PTH) levels since infusion of PTH failed to alter urinary calcium excretion. We conclude that chronic phosphate depletion leads to a reversible, sustained inhibition in proximal tubular reabsorptive fuction as well as a specific decrease in distal nephron calcium reabsorption. This latter reabsorptive defect is sensitive to phosplate infusion but not corrected by PTH.
S Goldfarb, G R Westby, M Goldberg, Z S Agus
Respiratory and arterial baroreceptor reflex interactions were studied in six healthy young adults. Carotid baroreceptors were stimulated with two intensities of neck suction during early inspiration or expiration at 100 or 150% of each subject's normal tidal volume. Sinus node responses to moderate baroreflex stimuli were inhibited by inspiration, but responses to intense stimuli were not influenced by the phase of respiration. Supranormal tidal volume did not diminish responses to inspiratory baroreflex stimuli, but significantly reduced responses to expiratory stimuli. These results provide evidence for a central respiratory baroreceptor reflex interaction in man whose quality is dependent upon the level of afferent baroreceptor activity and the depth of inspiration.
D L Eckberg, C R Orshan
Tubular fluid flow, urine osmolality, and pH were selectively altered to determine the relative protective roles of these factors in a rat model of acute urate nephrophathy. Various prehyper uricemic conditions were established in five groups of animals: (a) normopenic Wistar rats given no pretreatment (Group I); (b) Wistar rats given acetazolamide, 20 mg/kg, and isotonic NaHCO3 to produce urine alkalinization (Group II); (c) Wistar rats in which a moderate diuresis, similar to that observed in Group II but without urine alkalinization, was induced with furosemide, 2 mg/kg (Group III); (d) Wistar rats in which a high-flow solute diuresis was induced with furosemide, 15 mg/kg (Group IV); (e) Brattleboro rats, homozygous for pituitary diabetes insipidus, that had a spontaneous high-flow water diuresis (Group V). A comparable level of hyperuricemia (19.4+/-2.2 mg/100 ml) was achieved in all animals with intravenous urate infusion. Clearance and micropuncture studies were performed before and 1 h after induction of hyperuicemia. Group I rats had mean falls in renal plasma flow and glomerular filtration rate of 83 and 86%, respectively; nephron filtration rate decreased 66%, and tubular and microvascular pressures increased twofold. In Group II there were 45 and 47% declines in renal plasma flow and glomerular filtration rate, respectively, a 66% fall in nephron filtration rate, and a 30% increase in tubular and vascular pressures. Moderate amounts of urate were seen in the kidneys. Group III had changes in renal function identical to Group II suggesting that the moderate prehyperuricemic diuresis in the latter group and not urine alkalinization produced the partial protection observed. Groups IV and V were completely and comparably protected with renal function studies unchanged from controls. It is concluded that high tubular fluid flow, whether induced by a solute or water diuresis, is the primary mechanism of protection in acute urate nephropathy. At most, urine alkalinization plays a minor preventive role.
J D Conger, S A Falk
A new type of enzyme hydrolyzing the elastase substrate succinyl-L-alanyl-L-alanine-4-nitroanilide has been found in cell-free rheuma todi synovial fluid. Normal plasma and osteoarthritic synovial fluid contained relatively little enzyme. The pH optimum was 8.0. Unexpectedly, the enzyme activity was not due to leukocyte elastase or any proteinase bound to alpha2-macroglobulin. The enzyme activity was metal-dependent being inhibited by chelating agents but not by di-isopropylfluorophos phate or thiol-blocking reagents. Gel chromatography showed the enzyme activity was associated with material of high molecular weight. On Sepharose 4B chromatography two-thirds of the activity eluted in the void volume and one-third in a position of about 106 mol wt. Utracentrifugation showed that both components were associated with lipid. The buoyant density of the higher molecular weight material was 1.15-1.22 g/ml., and that of lower molecular weight material was 1.2-1.33 g/ml. No latency of the enzyme was revealed by freezing and thawing or treatment with detergents. The nature of the enzyme is discussed. It is likely to be a proteinase possibly bound to some kind of membrane fragment.
Serum from three patients with a complete, selective deficiency of the second component of complement (C2) did not promote optimal killing of Staphylococcus aureus, 502A by neutrophilic polymorphonuclear leukocytes (PMN) in vitro. The addition of C2 reagent or the presence of heat-stable opsonin in the C2-deficient serum corrected the defective killing of S. aureus that was observed with patient or control PMN. PMN from the patients or control subjects killed bacteria with equal efficiency under conditions of optimal opsonization (normal pooled serum). However, twice-washed control PMN were better than patient PMN in killing S. aureus under circumstances of suboptimal opsonization (C2-deficient serum, heated C2-deficient serum, heated normal pooled serum, or no replacement of serum). The latter finding was due to residual C2 on the surface of twice-washed control cells. As repeated washing of control PMN progressively removed cell-associated C2, the staphylocidal effectiveness of the control RMN decreased to the level of patient PMN. In contrast to the findings with S. aureus, triply-washed PMN from patients or controls killed normal numbers of Escherichia coli, ON2, in C2-deficient serum.
J E Repine, C C Clawson, P S Friend
The development of antibody in response to invasive infection with type III strains of group B Streptococcus was studied in sera from 31 infants and 4 adults by means of a quantitative radioactive antigen-binding assay. Low concentrations of antibody were consistently found in the acute sera of patients who developed clinical illness. Although adults with puerperal sepsis and infants with bone or joint infection uniformly demonstrated significant rises in serum antibody concentration after recovery, much lower levels of antibody were detected in convalescent sera from infants recovering from meningitis or sepsis. The median antibody concentration in sera from 43 parturients with type III strains of group B Streptococcus isolated from vaginal cultures whose neonates failed to develop symptomatic disease was significantly greater than that in sera from 29 mothers of infants with invasive, type III, group B streptococcal infection. Study of paired maternal and cord sera demonstrated a significant correlation between the antibody concentration in a mother's serum and that in her neonate.
C J Baker, D L Kasper, IRAB Tager, A Paredes, S Alpert, W M McCormack, D Goroff
Urimary excretion of hydroxyprolin (Hyp) is one index of total collagen degradation, from all sources. Since some of the Hyp released from collagen may be further metabolized before it is excreted, other markers are necessary to measure collagen breakdown. Excretion of the glycosides of hydroxylysine (Hyl), glucosyl galactosyl hydroxylysine (Hy1[Gl)cGa1]), and galactosyl hydroxylysine (Hyl[Ga)]), more accurately reflects collagen metabolism since these products occur in specificratios in different tissue collagens and are themselves metabolized only to a minor degree. The ratios of total Hy1/Hyp and Hyl(GlcGal)/Hyl(Ga1) were measured in the urine of norma. subjects and of patients with Paget's disease of bone, hyperphosphatasia, and extensive thermal burns. In patients with extensive thermal burns the pattern of urinary Hy1 and its glycosides was consistent with degradation of collagen in dermis and fascia. When bone collagen degradation was dominant, the pattern of urinary metabolites reflected that source. Pagetic bone collagen has an amino acid composition similar to normal bone and Hy1(G1cGa1/Hyl(G1) of 0.396-0.743,vs. normal of 0.474+/-0.088. In untreated patients with severe Paget's disease of bone or hyperphosphatasia (urinary Hyp greater than 2.0 micronmol/mg creatinine) urinary Hyl/Hyp averaged 0.052+/-0.042 (0.042+/-0.009 in normal bone) and Hy1(G1cGa1)/Hy1(Ga1) 0.601+/-0.017 (0.47+/-0.009 in normal bone). When bone resorption was decreased sufficiently with calcitonin or disodium etidronate in these patients, both the urinary ratios of Hy1/Hyp and Hy1(G1cGa1)/Hyl(Gal) rose. In normal subjects treated with calcitonin and excreting relatively little Hyp, the ratio of Hy1/H)P approached 0.7 and Hy1(G1ycGa1)/Hy1(Ga1) approached 3.5. There increased ratios reveal the existence of a source of collagen breakdown other than skin or bone. The first subcompoent of complement, Clq, which has collagen-like sequences, relatively high amounts of Hy1, and most of the glycosylated Hy1 as Hy1(G1cGa1), could be the source of these metabolites.
S M Krane, F G Kantrowitz, M Byrne, S R Pinnell, F R Singer
Cholesterol gallstone disease is initiated in a liver which produces abnormal bile with excess cholesterol relative to bile salts and phospholipid. To define the responsible secretory mechanism(s), the rate of biliary lipid secretion was measured by a duodenal marker perfusion technique, while the bile salt pool was simultaneously estimated by isotope dilution. Two groups of control patients expected to have normal biliary lipid composition--14 subjects without hepatobiliary disease and 6 patients with pigment gallstones, were compared to two experimental groups expected to have abnormal bile--10 nonobese patients with cholesterol gallstones and 7 obese subjects without gallstones. Both control groups had nearly identical biliary lipid secretion rates, and a corresponding low relative molar concentration of cholesterol. Two different secretory mechanisms were found to be responsible for the abnormal bile in the experimental groups. In the nonobese patients with cholesterol gallstones, bile salt and phospholipid secretion rates were both significantly reduced. Conversely, the grossly obese subjects had an increased cholesterol secretion. To determine how cholecystectomy improves biliary lipid composition, three groups of gallstone patients --6 with pigment stones, 4 grossly obese with cholesterol stones, and 13 nonobese with cholesterol stones --were all examined after full recovery from surgery. In the nonobese patients with cholesterol gallstones, both bile salt and phospholipid secretion significantly increased, causing a definite improvement in bile composition. Cholecystectomy produced a similar but less marked trend in the obese patients with cholesterol stones, and in the patients with pigment stones. Cholesterol secretion, however, was unaffected by surgery. The bile salt pool was definitely small in the nonobese patients with cholesterol gallstones and became slightly smaller after cholecystectomy. The pool was significantly reduced by cholecystectomyin the patients with cholesterol gallstones. Removal of the gallbladder in all three groups caused a greater fraction of the pool to cycle around the enterohepatic circulation each hour. This more rapid cycling produced the increase in bile salt and phospolipid secretion, and was responsible for the improved composition found after cholecystectomy.
E A Shaffer, D M Small
In the plasma clot culture system both normal and polycythemia vera (PV) bone marrow cells respond to erythropoietin (Ep), giving rise to large numbers of colonies of erythroid cells. In PV, but not in normal individuals, the marrow produced endogenous erythroid colonies (EED) in the absence of exogenous Ep. The number of EEC formed varied from patient to patient comprising anywhere from 6 to 29% of the total number of colonies formed in the presence of Ep. Exposure, before use in culture, of fetal calf serum and citrated bovine plasma to the gammaglobulin fraction of rabbit anti-Ep serum followed by treatment with goat anti-rabbit gamma-globulin re sulted in a significant decrease in EEC formation. Addition of anti-Ep directly to the culture medium produced similar results. In addition, the production of EEC in response to added Ep was inhibited in the presence of anti-Ep. Addition of very small doses of highly purified Ep to anti-Ep-treated cultures resulted in the reappearance of a significantnumber of EEC formation in PV may be due to a population of erythroid-committed precursors that are abnormally sensitive to small concentrations of Ep which may be present in fetal calf serum and citrated plasma. Although the mechanism of formation of these cells is not known, it appears that the final steps in the formation of red cells derived from this clone of precursors is subject to the usual Ep control.
E D Zanjani, J D Lutton, R Hoffman, L R Wasserman
Since phenobarbital administration produces a profound increase in bile flow without changing bile acid secretion, we examined whether this drug increases the activity of hepatic sodium-potassium-activated ATPase [Na+-K+)-ATPase], the postulated regulating enzyme in the secretion of bile salt independent bile flow. After freeze-thawing to increase substrate accessibility, (Na+-K+) ATPase activity was determined by ouabain inhibition of total ATPase activity. Its activity was highest in isolated liver surface membrane fractions enriched in bile canalicult. Phenobarbital administration significatly increased (Na+-K+)-ATPase activity in both liver surface membrane fractions as well as liver homogenates. This enhanced activity is apparently selective for other membrane phosphatases and the enzyme activity in other tissues is either unaltered or decreased. Kinetic analysis of (Ka+-K+)-ATPase indicates that phenobarbital treatment increased maximum velocity and half-maximum activation constant was unchanged, consistent with activation of latent molecules or an increased number of enzyme molecules. The latter process seems more likely because cycloheximide prevented phenobarbital induction and activators were not demonstrated in vitro. Examination of the full time course of phenobarbital induction to determine whether phenobarbital increased synthesis or decreased degradation was consistent with increased synthesis since the apparent degradation rates were similar with or without phenobarbital treatment. The apparent half-life for (Na+-K+)-ATPase was estimated to be approximately 2.5 days, consistent with liver surface membrane protein turnover. The correlation of changes in bile flow with (Na+-K+)-ATPase was examined under several experimental situations. Phenobarbital caused a parallel increase in each during the 1st 2 days of greatment: thereafter other factors become rate limiting for flow, since enzyme activity doesn't reach a new steady state until 4-days. Consistent with increased sodium-potassium exchange, bile sodium was unchanged while potasium concentrations were significantly reduced. Changes in both bile flow and (Na+-K+)-ATPase induced by phenobarbital are independent of thyroid hormone. These studies support the postulate that (Na+-K+)-ATPase is an important factor in regulation of bile flow. In addition, phenobarbital enhancement of both bile flow and (Na+-K+)-ATPase is dependent upon de novo protein synthesis.
F R Simon, E Sutherland, L Accatino
Quantitative determination of the small C3 breakdown product, C3d, was used to investigate complement activation in 45 plasma samples from 30 patients with rheumatoid arthritis (RA). The mean plasma C3e level in these samples (3.0 +/- 1.3 mg/100 ml) was significantly increased (P less than 0.001) as compared to patients with degenerative joint disease (0.9 +/- 0.4 mg/100 ml) and healthy blood donors (0.8 +/- 0.5 mg/100 ml). C3d levels were increased by more than s SD in 79% of RA samples. Plasma C3d levels were compared with C3d concentrations in synovial fluid. In most RA patients, the C3d levels were higher in synovial fluid than in plasma. A very significant correlation between plasma C3d levels and circulating immune complexes, as measured by determination of Clq binding activity (Clq BA), was observed (P less than 0.001). C3d levels were more elevated in RA patients with extra-articular disease manifestations (3.8 +/- 1.2 mg/100 ml) as compared to patients with joint disease alone (2.2 +/- 1.0 mg/100 ml). C3d levels and Clq BA were also significantly correlated (P less than 0.001) with the RA disease activity expressed by an index derived from sedimentation rate, joint score, and duration of morning stiffness. A close relationship between C3d levels, Clq BA, and the clinical activity further appeared during follow-up studies. The present observations suggest that a parallel but rather independent activation of the complement system may be induced by immune complexes in circulating blood and in the joint spaces during the course of rheumatoid arthritis.
U E Nydegger, R H Zubler, R Gabay, G Joliat, C H Karagevrekis, P H Lambert, P A Miescher
Chronic renal failure in rats leads to changes in hepatic protein synthesis and albumin metabolism at both the cellular and molecular level. In rats with chronic uremia (blood urea nitrogen greater than 45 mg/100 ml 1 mo after surgical reduction in renal mass), cell-free protein synthesis is reduced 30--40% in liver membrane-bound polyribosomes. Albumin synthesis by membrane-bound polysomes in uremia is reduced even more than the reduction in total protein synthesis. Activity of free polysomes remains norma. There is also intracellular accumulation of albumin in liver of uremic rats and a concomitant decrease in serum albumin. In normal liver, most intracellular albumin is located in the microsomal fraction, whereas in liver from uremic animals the excess albumin is found in the free cytosol fraction. These results can be explained either by a defect in synthesis of albumin by membrane-bound polysomes with release of newly synthesized albumin into the cytosol or by a reduced ability of polysomes synthesizing albumin to associate with the membrane fraction in rats with chronic renal failure.
S B Grossman, S H Yap, D A Shafritz
Acute leukopenia occurs in all patients during the first hour of hemodialysis with cellophanemembrane equipment. This transient cytopenia specifically involves granulocytes and monocytes, cells which share plasma membrane reactivity towards activated complement components. The present studies document that complement is activated during exposure of plasma to dialyzer cellophane, and that upon reinfusion of this plasma into the venous circulation, granulocyte and monocyte entrapment in the pulmonary vasculature is induced. During early dialysis, conversion of both C3 and factor B can be demonstrated in plasma as it leaves the dialyzer. Moreover, simple incubation of human plasma with dialyzer cellophane causes conversion of C3 and factor B, accompanied by depletion of total hemolytic complement and C3 but sparing of hemolytic C1. Reinfusion of autologous, cellophane-incubated plasma into rabbits produces selective granulocytopenia and monocytopenia identical to that seen in dialyzed patients. Lungs from such animals reveal striking pulmonary vessel engorgement with granulocytes. The activated complement component(s) responsible for leukostasis has an approximate molecular weight of 7,000-20,000 daltons. Since it is generated in C2-deficient plasma and is associated with factor B conversion, it is suggested that activation of complement by dialysis is predominantly through the altermative pathway.
P R Craddock, J Fehr, A P Dalmasso, K L Brighan, H S Jacob
In the present study, terminal deoxynucleotidyltransferase was examined in the peripheral blood and (or) bone marrow of 115 children with a variety of neoplastic, hematologic, and other unrelated disorders. Terminal deoxynucleotidyltransferase activity was present at 4.08+/-0.74 U/108 cells in 23 morphologicall normal bone marrow samples from childhood controls. Terminal transferase was present at greater than 23 U/108 nucleated cells and at greater than31 U/108 blasts in the bone marrow of all children with acute lymphoblastic leukemia studied at initial diagnosis and at disease relapse. Terminal deoxynucleotidyltransferase was detectable at low levels, less than 7.5 U/108 cells, in all remission marrow smaples. Bone marrow terminal transferase activity was markedly elevated in all untreated acute lymphoblastic leukemia patients, whereas low levels which were difficult to interpret were present in the peripheral blood samples of two patients at diagnosis and six patients at relapse who had low absolute lymphoblast counts. Because of greater variation in the lymphoblast content of peripheral blood, bone marrow assays are more reliable in detecting disease activity. Marrow terminal deoxynucleotidyltransferase values obtained during the active phase of acute lymphoblastic leukemia were significantly greater than those found in other types of leukemia, bone marrow malignancies, and hematologic disorders. Terminal transferase determinations in blast cells of two patients with leukemic conversion of non-Hodgkin's lymphoma and in tumor cells from one patient with Burkitt's lymphoma were within the control range. These dat further define the usefulness of terminal deoxynucleotidyltrnasferase assay in the differentiation and classication of hematologic malignancies.
M F Greenwood, M S Coleman, J J Hutton, B Lampkin, C Krill, F J Bolium, P Holland
Factor IX, isolated from normal human plasma, was homogenous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate. On the latter, it migrated as a single polypeptide chain with or without reducing agents and had an apparent mol wt of 62,000. After iodination by chloramine-T, a single peak of 125I was found on gels. Immunoelectrophoresis in agarose with rabbit antifactor IX sera gave a single arc against both isolated and partially purified factor IX preparations. The rabbit antibody was specific as it failed to inhibit the activities of prothrombin or factors VII or X in normal plasma. At an additional 20-fold dilution, factor IX activity was inhibited 50%. In a double-antibody radioimmunoassay, excess rabbit anti-human factor IX precipitated 90-95% of the 125I-human factor IX. Control without specific antibody gave 6-8%. Dilutions of a pool of normal human plasma paralleled dilutions of the isolated preparation and were used for the standard curve. Of 39 plasma samples from normal donors, the mean factor IX antigen level was 93% of that of a separate normal pool. The radioimmunoassay detected the abnormal factor IX produced in patients on warfarin therapy. After Al(OH)3 adsorption of warfarin treated patient's plasma, factor IX antigen, but not activity, was present in the supernate. Samples from 28 patients on warfarin gave a mean factor IX clotting activity of 27% with a mean antigen of 69%. The antigen level from the warfarin group was significantly lower than the antigen level of the normal group (P less than 0.001). The factor IX antigen level was then assessed in 36 patients from 29 pedigrees with hemophilia B. The median antigen level was 17% of normal. The distribution of the antigen level was wide with two patients around 100% of normal; only two had levels below the limits of resolution of the radioimmunoassay as currently performed (less than 2%). Within each of the five pedigrees in which more than one affected member was tested, activity and antigen levels were the same. The degree of neutralization of the antibody's inhibition of normal plasma by patient's plasma was highly correlated. Additional evidence for the detection of abnormal protein was provided by immunodiffusion of plasmas concentrated by lyophilization. Reactions of complete identity occurred between normal, a warfarin treated and a hemophilia B subject's plasmas.
A R Thompson
To determine the influence of cardiac ischemia on the activity and subcellular localization of lysosomal cathepsin D, anesthetized rabbits were subjected to ligation of the circumflex coronary artery. Total enzyme activity remained unchanged throughout the 2-h ischemic period, but the subcellular distribution of cathepsin D, as analyzed by biochemical and immunohistochemical techniques, was altered dramatically. A marked increase in nonsedimentable (i.e., 40,000-g supernate) activity developed by 30-45 min and increased further by 2 h. Simultaneously, the immunofluorescent localization of cathepsin D was also changed significantly. Within 30-60 min after occlusion, the fine, particulate staining observed in control myocytes was replaced by bright fluorescent patches composed of large granules. Many of these structures displayed prominent halos of diffuse fluorescent staining in the neighboring myocytic cytoplasm, apparently outside lysosomes per se. After 2 h, when nonsedimentable activity was maximally elevated, most of the fluorescent particles had disappeared completely. During this same interim there was no detectable change in the distribution of lysosomal cathepsin D within interstitial cells. These results are consistent with the hypothesis that an early feature of cardiac ischemia is the release of cathepsin D from myocytic lysosomes into the cytosol of damaged cells.
R S Decker, A R Poole, E E Griffin, J T Dingle, K Wildenthal
Antibodies reacting with neuronal cytoplasmic antigens present in normal human caudate and subthalamic nuclei were detected in 37 of 80 probands afflicted with Huntington's disease (HD). IgG antibodies were detected by immunofluorescence using frozen sections of unfixed normal human and rat brain. Specificity of IgG binding was confirmed using pepsin F(ab')2 fragments of IgG isolated from positive sera. In vitro complement fixation of IgG antibody was detected in 22 of 31 sera tested. Neuronal cytoplasmic antigens reacting with positive HD sera were diminished after trypsin or RNAase treatment of tissue sections but were not removed by DNAase, neuraminidase, EDTA, or dithiothreitol treatment. Antibody staining of neurons could be removed after absorption with isolated caudate nucleus neurons or by using perchloroacetic acid extracts of caudate nucleus. Prevalence of antibody reacting with neuronal cytoplasm was 3% in 60 normal controls and 6% among a wide variety of patients with diverse neurological disorders. However, one-third of 33 patients with Parkinson's disease showed presence of antineuronal antibody. Among patients with HD, a significant association was noted between duration of clinical disease greater than 7 yr and titers of antibody of 1:2 or greater (P less than 0.001). When 115 family members of HD probands were tested, 30% of unaffected spouses showed presence of antineuronal antibody. 23.2% of first-degree relatives at risk for developing HD was also positive (P less than 0.001). 10.5% of second-degree relatives showed presence of antineuronal antibody. These data may support an environmental or infectious factor somehow involved in the ultimate expression of HD.
G Husby, L Li, L E Davis, E Wedege, E Kokmen, R C Williams Jr
The intestinal absorption of [3H]-pteroylmonoglutamate (simple folic acid) and pteroyl-micron[14C]glutamyl-gamma-hexaglutamate ([14C]PG-7, conjugated folic acid) was assessed by the method of jejunal perfusion in five patients with proven celiac sprue who were studied after a gluten-containing or a gluten-free diet, and in nine normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. In each experiment, column chromatography of the luminal aspirates revealed similar spectra of hydrolytic products of [14C]PG-7, whereas the fraction of the distal aspirate chromatogram appearing as pteroyl-micron[14C]glutamyl-gamma-monoglutamate ([14C]-PG-1) was similar in all three groups. By accounting for the variable effects of absorption on the luminal appearance of [14C]PG-1 and by correcting for mucosal hydrolysis which was not followed by release of [14C]PG-1 to the luminal contents, the calculated rate of in vivo hydrolysis of [14C]PG-7 to [14C]PG-1 was found impaired in both celiac sprue groups, with significant improvement on treatment. In mucosal biopsies from the sprue patients, the in vitro activity of folate conjugase in whole homogenates was higher and the activity of disaccharidase lower than in a group of 12 normal mucosal biopsies. These in vitro data suggest that the predominant cellular location of mucosal folate conjugase is different from that of disaccharidase, whereas comparison with the results of in vivo hydrolysis suggests that measurement of the enzyme in whole mucosal homogenates overestimates its significant digestive activity. The present studies indicate that (a) the mucosal lesion of celiac sprue significantly limits the intestinal absorption of both simple and conjugated folate, and (b) malabsorption of conjugated folate results from a combination of impaired hydrolysis and decreased mucosal uptake of hydrolytic product.
C H Halsted, A M Reisenauer, J J Romero, D S Cantor, B Ruebner
The anti-helminthic drug levamisole hydrochloride has been reported to stimulate immune responses in humans and experimental animals. We have investigated levamisole effects on human leukocyte locomotion in vitro in studies of neutrophils and mononuclear cells from normal adults, from patients with Chediak-Higashi disease and from patients with the syndrome of hyperimmunoglobulin E, recurrent pyogenic infections, and defective leukocyte chemotaxis. Directed migration (chemotaxis) of neutrophils and mononuclear cells from normal adults and from the hyperimmunoglobulin E syndrome patients, but not from Chediak-Higashi patients, were stimulated by levamisole at concentrations of 0.01-1.0 micronM, with stimulation observed most consistently at 0.1 micronM. These concentrations of drug also increased cyclic GMP levels in mononuclear cells and enhanced hexose monophosphate shunt activity in neutrophils, but did not alter chemotactic factor-induced changes in the surface charge of neutrophils. Other concentrations of levamisole did not affect leukocyte locomotion except for a high concentration (5.0 mM) which stimulated both random and directed leukocyte migration. When patients with the hyperimmunoglobulin E syndrome took levamisole by mouth, the abnormal chemotactic responses of their neutrophils were significantly improved towards normal. These studies are the first to show pharmacologic improvement of in vitro leukocyte locomotion in patients in whom recurrent infections have been attributed to a defect of this leukocyte function.
D G Wright, C H Kirkpatrick, J I Gallin
Small amounts (10(-10) mol) of purified human chemotactic factor inactivator (CFI) suppress leukocytic infiltration, permeability changes, and hemorrhage associated with acute immune complex-induced injury in rats. The reversed passive dermal Arthus reaction and acute immune complex-induced alveolitis in rats have served as the model systems of inflammation. The mechanism of inhibition does not appear to relate to interference with formation and deposition of immune complexes, or with fixation of complement in vitro or iv vivo. Human CFI inhibits in vitro the chemotactic activity generated in complement-activated rat serum. The inhibitory effects of human CFI are not seen if it is first heat inactivated. The data provide the first direct support for the conclusion that CFI has anti-inflammatory activity.
K J Johnson, T P Anderson, P A Ward
During the aerobic conversion of xanthine to uric acid by xanthine oxidase, superoxide anion and hydrogen peroxide are produced along with the hydroxyl radical. Our studies demonstrate that washed human platelets incubated with xanthine and xanthine oxidase aggregated and released [14C]serotonin. Aggregation and release were dependent on the duration of exposure to xanthine oxidase as well as the concentration of enzyme. Both reactions were inhibited by the superoxide scavenger enzyme superoxide dismutase but not by catalase, or the free radical scavenger mannitol, suggesting that they were induced by superoxide anion. Superoxide-dependent release was inhibited by prior incubation of platelets with 1 mM EDTA, 1 micronM prostaglandin E1, or 1 mM dibutyryl cyclic AMP, but was unaffected by 1 mM acetylsalicylic acid or 1 micronM indomethacin. After prolonged incubation with xanthine and xanthine oxidase there was also efflux of up to 15% of intraplatelet 51Cr, a cytosol marker. This leakage was prevented by the addition of catalase to the media but not by superoxide dismutase. Incubation with xanthine and xanthine oxidase did not produce malonyldialdehyde, the three-carbon fatty acid fragment produced during prostaglandin endoperoxide synthesis and lipid peroxidation. Prior exposure of platelets to low fluxes of superoxide anion lowered the threshold for release by subsequent addition of thrombin, suggesting a synergistic effect. We conclude that superoxide-dependent aggregation and release may be a physiologically important method to modulate hemostatic reactions particularly in areas of inflammation or vessel injury which could have high local concentrations of superoxide anion.
R I Handin, R Karabin, G J Boxer
Viable suspensions of human colonic mucosal lymphoid cells have been prepared by sequential treatment of tissue with dithiothreitol, EDTA in calcium- and magnesium-free salt solutions, and purified collagenase. The intestinal lymphocyte population, in comparison with that of peripheral blood, had greater numbers of bone marrow-derived cells, particularly cells bearing membrane IgA; showed spontaneous association with macrophages; underwent rapid rosette formation with sheep erythrocytes; and demonstrated increased in vitro synthesis of immunoglobulin. Total thymus-derived cells were equal in the two populations. Decreases were found in "null" cell numbers, in cells bearing membrane IgD and IgM, and in responsiveness to phytohemagglutinin. Macrophage/monocytes in the intestinal population were increased in size, granularity, motility, sustained glass adherence, and phagocytic activity. Human intestinal lymphoid cells appear to constitute a cell population that is more "mature" and/or "activated", in comparison with the lymphoid cells of peripheral blood. The method of preparation should lend itself to the study of inflammatory bowel disease, gastrointestinal cancer, and the intestinal secretory immune system.
D M Bull, M A Bookman
Calcium and phosphate transport was examined in rabbit thin descending, thin ascending, and thick ascending limbs of Henle by in vitro perfusion of isolated tubular segments. Permeability coefficients for these segments with 45Ca and 32PO4 were determined for both lumen-to-bath and bath-to-lumen directions. Both the thin descending and thin ascending limbs were found to be relatively impermeable to both 45Ca and 32PO4. In neither segment were we able to show evidence for net transport of calcium or phosphate. In contrast, the thick ascending limb of Henle showed a decrease in calcium lumen-to-bath concentration from 0.97 +/- 0.02 to 0.88 +/- 0.02 when perfused at 4.8 nl min-1. 45Ca lumen-to-bath and bath-to-lumen fluxes were 19.96 +/- 1.05 and 9.89 +/- 0.02 peq-min-1-cm-1, respectively, and the potential difference was +3.8 +/- 0.3 mV (lumen positive). The observed calcium flux ratio was significantly higher than that predicted by Ussing's equation. When ouabain was added to the bath the potential difference fell to +1.1 +/- 0.3 mV, whereas the calcium efflux was only slightly diminished (29.5 +/- 5.3-23.7 +/- 5.1 peq-cm-1-min-1). Ouabain had no effect on the influx of Ca across the thick ascending limb of Henle. There was no net transport of phosphate across the thick ascending limb. Phosphate permeability was exceedingly low bidirectionally across the thick ascending limb. Our findings indicate: (a) all segments of Henle's loop are relatively impermeable to calcium and phosphate; (b) net transport of phosphate seems to be absent in Henle's loop; (c) net calcium reabsorption, which cannot be explained by passive mechanisms, occurs in the thick ascending limb.
A S Rocha, J B Magaldi, J P Kokko
By utilizing a simple modification of previous immunological assays, we have demonstrated that most, if not all, hemophilic plasmas contain antigen reactive with human antibodies directed against Factor VIII procoagulant activity (VIIIc). Antibodies developing in a nonhemophiliac patient and in a hemophiliac patient gave similar results. The VIIIc antigen so identified was removed from hemophilic plasmas with immobilized rabbit antibody which reacted with normal VIIIc and von Willebrand's disease antigen. These data suggest that there are greater antigenic similarities between normal and hemophilic Factor VIII than previously thought.
T S Zimmerman, L de la Pointe, T S Edgington
This study describes two sensitive, rapid, relatively simple, competitive inhibition radioimmunoassays for detecting immune complex. The tests are based on the inhibition of I125-Clq or I125-monoclonal rheumatoid factor (mRF) binding to an insoluble substrate, IgG-Sepharose. The assays can be performed in 5 h utilizing 10 micronl of serum. Heating of serum is not required and polyclonal rheumatoid factors do not interefere. With the two assays, a wide range of complexes of various size and complement fixing activity can be detected. The Clq test can detect complement fixing Ig complexes larger than 19S, while the mRF tests detect complexes of IgG as small as 8S irrespective of their complement fixing activity. Mouse, rabbit, and human aggregated IgG (agg IgG) can be detected in the Clq test, and human and rabbit agg IgG in the mRF test. As low as 4 microng/ml of isolated human agg IgG can be detected in the Clq test and 0.5 microng/ml in the rheumatoid factor test. Sensitivity is greater for mouse agg IgG. For pathologic sera which must be diluted to eliminate interfering factors, the sensitivity of the assay is approximately 10 times less. The Clq test showed marked inhibition by systemic lupus erythematosus sera with close correlation with CH50 levels and disease activity. The mRF test showed better correlation with rheumatoid arthritis sera. In addition, anionic macromolecules known to react with Clq and other Clq reactants that occur in pathologic sera such as the "low molecular weight" substances in systemic lupus erythematosus are also detected. These reactants are not detectable in the mRF test and can be eliminated in the Clq test by performing the test at higher ionic strength. The tests can be applied to the study of a variety of pathologic states where immune complexes appear to play a role.
A Gabriel Jr, V Agnello
1 mg ovine prolactin was injected intramuscularly into rabbit fetuses (24th day of gestation) located in one of the two uterine horns exposed by laparotomy (n = 12). Fetuses in the other uterine horn were injected with an identical volume of vector and served as controls (n = 13). 2 days later the fetuses were removed by a second laparotomy and sacrificed. Analysis of lung tissue composition yielded the following results: (a) the prolactin-treated group of fetuses showed 40% higher total lung phospholipid content (17.0 +/- 0.8 micronmol/g) than the control group (12.2 +/- 0.5 micronmol/g); (b) the prolactin-treated group had a 67% higher lung lecithin content (8.7 +/- 0.8 micronmol/g) than the control group (5.2 +/- 0.4 micronmol/g); (c) dipalmitoyllecithin accounted for 67% of total lung lecithin in the prolactin-treated group and 44% in the control group. These differences were statistically highly significant (P less than 0.001). However, between the prolactin-treated and the control groups, there were no statistically significant differences in body weight and length, lung weight, the ratio of lung weight to body weight, DNA, protein and, water content. These results suggest that prolactin might be a trigger of lung surfactant synthesis in the rabbit fetus.
M Hamosh, P Hamosh