S J Korsmeyer
Only antibodies of the IgM class support the lytic effect of complement on Giardia lamblia (GL). We sensitized GL trophozoites (SGL) at 4 degrees C with serum containing anti-GL antibodies or IgM purified from this serum, and either normal human serum (NHS), complement 2-deficient human serum (C2d-HS), or C4-deficient guinea pig serum was used as source of complement. SGL were killed by NHS (86%) and by the deficient sera (50 and 40%, respectively), suggesting activation of the alternative pathway. However, the reaction was inhibited by Mg-EGTA. These observations led to studies of the role of C1. The lytic effect of NHS and C2d-HS on SGL was abolished by immunochemically depleting C1 from these sera, and reconstituted by adding purified C1q plus C1r and C1s. Factor B-depleted C2d-HS also lost its capacity to mediate killing, but reconstitution with factor B led to a dose-dependent increase in the killing of SGL. We next investigated the participation of the membrane attack complex in this system. SGL carrying C5b to C7 were lysed when incubated with C8 alone (56%); the addition of C9 further increased killing (98%), while C9 in the absence of C8 had no effect. We concluded that although activation of the classical pathway produces lysis of SGL, lysis may also proceed through a unique pathway of complement activation that requires C1 and factor B, but is independent of C4 and C2. Lysis of SGL can be accomplished by C5b to C8 in the absence of C9.
M Deguchi, F D Gillin, I Gigli
We describe two patients with short-chain acyl-coenzyme A (CoA) dehydrogenase (SCADH) deficiency. Neonate I excreted large amounts of ethylmalonate and methylsuccinate; ethylmalonate excretion increased after a medium-chain triglyceride load. Neonate II died postnatally and excreted ethylmalonate, butyrate, 3-hydroxybutyrate, adipate, and lactate. Both neonates' fibroblasts catabolized [1-14C]butyrate poorly (29-64% of control). Neonate I had moderately decreased [1-14C]octanoate catabolism (43-60% of control), while neonate II oxidized this substrate normally; both catabolized radiolabeled palmitate, succinate, and/or leucine normally. Cell sonicates from neonates I and II dehydrogenated [2,3-3H]butyryl-CoA poorly (41 and 53% of control) and [2,3-3H]octanoyl-CoA more effectively (59 and 95% of control). Mitochondrial acyl-CoA dehydrogenase (ADH) activities with butyryl- and octanoyl-CoAs were 37 and 56% of control in neonate I, and 47 and 81% of control in neonate II, respectively. Monospecific medium-chain ADH (MCADH) antisera inhibited MCADH activity towards both butyryl- and octanoyl-CoAs, revealing SCADH activities to be 1 and 11% of control for neonates I and II, respectively. Fibroblast SCADH and MCADH activities were normal in an adult female with muscular SCADH deficiency.
B A Amendt, C Greene, L Sweetman, J Cloherty, V Shih, A Moon, L Teel, W J Rhead
99mTc-Pertechnetate (99mTcO4-) has widespread clinical use in the diagnosis and evaluation of dysfunctions in many different tissues. However, despite the broad clinical application of this radionuclide, very little is known about the mechanism by which 99mTcO4- enters a cell. We report evidence here that 99mTcO4- shares the Na+/K+/Cl- co-transport system localized to the basolateral membrane of rat parotid acinar cells. 99mTcO4- uptake by these cells was quite rapid (t1/2 approximately 30 s), was completely inhibited by the loop diuretics furosemide and bumetanide, and was markedly dependent on the presence of Na+, K+, and Cl- in the extracellular medium. Relative to uptake measured in the presence of physiological extracellular salt concentrations (Hanks' salts), 99mTcO4- uptake was inhibited 80% by sodium replacement and 50% by potassium replacement. When Cl- was replaced with the physiologically inert anion gluconate a threefold stimulation in 99mTcO4- uptake resulted. These observations provide strong evidence that 99mTcO4- can substitute for Cl- as a substrate for the Na+/K+/Cl- co-transporter and indicate that 99mTcO4- uptake by salivary glands (e.g., as seen with salivary scintiscans), and possibly by a variety of other tissues, reflects the functional activity of this co-transport mechanism.
J Helman, R J Turner, P C Fox, B J Baum
Native and oxidized alpha 1-proteinase inhibitor (alpha 1-PI) were compared as substrates for the metalloproteinase macrophage elastase. At substrate concentrations at which native alpha 1-PI was readily degraded by macrophage elastase, oxidized alpha 1-PI was hardly degraded at all. Incubation of macrophage elastase with oxidized alpha 1-PI before the addition of native alpha 1-PI showed that oxidized alpha 1-PI was not an inhibitor of macrophage elastase. Competition experiments with up to twofold excess oxidized alpha 1-PI did not interfere with the degradation of native alpha 1-PI by macrophage elastase. Sequence analysis of amino acids in degraded native alpha 1-PI showed that macrophage elastase attacked a single peptide bond between Pro-357 and Met-358, the latter representing the P1 reactive-site residue of alpha 1-PI. In oxidized alpha 1-PI, Met-358 was converted to methionine sulfoxide and macrophage elastase hydrolyzed the bond between Phe-352 and Leu-353. These data suggest that methionine may be the primary cleavage site for macrophage elastase and not leucine, as previously thought.
M J Banda, E J Clark, S Sinha, J Travis
Transient exposure to inflammation-associated, fibroblast-stimulatory factors appears to initiate fibrosis by inducing the persistently activated phenotypes displayed by fibroblast cultures derived from scleroderma skin and other fibrotic tissues. To determine whether one class of fibroblast-inhibitory factors, the interferons (IFNs), plays a role in terminating fibrosis by acting as persistent fibroblast deactivators, we inhibited (40-60%) the growth and collagen production of normal dermal fibroblasts and hypercollagen-producing scleroderma fibroblasts by short-term exposure to IFN-alpha, beta, or gamma. During subsequent subculture in the absence of IFNs, the growth and collagen production of normal fibroblasts and the growth of scleroderma fibroblasts increased to untreated control levels after two to three passages. In contrast, collagen production by scleroderma fibroblasts remained inhibited for at least five passages (18 cell doublings) and was not further suppressed by subsequent IFN exposure. These data suggest that IFNs may help terminate fibrosis by suppressing persistently activated fibroblast functions.
M R Duncan, B Berman
We undertook a study of fetal synthesis, storage, and release of atriopeptin (AP). Plasma levels of both atriopeptin immunoreactivity (APir) and the NH2-terminal fragment of the prohormone immunoreactivity (NTFir) were very high in the fetus (4 and 20 times the maternal plasma, respectively). However, the atrial content of the AP was low, but surprisingly, ventricular content of AP was quite high (relative to the adult) in the fetus and fell postnatally. Atrial AP messenger RNA (mRNA) increased with postnatal age, whereas ventricular mRNA was extremely high in the fetus and fell rapidly after birth. High fetal plasma peptide levels may derive from the mother since infusion of exogenous atriopeptin 24 into the mother resulted in parallel increases in fetal and maternal peptide levels. Fetal plasma APir and NTFir levels partially reflect the markedly reduced total renal metabolic capacity compared with that of the adult. Plasma levels fell progressively after birth; whereas neonatal atrial content rose substantially. Plasma AP and NTF were simultaneously elevated in both the maternal and fetal circulation after vasopressin injection of the mother. The fetus can also respond to exogenous stimuli (vasopressin or indomethacin--presumably via ductal closure) and promptly release substantial amounts of peptide into its circulation. Thus, it appears that the AP hormonal system is functional during fetal life and responds avidly to increases in intracardiac pressure as does the mature animal.
Y F Wei, C P Rodi, M L Day, R C Wiegand, L D Needleman, B R Cole, P Needleman
We have studied the structure and function of the insulin receptors in obese patients with and without noninsulin dependent diabetes mellitus (NIDDM) and in nonobese controls using partially purified receptors from muscle biopsies. Insulin binding was decreased in obesity due to reduced number of binding sites but no differences were observed in insulin binding between obese subjects with or without NIDDM. The structural characteristics of the receptors, as determined by affinity labeling methods and electrophoretic mobility of the beta-subunit, were not altered in obese or NIDDM compared to normal weight subjects. Furthermore, the ability of insulin to stimulate the autophosphorylation of the beta-subunit and the phosphoamino acid composition of the phosphorylated receptor were the same in all groups. However, insulin receptor kinase activity was decreased in obesity using Glu4:Tyr1 as exogenous phosphoacceptor without any appreciable additional defect when obesity was associated with NIDDM. Thus, our data are supportive of the hypothesis that in muscle of obese humans, insulin resistance is partially due to decreased insulin receptors and insulin receptor kinase activity. In NIDDM the defect(s) in muscle is probably distal to the insulin receptor kinase.
J F Caro, M K Sinha, S M Raju, O Ittoop, W J Pories, E G Flickinger, D Meelheim, G L Dohm
Menkes kinky hair syndrome is an X-linked neurodegenerative disorder, causing tissue-specific increases in copper and metallothionein content. A mouse model is provided by hemizygotes for mutant alleles at the X-linked mottled locus. Herein we test the possibility that the primary defect in both species is in metallothionein gene regulation. We show that metallothionein-I messenger RNA (mRNA) (mouse) and metallothionein-II mRNA (human) are elevated in mutant fibroblasts. However, comparable dose-response curves in mutant and control cells are generated when mouse metallothionein-I mRNA concentrations are measured in cells exposed to varying concentrations of cadmium or copper (metallothionein inducers). Furthermore, when mutant and control cells are grown to achieve overlapping intracellular copper concentrations in the two cell types, metallothionein-I (mouse) and metallothionein-II (human) mRNA levels are proportional to the intracellular copper concentrations. Finally, in paired determinations in blotchy hemizygote and littermate kidneys containing comparable copper levels, metallothionein-I mRNA contents are very similar. The observations suggest that elevated intracellular copper in these mutants induces metallothionein synthesis by normal regulatory mechanisms.
S Packman, R D Palmiter, M Karin, C O'Toole
Chronic alcoholism is associated with a high prevalence of riboflavin deficiency. Experiments were designed in an animal model to determine whether ethanol alters selectively the absorption of riboflavin and flavin adenine dinucleotide (FAD), the predominant dietary form of the vitamin. Rats received by gavage a liver homogenate to which either [14C]riboflavin or [14C]FAD was added with either ethanol or isocaloric sucrose solutions. Ethanol markedly diminished the bioavailability of [14C]FAD to a greater degree than that of [14C]riboflavin. Corroboration of an ethanol-impaired intraluminal hydrolysis of FAD was provided by using everted jejunal segments and measuring mucosal uptake of [14C]riboflavin together with nonradiolabeled FAD. In subsequent studies with mucosal cell extracts, ethanol markedly inhibited activities of FAD pyrophosphatase and flavin mononucleotide (FMN) phosphatase. These findings suggest that dietary sources of riboflavin (FMN and FAD) are not absorbed as well in the presence of ethanol than are vitamin preparations containing riboflavin, which is utilized more readily.
J Pinto, Y P Huang, R S Rivlin
The effects of dietary protein on the activity of skeletal muscle branched-chain alpha-keto acid dehydrogenase (BCKAD) were investigated. BCKAD is rate-limiting for branched-chain amino acid (BCAA) catabolism by muscle; its activity is modulated by phosphorylation-dephosphorylation. In rats fed an adequate protein (25% casein) diet, BCKAD was approximately 2% active postabsorptively and increased to 10% or 16% active after a 25% or 50% protein meal, respectively. Prolonged feeding of a 50% protein diet increased postabsorptive BCKAD activity to 7% with further increases to 40% active postprandially. On a low protein (9% casein) diet BCKAD remained approximately 2% active regardless of meal-feeding. Dose-dependent activation of BCKAD by intravenous leucine in postabsorptive rats was blunted by a low protein diet. We conclude that excesses of dietary protein enhance the capacity of skeletal muscle to oxidize BCAA, muscle conserves BCAA when protein intake is inadequate, and skeletal muscle may play an important role in whole-body BCAA homeostasis.
K P Block, R P Aftring, W B Mehard, M G Buse
Formyl-methionylleucylphenylalanine (fMLP) activation of neutrophils causes an increase in intracellular Ca2+, activation of protein kinase C and an increase in F-actin content. To examine the role of Ca2+ and protein kinase C activation as determinants of change in F-actin content of neutrophils, we used the NBD-phallacidin extraction assay to compare the kinetics and extent of change in F-actin content of cells activated with fMLP, the calcium ionophore A23187 or phorbol myristate acetate (PMA). All stimuli increase the F-actin content in a dose-dependent manner; however, the rate of increase is slower and the maximum F-actin content is less for calcium ionophore and PMA than for fMLP-activated cells. The A23187-induced increase in F-actin content, but not that of fMLP, depends upon external free [Ca2+]. In A23187-activated cells, F-actin content increases at [Ca2+]free greater than or equal to 5 microM, is maximal at [Ca2+]free greater than or equal to 10 microM and is negligible at physiologic free [Ca2+] (10(-7)-10(-6) M). Combinations of PMA with A23187 or fMLP inhibit the A23187, but not the fMLP, activated actin polymerization. Comparison and combination of these activators shows that neither Ca2+-dependent activation with A23187 nor activation with PMA alone or in combination mimic the fMLP-induced changes in cytoskeleton organization of neutrophils.
T H Howard, D Wang
Plasma vitamin D binding protein (DBP) may scavenge actin released during cell lysis. We examined the plasma disappearance and tissue appearance of 125I-DBP, 125I-G-actin, and the DBP-G-actin complex after their intravenous administration to rats. The plasma disappearance of DBP and DBP-actin were indistinguishable, with rapid initial (t1/2 = 2.6 h) and slower second (t1/2 = 7 h) slopes. After 125I-G-actin (nanomole) injection, plasma disappearance paralleled that of DBP and DBP-actin. All injected actin was associated with DBP, without evidence of free actin, actin-gelsolin complexes or actin oligomers. Tissue appearances of 125I-apo-DBP (apo) or holo-DBP were similar, with highest accumulations in perfused liver, kidney, and skeletal muscle. Although more complex phenomena (plasma entry of F-actin and intracellular actin binding proteins) would occur in vivo after cell lysis, our results suggest a role for DBP in the sequestration and disposition of actin monomers in the circulation.
K D Harper, J F McLeod, M A Kowalski, J G Haddad
Fetal rabbits were treated with corticosteroids by maternal administration for 48 h before delivery at 27 d gestational age. The treated and control rabbits were placed on ventilator-plethysmographs so that ventilation could be adjusted by regulation of tidal volumes to 10-13 ml/kg body wt. [125I]albumin was mixed with fetal lung fluid at birth, alternate rabbits from each litter were treated with Surfactant-TA, and [131I]albumin was injected intravascularly. The movement of the labeled albumins into and out of the alveolar wash and lung tissue was measured after 30 min of ventilation. Corticosteroid treatment (total dose, 0.2 mg/kg betamethasone) significantly decreased the protein leak across the endothelium (P less than 0.001) but increased the protein leak across the epithelium (P less than 0.001). Surfactant treatment decreased both the endothelial and epithelial leaks, and the combination of surfactant and corticosteroid treatments decreased endothelial leaks to 29% of control values and increased compliance more than either treatment alone. The 48-h corticosteroid treatment did not increase alveolar surfactant pool sizes. Corticosteroids significantly changed lung protein leaks independently of surfactant, and improved the response of the preterm lung to surfactant treatments.
M Ikegami, D Berry, T elKady, A Pettenazzo, S Seidner, A Jobe
Reactive oxygen species, particularly hydrogen peroxide (H2O2), participate in neutrophil-mediated glomerulonephritis. However, the mechanism of H2O2 neptrotoxicity is unknown. Myeloperoxidase (MPO), a neutrophil cationic enzyme that localizes in glomeruli, can react with H2O2 and halides to form highly reactive products. We tested the hypothesis that the MPO-H2O2-halide system may induce glomerular injury by infusing MPO followed by H2O2 in a chloride-containing solution into the renal artery of rats. Controls received MPO or H2O2 alone. MPO-H2O2-perfused rats developed significant proteinuria, endothelial cell swelling, and epithelial cell foot process effacement, whereas control kidneys were normal. In the presence of free 125I, MPO-H2O2-perfused rats incorporated large amounts of 125I, localized to the glomerular basement membrane and mesangium by autoradiography, into glomeruli. Glomerular iodination was greatly decreased or absent in controls. The MPO-H2O2-halide system causes glomerular injury and may be important in neutrophil-mediated glomerulonephritis.
R J Johnson, W G Couser, E Y Chi, S Adler, S J Klebanoff
We have developed a new bioassay for thyrotropin (TSH) in human serum to evaluate bioactivity in normal individuals and patients with different degrees of primary hypothyroidism. Unpurified TSH in serum showed no stimulation of cyclic AMP production in cultured FRTL-5 rat thyroid cells, but after immunopurification showed potent stimulatory activity. Immunoaffinity purification permitted up to 400-fold concentration of serum TSH, allowing bioactivity measurements even in certain normal sera. The limit of detection in the FRTL-5 bioassay was 10 microU of human TSH per 0.5 ml incubate, and half-maximal responses for standard human TSH was 102 +/- 26 (+/- SE) microU/0.5 ml. Immunoaffinity-purified serum TSH varied in bioactivity-to-immunoactivity (B/I) ratios from less than 0.25 to 1.21 among four euthyroid subjects and eight primary hypothyroid patients. An inverse correlation was found between B/I ratios of immunopurified basal TSH and the serum-free T4 (r = -0.7237, P less than 0.01), T4 (r = -0.6650, P less than 0.05), and T3 (r = -0.6382, P less than 0.05). B/I ratios of immunopurified TSH from three hypothyroid patients before and after acute stimulation by thyrotropin-releasing hormone showed no significant change, despite major changes in serum TSH. In summary, the present study shows an inverse relationship between the metabolic status of an individual and the intrinsic bioactivity of TSH.
P A Dahlberg, P A Petrick, M Nissim, M M Menezes-Ferreira, B D Weintraub
The X-linked form of severe combined immunodeficiency (XSCID) is underdiagnosed because no methods have been available for detecting carriers. Although boys with XSCID are deficient in T cells, female carriers are immunologically normal. Carriers' normal immune function would be expected if all their T cells were derived from precursors whose X chromosome bearing the XSCID mutation was inactivated early in embryogenesis. Using somatic cell hybridization to separate the active and inactive X chromosomes and restriction fragment length polymorphisms to distinguish them, we have determined the lymphocyte X inactivation pattern in XSCID carriers and their female relatives. In the T cells of three carriers, the X chromosome bearing the XSCID mutation was consistently inactive. Nonrandom X inactivation was also found in the T cells of one at-risk female, while two others had normal, random X inactivation. This method constitutes a generally applicable carrier test for XSCID.
J M Puck, R L Nussbaum, M E Conley
We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.
H M Lokhorst, S E Boom, B J Bast, P J Peters, T F Tedder, J Gerdes, E Petersen, R E Ballieux
Using a small cell lung cancer (SCLC) cDNA library, we obtained clones for the creatine kinase-B (CK-B) gene and determined the nucleotide sequence for the protein coding and 3' untranslated region (3' UT). The human translated protein spans 381 residues and the amino acid homology with rabbit CK-B is greater than 98%. We have demonstrated that a nucleic acid probe encompassing the protein coding region will also hybridize to CK-M sequences while a probe derived from the 3' UT region is CK-B specific. When a B-isoenzyme specific sequence is hybridized to Eco RI cut genomic DNA, two independent restriction fragment polymorphisms are detected. We have subsequently localized these two CK-B homologous sequences to chromosomes 14q32 and 16. Finally, we show that increased levels of CK-B seen in SCLC are not accompanied by gene amplification or rearrangement, but reflect a greatly enhanced level of CK-B specific mRNA that is not seen in non-SCLC lines thus far examined.
F J Kaye, O W McBride, J F Battey, A F Gazdar, E A Sausville
Epstein-Barr virus (EBV)-transformed human B lymphocytes were fused with a murine-human heteromyeloma to produce stable hybrid cell lines that secreted human monoclonal antibodies (mAbs) of the IgM class that recognized conserved epitopes in the core-lipid A region of lipopolysaccharides (LPS). Three of the mAbs reacted with epitopes on the lipid A moiety, while a fourth recognized a determinant in the core oligosaccharide. The lipid A-specific mAbs cross-reacted with heterologous rough LPS and with lipid As released by acid hydrolysis of different intact (smooth) LPS. Carbohydrate groups in the O-side chain and core oligosaccharide of isolated, smooth LPS restricted antibody access to antigenic sites on lipid A. Yet, one lipid A-reactive mAb recognized its epitope on the surfaces of a variety of intact bacteria. These findings confirm the presence of highly conserved epitopes in the core-lipid A complex and prove the existence of human B cell clones with the potential for secreting high avidity IgM antibodies that react with these widely shared determinants. Such human mAbs might provide protective activity against disease caused by diverse gram-negative bacteria.
M Pollack, A A Raubitschek, J W Larrick
Whether steroids lead to thinner scars and larger aneurysms by delaying collagen deposition or worsening infarct expansion before significant collagen deposition begins is unknown. Rats underwent either transmural infarction by left coronary ligation or sham operation. Both infarct and sham rats were randomized to methylprednisolone 50 mg/kg i.p. X 4 or saline treatment within 24 h after operation. Sacrifice occurred before (3 d) or after (7 d) collagen deposition typically begins. Despite similar infarct size, infarct wall thickness was 1.35 +/- 0.08 mm in the saline and 0.99 +/- 0.12 mm in the methylprednisolone group (P less than 0.001) at 3 d. This decrease in wall thickness was explained by a decrease in the number of myocytes across the infarct wall (r = 0.99; P less than 0.001), suggesting that steroids promote myocyte slippage. Furthermore, methylprednisolone caused no further infarct thinning or cavity dilatation beyond 3 d. Thus, high-dose methylprednisolone given within 24 h after transmural infarction worsens infarct expansion before collagen is laid down by promoting the slippage of necrotic myocytes.
J A Mannisi, H F Weisman, D E Bush, P Dudeck, B Healy
Neovascularization has a role in the propagation of rheumatoid synovitis because the spread of mononuclear cell infiltration and the growth of pannus are dependent on the growth of new blood vessels. Growth of such vessels requires local endothelial cell (EC) proliferation. Inhibition of synovial EC proliferation, therefore, would have the potential to diminish rheumatoid inflammation. We have, therefore, studied the effects of gold sodium thiomalate (GST), auranofin, and gold chloride on the proliferation of human umbilical vein EC. GST suppressed both basal and EC growth factor-induced tritiated thymidine incorporation into EC in a dose-dependent fashion. Inhibition was observed with concentrations as low as 1 microgram/ml GST, 5 micrograms/ml gold chloride, and 0.1 microgram/ml auranofin, levels attainable in blood and synovium of patients. These results suggest that gold compounds have an antiangiogenic effect. The low concentrations inhibiting EC proliferation suggest that gold compounds may suppress rheumatoid synovitis by reducing the number of small blood vessels available for mononuclear cell infiltration and synovial tissue proliferation.
T Matsubara, M Ziff
Chronic potassium deficiency results in progressive tubulointerstitial injury, associated with augmented renal ammoniagenesis. We investigated the role of elevated renal ammonia levels and the interaction of ammonia with the complement system in this injury. Potassium deficiency was induced in rats by feeding a low potassium diet. Experimental animals received 150 mM NaHCO3 or equimolar NaCl, as drinking water. After 3 wk, NaHCO3 supplemented rats demonstrated decreased ammonia production, less renal hypertrophy, less histologic evidence of injury, and less proteinuria. In in vitro studies on normal cortical tubular fragments, the addition of ammonia to serum in concentrations comparable to renal cortical levels in potassium-deficient animals significantly increased tubular deposition of C3 as quantitated by a radiolabeled antibody binding technique. Thus, alkali supplementation reduced chronic tubulointerstitial disease in a rat model of hypokalemic nephropathy. We propose that increased cortical ammonia levels contribute to hypokalemic nephropathy through ammonia-mediated activation of the alternative complement pathway.
J P Tolins, M K Hostetter, T H Hostetter
Among all patients with von Willebrand disease (vWD), alloantibodies to von Willebrand factor (vWF) have been described only in severe vWD (type III). The relationship between the development of alloantibodies and the nature of the genetic lesion in vWD is not known. In hemophilia B, large deletions within the factor IX gene appear to correlate with the occurrence of alloantibodies, whereas in hemophilia A no such correlation is apparent. We have studied 19 patients with severe recessive vWD (type III) and 19 with autosomal dominant vWD (type I) by Southern blotting with probes encompassing the full 9 kilobases (kb) of the vWF cDNA. Two apparently unrelated patients were shown to have large deletions within the vWF gene. Both patients had severe vWD (type III) and were the only patients among those studied that had inhibitory alloantibodies to vWF. The extent of deletion was similar in both patients, corresponding to at least the 3'-7.4 kb of the vWF cDNA. The deletion in each patient was estimated to exceed 110 kb. In addition, the localization of the vWF gene to chromosome 12 was confirmed, and a homologous sequence on chromosome 22 was identified.
B B Shelton-Inloes, F F Chehab, P M Mannucci, A B Federici, J E Sadler
A single infusion of phospholipid liposomes promptly and persistently abolished the ability of hypercholesterolemic rabbit plasma to cause cholesteryl ester loading in cultured macrophages. This phospholipid enrichment of plasma caused moderate stimulation of cellular cholesterol efflux and, unexpectedly, almost complete inhibition of cellular uptake of beta-very low density lipoprotein (beta-VLDL), the major cholesteryl ester-rich particle in hypercholesterolemic rabbit plasma. Cell viability and LDL receptor activity were unaffected. Incubation of liposomes with beta-VLDL resulted in transfer of apolipoprotein-E (apoE) to the liposomes; reisolated apoE-phospholipid liposomes then competed efficiently for cellular apoprotein receptors. Thus, a major mechanism by which phospholipid infusions result in diminished accumulation of cholesteryl ester in cultured macrophages is by blocking cellular uptake of beta-VLDL. The liposomes deplete beta-VLDL of apoE, then compete for receptor-mediated uptake. These results suggest a novel mechanism contributing to the known antiatherogenic effect of phospholipid infusions: infused liposomes acquire apoE, then block uptake of atherogenic lipoproteins by arterial wall macrophages.
K J Williams, A R Tall, C Bisgaier, R Brocia
To determine the relation between stenosis anatomy and perfusion in man, 31 patients had quantitative coronary arteriography and positron imaging (PET) with Rb-82 or N-13 ammonia at rest and after dipyridamole-handgrip stress. 10 patients were also studied after angioplasty (total stenoses = 41). Percent narrowing and absolute cross-sectional luminal area were related through a quadratic function to myocardial perfusion reserve determined with PET. Arteriographically determined coronary flow reserve was linearly related to relative myocardial perfusion reserve as expected, based on the derivation of equations for stenosis flow reserve. All of the correlations had considerable scatter, indicating that no single measurement derived by coronary arteriography was a good indicator of perfusion reserve by PET in individual patients. This study provides the relation between all anatomic dimensions of coronary artery stenoses and myocardial perfusion reserve in man, and suggests that PET indicates the functional significance of coronary artery stenoses for clinical purposes.
R A Goldstein, R L Kirkeeide, L L Demer, M Merhige, A Nishikawa, R W Smalling, N A Mullani, K L Gould
Axo-glial dysjunction refers to the disruption of important junctional complexes that anchor terminal loops of myelin to the paranodal axolemma in diabetic human and animal peripheral nerve. Neither axo-glial dysjunction nor the preceeding acute localized paranodal swelling has been specifically attributed to discrete metabolic consequences of insulin deficiency or hyperglycemia. Two metabolic sequelae of hyperglycemia in diabetic nerve, sorbitol accumulation via aldose reductase, and (Na,K)-ATPase deficiency related to myo-inositol depletion, were explored as possible underlying causes of acute paranodal swelling in the spontaneously diabetic bio-breeding rat. 3 wk of insulin replacement, or therapy with an aldose reductase inhibitor or myo-inositol completely reversed paranodal swelling in sural nerve fibers after 3 wk of untreated insulin deficiency. These observations suggest that insulin deficiency and hyperglycemia cause reversible paranodal swelling, and ultimately poorly reversible axo-glial dysjunction, via the myo-inositol-related (Na,K)-ATPase defect rather than by the osmotic effects of sorbitol accumulation within nerve fibers.
D A Greene, S Chakrabarti, S A Lattimer, A A Sima
Infection of normal individuals with human parvovirus (B19) results in a mild disease (erythema infectiosum) but gives rise to aplastic crises in patients with chronic hemolytic anemias. The effects of this disease on hemopoiesis were investigated following intranasal inoculation of the virus into three volunteers. A typical disease ensued with a viremia peaking at 9 d. Marrow morphology 6 d after inoculation appeared normal but at 10 d there was a severe loss of erythroid precursors followed by a 1-2-g drop in hemoglobin, and an increase in serum immunoreactive erythropoietin. Erythroid burst-forming units (BFU-E) from the peripheral blood were considerably reduced, starting at the time of viremia and persisting for 4-8 d depending on the individual. Granulocyte-macrophage colony-forming units (CFU-GM) were also affected but the loss started 2 d later. Both CFU-GM and BFU-E showed a sharp overshoot at recovery. In the marrow, BFU-E and CFU-E were reduced at 6 and 10 d in the individual having the longest period of peripheral progenitor loss. In contrast, there was an increase in BFU-E and CFU-E in the subject with least change in peripheral progenitors. In the third subject, with an intermediate picture, there was a loss at 6 d but an increase at 10 d of erythroid progenitors. It is suggested that the architecture of the marrow might partially isolate progenitors from high titers of virus in the serum and individual variation in this respect might give the results observed.
C G Potter, A C Potter, C S Hatton, H M Chapel, M J Anderson, J R Pattison, D A Tyrrell, P G Higgins, J S Willman, H F Parry
The effects of the short-chain aliphatic carboxylic acid, n-butyrate, on glycosaminoglycan (GAG) accumulation were studied in cultured human skin fibroblasts. Normal fibroblast cultures were grown to confluence, shifted to a medium without or with n-butyrate for 24 h, labeled with either [3H]acetate or [3H]glucosamine and analyzed for [3H]GAG and [3H]hyaluronate accumulation. Accumulation was stimulated at low concentrations (0.1-1 mM) by up to 27%. Higher concentrations of n-butyrate (greater than 1 mM) inhibited [3H]GAG by up to 70-90%. This effect was maximal at 10 mM and half-maximal at 3 mM. Propionate had similar effects but was less potent. Parallel studies conducted in colonic fibroblasts revealed that n-butyrate could markedly inhibit [3H]GAG accumulation in that cell type as well. These effects were rapid, occurring within 3 h of treatment, and were reversible. Chondroitin sulfate accumulation was unaffected by the compound. A pulse-chase study failed to demonstrate any effect on [3H]GAG degradation.
T J Smith
Within 20 min after intraperitoneal injection of Salmonella enteritidis endotoxin in rats, blood platelet-activating factor (PAF) increased from 4.3 +/- 1.3 to 13.7 +/- 2.0 ng/ml (P less than 0.01) and lung PAF from 32.3 +/- 4.9 to 312.3 +/- 19.6 ng (P less than 0.01), but not lung lavage PAF. We tested the effect of PAF receptor antagonists, CV 3988 and SRI 63-441, on endotoxin-induced hemodynamic changes and lung vascular injury. Pretreatment with CV 3988 attenuated systemic hypotension, preserved hypoxic pulmonary vasoconstriction, and prolonged survival of awake catheter-implanted endotoxin-treated (20 mg/kg) rats. Pretreatment with SRI 63-441 prevented the depressed hypoxic pulmonary vasoconstriction after low dose (2 mg/kg) endotoxin. Both CV 3988 and SRI 63-441 blocked the increased extravascular accumulation of 125I-albumin and water in perfused lungs isolated from endotoxin-treated rats. We conclude that PAF is produced in the lung during endotoxemia and may be an important mediator of the systemic and pulmonary hemodynamic changes as well as the acute lung vascular injury after endotoxemia.
S W Chang, C O Feddersen, P M Henson, N F Voelkel
Insulin resistance is characteristic of the diabetic state. To define the role of hyperglycemia in generation of the insulin resistance, we examined the effect of phlorizin treatment on tissue sensitivity to insulin in partially pancreatectomized rats. Five groups were studied: group I, sham-operated controls; group II, partially pancreatectomized diabetic rats with moderate glucose intolerance; group III, diabetic rats treated with phlorizin to normalize glucose tolerance; group IV, phlorizin-treated controls; and group V, phlorizin-treated diabetic rats restudied after discontinuation of phlorizin. Insulin sensitivity was assessed with the euglyemic hyperinsulinemic clamp technique in awake, unstressed rats. Insulin-mediated glucose metabolism was reduced by approximately 30% (P less than 0.001) in diabetic rats. Phlorizin treatment of diabetic rats completely normalized insulin sensitivity but had no effect on insulin action in controls. Discontinuation of phlorizin in phlorizin-treated diabetic rats resulted in the reemergence of insulin resistance. These data demonstrate that a reduction of beta-cell mass leads to the development of insulin resistance, and correction of hyperglycemia with phlorizin, without change in insulin levels, normalizes insulin sensitivity. These results provide the first in vivo evidence that hyperglycemia per se can lead to the development of insulin resistance.
L Rossetti, D Smith, G I Shulman, D Papachristou, R A DeFronzo
The antigen-specific immune unresponsiveness seen in bancroftian filariasis was studied by examining lymphokine production in peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from 10 patients with asymptomatic microfilaremia, 13 patients with elephantiasis and 6 normal North Americans. In each group of patients, the kinetics of the lymphokine response and the response to mitogens and nonparasite antigens did not differ significantly. In marked contrast, when antigen-induced lymphokine production was examined, most patients with microfilaremia were unable to produce either interleukin 2 (IL-2) or gamma-interferon (i.e., were nonresponders), and the few who could (hyporesponders, generally with quite low microfilaremia levels) did so at levels significantly less than those of patients with elephantiasis, all of whom showed strong responses to parasite antigen. Removal of neither adherent cells or T8+ cells affected the parasite-specific anergy seen in those with microfilaremia, suggesting a state of T cell tolerance to the parasite in patients with this most common clinical manifestation of bancroftian filariasis.
T B Nutman, V Kumaraswami, E A Ottesen
DNA-DNA crosslinks are the lethal cellular mechanism of bifunctional alkylating agent cytotoxicity. Novobiocin, an inhibitor of DNA topoisomerase II, impairs eukaryotic DNA repair of alkylating agent adducts and may increase the number of adducts and their resultant cytotoxicity in malignant cells. The effect of novobiocin on clonogenic survival and DNA crosslinking due to cisplatin (cDDP) and carmustine (BCNU) was studied. Novobiocin caused synergistic cytotoxicity in Chinese hamster ovary cells exposed to cDDP or BCNU. Novobiocin and cDDP increased the formation of DNA-DNA interstrand crosslinks six-fold greater than cDDP alone. The effect was schedule dependent. Novobiocin and cDDP or BCNU markedly reduced in vivo growth of a murine fibrosarcoma without increased host toxicity. As a modulating agent of cytotoxicity due to DNA-DNA crosslinking, novobiocin may enhance the clinical effectiveness of the alkylating agents in human cancer and offer insight into new therapeutic strategies.
J P Eder, B A Teicher, S A Holden, K N Cathcart, L E Schnipper
We have attempted to identify a role for mast cells in autonomic ganglia by examining the effects of antigen challenge on mast cell-associated mediator release and synaptic transmission through the superior cervical ganglion isolated from ovalbumin-sensitized guinea pigs. Ovalbumin induced the release of 7.9 ng of histamine, 40 pg of immunoreactive sulfidopeptide-leukotriene, and 140 pg of immunoreactive-PgD2 per ganglion. Ovalbumin produced long-lasting potentiation (51 +/- 4%, mean +/- SEM, n = 66) of synaptic transmission, the protracted nature of which could not be mimicked by exogenous histamine (10(-5) M). Selective histamine H1 antagonists inhibited the antigen-induced potentiation, but did not reverse it when added any time after antigen exposure. These results indicate that immunologic activation of mast cells can directly potentiate neurotransmission in sympathetic ganglia. Histamine appears to be a mediator involved in the induction of antigen-induced potentiation of synaptic transmission, but alone cannot account for the long term nature of this phenomenon.
D Weinreich, B J Undem
Hepatoerythropoietic porphyria (HEP) is due to a marked deficiency of uroporphyrinogen (URO) decarboxylase, a cytosolic enzyme in the heme biosynthetic pathway. Using a radioimmunoassay method, we determined the concentration of URO decarboxylase protein in erythrocytes from a patient with mild HEP and found that the enzyme protein concentration had markedly decreased to less than 7% of the normal controls. This finding, however, was in contrast to the enzyme activity in the patient's erythrocytes, which was 16% of normal control levels and different from previously reported HEP cases in that erythrocytes in our patient contained disproportionately elevated URO decarboxylase activity in comparison to its immunoreactive material. Our findings suggests the possibility of a mutant isozyme in this patient that is not immunoreactive with an antibody raised against the normal enzyme.
H Fujita, S Sassa, A C Toback, A Kappas
Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), a Ca2+-mobilizing messenger, can be phosphorylated by a cytoplasmic kinase, yielding inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P3). We observed that stimulation of the antigen receptor on a malignant human T cell line, Jurkat, led to substantial, sustained increases in Ins-1,4,5-P3 and InsP4. The Ins-1,4,5-P3 kinase partially purified from resting Jurkat cells had a maximum velocity (Vmax) of 0.09 nmol/min/mg protein and an apparent Michaelis constant (Km) of 0.2 microM. When the kinase was partially purified 10 min after stimulation of the antigen receptor or after the addition of phorbol myristate acetate, the Vmax was increased twofold. The activity of the Ins-1,4,5-P3 kinase obtained from either resting or stimulated Jurkat cells was enhanced in vitro by increasing the concentration of free Ca2+ from 0.1 to 0.5 microM. These results indicate that the activity of the Ins-1,4,5-P3 kinase is regulated as a consequence of stimulating the T cell antigen receptor.
J B Imboden, G Pattison
Pharmacologic elevation of cyclic AMP (cAMP) promotes growth arrest and differentiation in a variety of transformed mammalian cells, including the HL-60 human promyelocytic leukemia cell line. However, mechanisms underlying this phenomenon are poorly understood. Because cellular oncogenes play a pivotal role in regulating proliferation and differentiation, we examined whether cAMP-promoted differentiation of HL-60 was preceded by a decrease in the expression of c-myc, a cellular oncogene both amplified and constitutively expressed in HL-60. We find that cyclic AMP elevation in HL-60 caused by three different pharmacologic regimens is followed by an abrupt, greater than 90% decrease in steady state c-myc mRNA levels within 3 h, well before detectable changes in proliferation and differentiation. This decrease, which occurs despite protein synthetic blockade, is attributable to transcriptional down-regulation of c-myc and is accompanied by changes in chromatin structure near c-myc promoter sites. Our findings establish that cAMP, a ubiquitous intracellular regulatory messenger previously known only to enhance gene transcriptional activity in higher eukaryotic cells, can also suppress transcription of a cellular oncogene, thereby suggesting a potential mechanism for cAMP-promoted differentiation.
A Slungaard, D L Confer, W H Schubach