R G Crystal
Individuals with sarcoidosis were evaluated for preferential usage of T cells with the gamma delta-positive (+) type of T cell antigen receptor. Compared with normal subjects (n = 19), the group with sarcoidosis had increased numbers of CD3+ alpha beta-negative (-) T cells in the blood (normal, 58 +/- 12 cells/microliters; sarcoid, 192 +/- 45 cells/microliters, P less than 0.05) and in the epithelial lining fluid of the lung (normal, 78 14 cells/microliters; sarcoid, 240 +/- 60 cells/microliters, P less than 0.04) and a concomitant elevated number of blood and lung CD3+ gamma delta+ T cells, owing to a striking increase in the number of CD3+ gamma delta+ T cells in a subgroup (7 of 20) of sarcoid individuals. The elevated numbers of sarcoid blood gamma delta+ T lymphocytes were mostly Ti gamma A+ and delta TCS1-, a pattern also seen in normal individuals, consistent with the majority of gamma delta+ T cells expressing one gamma-chain variable region, V gamma 9. The observation of an increase in the total gamma delta+ T cell numbers in a sarcoid subgroup suggests that various specific stimuli may trigger the expansion of different T cell subpopulations within different groups of individuals with sarcoidosis.
B Balbi, D R Moller, M Kirby, K J Holroyd, R G Crystal
We conducted this study in an effort to characterize and understand vagal abnormalities in heart failure patients whose sympathetic activity is known. We measured sympathetic (peroneal nerve muscle sympathetic recordings and antecubital vein plasma norepinephrine levels) and vagal (R-R intervals and their standard deviations) activities in eight heart failure patients and eight age-matched healthy volunteers, before and after parasympathomimetic and parasympatholytic intravenous doses of atropine sulfate. At rest, sympathetic and parasympathetic outflows were related reciprocally: heart failure patients had high sympathetic and low parasympathetic outflows, and healthy subjects had low sympathetic and high parasympathetic outflows. Low dose atropine, which is known to increase the activity of central vagal-cardiac motoneurons, significantly increased R-R intervals in healthy subjects, but did not alter R-R intervals in heart failure patients. Thus, our data document reciprocal supranormal sympathetic and subnormal parasympathetic outflows in heart failure patients and suggest that these abnormalities result in part from abnormalities within the central nervous system.
T R Porter, D L Eckberg, J M Fritsch, R F Rea, L A Beightol, J F Schmedtje Jr, P K Mohanty
Vitronectin (Vn) is a multifunctional 75-kD glycoprotein that is present in plasma and the extracellular matrix. Vn functions as a complement regulatory protein in plasma, and promotes the growth and attachment of cells in tissue culture. Recent cDNA cloning reveals that like other adhesive proteins, Vn contains the sequence Arg-Gly-Asp and binds to some members of the integrin class of adhesive membrane receptors. In liposomes, the platelet membrane glycoprotein complex IIb/IIIa binds Vn, as well as fibrinogen, von Willebrand factor, and fibronectin. We examined the binding of purified Vn to resting and stimulated human platelets. Vn bound to thrombin-stimulated platelets in a calcium-dependent, specific, and saturable manner with a Kd of 320 nM and 8,000 sites per platelet. Epinephrine or ADP stimulation led to specific binding with KdS of 93 and 116 nM, respectively. Binding was inhibited by the tetrapeptide Arg-Gly-Asp-Ser and by monoclonal and polyclonal antibodies to GPIIb/IIIa. Endogenous platelet Vn stores were identified in immunoblots of gel-filtered platelets and the surface expression of endogenous platelet Vn was thrombin inducible. Monoclonal as well as polyclonal antibodies to Vn inhibited platelet aggregation, suggesting that Vn plays a role in the formation of stable platelet aggregates.
E Asch, E Podack
Ro/SS-A antibodies are found in a number of human autoimmune disorders including Sjogren's syndrome and several systemic lupus erythematosus-related disorders. These heterogeneous autoantibodies are known to recognize several distinct cellular antigens. With synthetic oligonucleotides corresponding to amino acid sequence information we have isolated a full-length cDNA clone which encodes a human Ro ribonucleoprotein autoantigen. The 1,890-base pair clone contains an open reading frame that encodes a 417-amino acid, 48-kD polypeptide that migrates aberrantly at 60 kD by SDS-PAGE. Rabbit antibodies raised against this protein's recently described amino-terminal epitope react with a previously identified 52-kD human Ro protein and immunoprecipitate the human cytoplasmic RNAs. Ultraviolet light cross-linking studies suggest that this Ro protein binds each of the four major human cytoplasmic RNAs. The deduced amino acid sequence is 63% homologous to an Onchocerca volvulus antigen. Southern filter hybridization analysis indicates that this gene is not highly polymorphic and exists as a single copy in the human genome. Chromosomal localization studies place this gene on the short arm of chromosome 19 near the gene encoding the low density lipoprotein receptor.
D P McCauliffe, F A Lux, T S Lieu, I Sanz, J Hanke, M M Newkirk, L L Bachinski, Y Itoh, M J Siciliano, M Reichlin
To evaluate whether IL-6 participates in the host response to intrauterine infection, we studied IL-6 bioactivity and isoforms in amniotic fluid (AF). Two different assays for IL-6 were used: the hepatocyte stimulating factor assay (in Hep3B2 cells) and the SDS-PAGE/immunoblot assay. IL-6 determinations were performed in 205 AF samples. Samples were obtained from patients in the midtrimester of pregnancy (n = 25), at term with no labor (n = 31), at term in active labor (n = 40), and from patients in preterm labor (n = 109). Higher AF IL-6 levels were observed in women in preterm labor with intraamniotic infection than in women in preterm labor without intraamniotic infection (median = 375 ng/ml, range = 30-5000 ng/ml vs. median = 1.5 ng/ml, range = 0-500, respectively, P less than 0.0001). The 23-25- and 28-30-kD IL-6 species could be readily detected in SDS-PAGE immunoblots performed directly on 10-microliters aliquots of AF from patients with intraamniotic infection. Among women in preterm labor with culture-negative AF, those who failed to respond to subsequent tocolytic treatment had higher AF IL-6 concentrations than those who responded to therapy (median = 50 ng/ml vs. median = 1.2 ng/ml, respectively, P less than 0.05). Only low levels of IL-6 were detected in AF obtained from normal women in the midtrimester and third trimester of pregnancy. Decidual tissue explants obtained from the placentas of women undergoing elective cesarean section at term without labor (n = 11) produced IL-6 in response to bacterial endotoxin. In a pilot study, AF IL-6 was determined in 56 consecutive women admitted with preterm labor. All patients (n = 10) with elevated AF IL-6 (cutoff = 46 ng/ml) delivered a premature neonate. 4 of these 10 patients had positive AF cultures for microorganisms. These studies implicate IL-6 in the host response to intrauterine infection and suggest that evaluation of AF IL-6 levels may have diagnostic and prognostic value in the management of women in preterm labor.
R Romero, C Avila, U Santhanam, P B Sehgal
We report the molecular characterization of 2A4, an IgG, DNA-binding antibody bearing the 3I and F4 idiotypes which are associated with anti-DNA antibodies in serum of patients with systemic lupus erythematosus (SLE). The antibody is produced by an EBV-transformed B cell line derived from a patient with multiple myeloma whose myeloma protein is also an IgG, 3I-reactive, F4-reactive, DNA-binding immunoglobulin, although the 2A4 antibody does not itself represent the myeloma protein. The 2A4 heavy chain is encoded by a VH4 gene, a D-D gene fusion and the JH6 gene; the light chain is derived from a Vk1 gene and the Jk2 gene. This is the first human antibody shown to have a CDR3 encoded by a D-D fusion. DNA sequence analysis of the 2A4 VH gene together with a Southern blot of genomic DNA probed with a 2A4 VH-specific oligonucleotide strongly suggest it to be somatically mutated. The data provide evidence that human autoantibodies can be products of somatically mutated genes and suggest that the 2A4 antibody may reflect the selective pressure of antigen.
A Davidson, A Manheimer-Lory, C Aranow, R Peterson, N Hannigan, B Diamond
To test the hypothesis that aldose reductase inhibition may prevent or delay the development of functional and structural neuropathy in the insulin-deficient diabetic Bio-Breeding rat (BB-rat), hyperglycemic rats were begun on the aldose reductase inhibitor (ARI) ponalrestat 25 mg/kg body wt soon after the onset of diabetes and followed for 4 or 6 mo. Ponalrestat treatment completely prevented the characteristic nerve conduction slowing and structural abnormalities of the node of Ranvier for 4 mo despite only partial preservation of axonal integrity. Ponalrestat treatment for 6 mo achieved a partial but significant prevention of nerve conduction slowing, axoglial dysjunction, and axonal degenerative changes. This incomplete but significant prevention of neuropathy by ponalrestat suggests that additional mechanisms besides polyol-pathway activation may be of importance in the pathogenesis of diabetic neuropathy. Alternatively, the dosage used in the present study may not have been sufficient to achieve a complete prevention. Despite the only partial protective effect of ARI treatment on degenerative peripheral nerve changes in hyperglycemic BB-rats, 6 mo of treatment resulted in a more than threefold increase in regenerating nerve fibers. These data suggest that prophylactic ARI treatment may be efficacious in delaying the development of diabetic neuropathy.
A A Sima, A Prashar, W X Zhang, S Chakrabarti, D A Greene
Activated immune cells release cytokines which modulate the activity of bone cells in vitro. Expression of major histocompatibility complex (HLA in humans) class II determinants on bone surface cells may be important in local immune cell activation. In this study, expression of HLA-DR and DQ by cultured human bone cells (HBC) derived from normal trabecular bone surfaces was assessed by fluorescence-activated cell sorter (FACS) analysis and immunoperoxidase techniques using monoclonal antibodies. A subset of HBC (10-30%) expressed DR constitutively while 5-15% displayed DQ during long-term culture. HBC lacked a number of monocyte and lymphocyte markers. In addition, both DR+ and DR- HBC (FACS separated) produced osteocalcin stimulated by 1,25-dihydroxyvitamin D2 (1,25(OH)2D3). This suggests that both phenotypes belong to the osteoblast lineage. The number of DR+ HBC was increased by interferon-gamma (IFN gamma; 40-95% DR+ cells) whereas DQ+ HBC remained unchanged or was slightly increased (5-20% DQ+ cells). Moreover, 1,25(OH)2D3 enhanced IFN gamma-induced DR expression and at high concentration (10(-7) M) augmented DR expression by itself. Other major osteotropic factors, parathyroid hormone, interleukin 1, and calcitonin, did not affect HBC DR expression. The findings suggest that HBC may participate in activation of the immune system and that some osteotropic factors may regulate this function.
H Skjødt, D E Hughes, P R Dobson, R G Russell
We studied the effects of varying degrees and durations of hypernatremia on the brain concentrations of organic compounds believed to be important, so-called "idiogenic" osmoles in rats by means of conventional biochemical assays, nuclear magnetic resonance spectroscopy, and high-performance liquid chromatography. There were no changes in the concentrations of these osmoles (specifically myoinositol, sorbitol, betaine, glycerophosphorylcholine [GPC], phosphocreatine, glutamine, glutamate, and taurine) in rats with acute (2 h) hypernatremia (serum Na 194 +/- 5 meq/liter). With severe (serum Na 180 +/- 4 meq/liter) chronic (7 d) hypernatremia, the concentrations of each of these osmoles except sorbitol increased significantly: myoinositol (65%), betaine (54%), GPC (132%), phosphocreatine (73%), glutamine (143%), glutamate (84%), taurine (78%), and urea (191%). Together, these changes account for 35% of the change in total brain osmolality. With moderate (serum Na 159 +/- 3 meq/liter) hypernatremia, more modest but significant increases in the concentrations of each of these osmoles except betaine and sorbitol were noted. When rats with severe chronic hypernatremia were allowed to drink water freely, their serum sodium as well as the brain concentrations of all of these organic osmoles except myoinositol returned to normal within 2 d. It is concluded that: idiogenic osmoles play an important role in osmoregulation in the brain of rats subjected to hypernatremia; the development of these substances occur more slowly than changes in serum sodium; and the decrease in concentration of myoinositol occurs significantly more slowly than the decrease in serum sodium which occurs when animals are allowed free access to water. These observations may be relevant to the clinical management of patients with hypernatremia.
Y H Lien, J I Shapiro, L Chan
Thrombin increases intracellular calcium ([Ca++]i) in several cell types and causes a positive inotropic effect in the heart. We examined the mechanism of the thrombin-induced [Ca++]i increase in chick embryonic heart cells loaded with the fluorescent calcium indicator, indo-1. Thrombin (1 U/ml) increased both systolic and diastolic [Ca++]i from 617 +/- 62 and 324 +/- 46 to 1041 +/- 93 and 587 +/- 38 nM, respectively. An initial rapid [Ca++]i increase was followed by a more sustained increase. There were associated increases in contraction strength, beat frequency, and action potential duration. The [Ca++]i increase was not blocked by tetrodotoxin or verapamil, but was blocked by pretreatment with pertussis toxin (100 ng/ml). The thrombin-induced [Ca++]i increase was partly due to intracellular calcium release, since it persisted after removal of external calcium. The [Ca++]i increase in zero calcium was more transitory than in normal calcium and was potentiated by 10 mM Li+. Thrombin also induced influx of calcium across the surface membrane, which could be monitored using Mn++ ions, which quench indo-1 fluorescence when they enter the cell. Thrombin-induced Mn++ entry was insensitive to verapamil, but was blocked by 2 mM Ni++. Thrombin increased inositol trisphosphates by 180% at 90 s and this effect was also blocked by pretreatment with pertussis toxin. Conclusion: thrombin promotes calcium entry and release in embryonic heart cells even when action potentials are inhibited. Both modes of [Ca++]i increase may be coupled to the receptor by pertussis toxin-sensitive G proteins.
W W Chien, R Mohabir, W T Clusin
Static exercise in normal humans causes reflex increases in muscle sympathetic nerve activity (MSNA) that are closely coupled to the contraction-induced decrease in muscle cell pH, an index of glycogen degradation and glycolytic flux. To determine if sympathetic activation is attenuated when muscle glycogenolysis is blocked due to myophosphorylase deficiency (McArdle's disease), an inborn enzymatic defect localized to skeletal muscle, we now have performed microelectrode recordings of MSNA in four patients with McArdle's disease during static handgrip contraction. A level of static handgrip that more than doubled MSNA in normal humans had no effect on MSNA and caused an attenuated rise in blood pressure in the patients with myophosphorylase deficiency. In contrast, two nonexercise sympathetic stimuli, Valsalva's maneuver and cold pressor stimulation, evoked comparably large increases in MSNA in patients and normals. The principal new conclusion is that defective glycogen degradation in human skeletal muscle is associated with a specific reflex impairment in sympathetic activation during static exercise.
S L Pryor, S F Lewis, R G Haller, L A Bertocci, R G Victor
Hyp mice exhibit increased renal catabolism of vitamin D metabolites by the C-24 oxidation pathway (1988. J. Clin. Invest. 81:461-465). To examine the regulatory influence of dietary phosphate on the renal vitamin D catabolic pathway in Hyp mice, we measured C-24 oxidation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in renal mitochondria isolated from Hyp mice and normal littermates fed diets containing 0.03% (low-Pi), 1% (control-Pi), and 1.6% (high-Pi) phosphate. In normal mice the low-Pi diet led to a rise in serum 1,25(OH)2D (22.2 +/- 1.8 to 48.1 +/- 6.8 pg/ml, P less than 0.05) and no change in C-24 oxidation products (0.053 +/- 0.006 to 0.066 +/- 0.008 pmol/mg protein per min) when compared with the control diet. In Hyp mice the low-Pi diet elicited a fall in serum 1,25(OH)2D (21.9 +/- 1.2 to 8.0 +/- 0.2 pg/ml, P less than 0.05) and a dramatic increase in C-24 oxidation products (0.120 +/- 0.017 to 0.526 +/- 0.053 pmol/mg protein per min, P less than 0.05) when compared with the control diet. The high-Pi diet did not significantly alter serum levels of 1,25(OH)2D or C-24 oxidation products in normal mice. Hyp mice on the high-Pi diet experienced a rise in serum 1,25(OH)2D (21.9 +/- 1.2 to 40.4 +/- 7.3, P less than 0.05) and a fall in C-24 oxidation products (0.120 +/- 0.017 to 0.043 +/- 0.007 pmol/mg protein per min, P less than 0.05). The present results demonstrate that the defect in C-24 oxidation of 1,25(OH)2D3 in Hyp mice is exacerbated by phosphate depletion and corrected by phosphate supplementation. The data suggest that the disorder in vitamin D metabolism in the mutant strain is secondary to the perturbation in phosphate homeostasis.
H S Tenenhouse, G Jones
In paroxysmal nocturnal hemoglobinuria (PNH), impaired glycosyl-phosphatidylinositol (PI)-anchoring of membrane proteins such as decay-accelerating factor has been known to lead to increased susceptibility to complement. Moreover, abnormal expression of non-PI-anchoring glycoproteins such as C3b/C4b receptor (CR1) or glycophorin-alpha also has been shown in PNH. Therefore, we biochemically analyzed glycosphingolipids (GSL) as one of the membrane glycoconjugates of PNH erythrocytes. Erythrocytes of all seven PNH patients showed altered expression of sialosyl GSL (gangliosides) as compared with the control erythrocytes of healthy donors. Both a sialosylparagloboside (IV6NeuAc-nLc4Cer) among four major gangliosides and some minor gangliosides in normal erythrocytes variably disappeared in erythrocytes from the peripheral blood of PNH patients. As one of the possible mechanisms of altered expression of gangliosides in PNH erythrocytes, structural analysis suggested impaired sialylation of GSL. These results suggest not only the altered metabolism of gangliosides in PNH erythrocytes, but also a metabolic disorder of membrane glycoconjugates as a new feature of PNH.
H Nakakuma, T Kawaguchi, K Horikawa, M Hidaka, Y Yonemura, M Kawakita, T Kagimoto, M Iwamori, Y Nagai, K Takatsuki
Moderate alcohol intoxication in man, a ubiqitious social event, causes acute but reversible myocardial depression, the mechanism of which is unknown. We investigated whether this depression could be due to a direct effect of ethanol on the process of electromechanical coupling by simultaneously measuring the transmembrane action potential and contraction, or the cytosolic calcium transient (via aequorin photoluminescence) and contraction in isolated ferret right ventricular papillary muscle. Ethanol, in concentrations that are similar to plasma levels in man during intoxication (0.15 vol %), depressed the force of contraction approximately 10%. The step in the electromechanical process that was affected appeared to be the calcium-myofilament interaction, as there was no change in the transmembrane action potential or cytosolic calcium transient. This inhibition was quickly reversed by removal of the ethanol from the perfusate. On the other hand, higher concentrations of ethanol produced changes in contraction, the calcium transient, and the action potential, suggesting multiple levels of inhibition of electromechanical coupling. Increasing the perfusate calcium or use of the calcium channel agonist, BAY-K 8644, increased cytosolic calcium to near maximum but had little effect on contractility, confirming that the relationship between calcium and the myofilaments had been altered. These data suggest that the acute depression in ventricular function seen with alcohol consumption may be due to a direct effect on electromechanical coupling through inhibition of the calcium myofilament interaction.
T Guarnieri, E G Lakatta
CAP37, an antimicrobial protein of human neutrophil granules, is a specific chemoattractant for monocytes. Purified to homogeneity by sequential chromatography over carboxymethyl Sephadex, G-75 Sephadex, and hydrophobic interaction HPLC, demonstratively endotoxin-free CAP37 was maximally chemotactic over a range of 1.3 X 10(-9)-10(-8) M. Thus it was active in the same molar concentrations as formyl-methionyl-leucyl-phenylalanine. CAP37 lacked chemotactic activity for neutrophils and lymphocytes. In checkerboard assays CAP37 had some chemokinetic activity as well. It was also chemotactic for rabbit mononuclear cells. Higher concentrations (2.7 X 10(-8) M) were required for activity with rabbit cells than with human. Sequence analysis of the first 42 NH2-terminal amino acid residues of CAP37 showed strong homologies with known serine proteases that mediate various functions in inflammation. However, a critical substitution of a serine for a histidine at position 41 suggested that CAP37 lacked serine protease action. This impression was supported by the failure of CAP37 to bind tritiated diisopropyl fluorophosphate. 89% of total CAP37 was released extracellularly from human neutrophils while they phagocytized Staphylococcus aureus. We propose that CAP37 released from neutrophils during phagocytosis and degranulation may mediate recruitment of monocytes in the second wave of inflammation.
H A Pereira, W M Shafer, J Pohl, L E Martin, J K Spitznagel
Molecular cloning of the human complement inhibitor SP-40,40, has revealed strong homology to a major rat and ram Sertoli cell product, sulfated glycoprotein-2, known also as clusterin. This study reports the purification and characterization of human seminal clusterin. Two-dimensional gel electrophoresis revealed charge differences between clusterin purified from semen and the serum-derived material. Both preparations demonstrate comparable hemagglutination (clustering) activity and inhibition of C5b-6 initiated hemolysis. The average clusterin concentration in normal seminal plasma is considerably higher than that found in serum. Mean seminal plasma clusterin concentrations were significantly lower in azoospermia caused by obstruction or seminiferous tubule failure than with oligospermia or normospermia. Only men with vasal agenesis had undetectable seminal clusterin, suggesting that some of the seminal clusterin is produced by the seminal vesicles. Immunofluorescence of human spermatozoa revealed that clusterin was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology. A pilot study of 25 patients suggests that seminal clusterin concentration, together with sperm motility and morphology, is correlated with the fertilization rate in vitro. The function of seminal clusterin is unknown. Its extensive distribution in the male genital tract and its high concentration in seminal plasma suggests an important role in male fertility.
M K O'Bryan, H W Baker, J R Saunders, L Kirszbaum, I D Walker, P Hudson, D Y Liu, M D Glew, A J d'Apice, B F Murphy
The cell-free supernatants of normal spleen and thymus lymphocytes in short-term culture release low molecular weight (LMW) DNA protein molecules that have an immunoproliferative effect (polyclonal B cell activation) in vitro. We have determined that the protein-LMW DNA complexes responsible for these effects are nucleosomal constituents of chromatin, since the mitogenically active fractions of these cell-free supernatants contain the constituents of core histones (H3, H2A, H2B, H4) together with LMW DNA (140-180 bp), and since the immunoproliferative effects of these cell-free supernatants could be mimicked by various other nucleoprotein preparations (including calf thymus and chicken erythrocyte nucleosomes). The spontaneous cellular release of cleaved chromatin constituents in vitro can be attributed to a form of programmed cell death termed apoptosis, since the cultured spleen cells exhibited (a) morphologic evidence consistent with this process by electron microscopy, and (b) evidence of intracellular cleavage of chromatin which, like apoptosis, could be blocked with ZnSO4. This resulted in inhibition of the extracellular release of nucleosomal constituents as well as the immunoproliferative effects of the cell-free supernatants. The immunoproliferative effect of nucleosomes released from cells during apoptosis could be responsible for previously observed spontaneous in vitro anti-DNA and anti-histone antibody responses of murine spleen cells, and in vivo in normal lymphoid tissues, resulting in renewed cellular proliferation after cell death. In pathological states, this could result in abnormal polyclonal B cell proliferation and autoantibody formation.
D A Bell, B Morrison, P VandenBygaart
Cardiac myocytes were isolated from adult dogs and incubated with isolated canine neutrophils (PMN). Intercellular adhesion was low and unchanged by stimulation of the PMN with zymosan activated serum or platelet activating factor (PAF) at concentrations that significantly enhance PMN adhesion to protein-coated glass and canine endothelial cell monolayers. Intercellular adhesion was significantly increased only when both myocytes and PMN were stimulated (e.g., myocytes incubated with IL-1, tumor necrosis factor, or phorbol myristate acetate, and PMN were chemotactically stimulated). Inhibitors of protein synthesis diminished the IL-1 beta-induced effect by greater than 80%. The IL-1 beta, PAF-stimulated PMN-myocyte adhesion was associated with substantial H2O2 production. Under conditions with low PMN-myocyte adhesion (i.e., IL-1 beta alone, PAF alone, or no stimulus) H2O2 production was generally less than 5% of that occurring with high adhesion. An anti-CD18 monoclonal antibody (R15.7) inhibited stimulated PMN-myocyte adhesion by greater than 95% and reduced H2O2 production by greater than 90%. Control isotype-matched, binding, and nonbinding antibodies were without effect on adherence or H2O2 production. The results indicate that cytokine stimulation of adult myocytes induces expression of a ligand involved in CD18-dependent adherence of canine neutrophils.
M L Entman, K Youker, S B Shappell, C Siegel, R Rothlein, W J Dreyer, F C Schmalstieg, C W Smith
The mechanisms that allow circulating basement membrane antibodies (Ab) to interact with the alveolar basement membrane (ABM) inducing Goodpasture's hemorrhagic pneumonitis are unknown. In laboratory animals the ABM is inaccessible to phlogogenic amounts of ABM Ab unless the permeability of the unfenestrated alveolar endothelium is increased. This study was designed to test the hypothesis that in the mouse polypeptide mediators, generated by activated lymphoid cells or cells infected by viruses, contribute to the pathogenesis of passive Goodpasture's hemorrhagic pneumonitis. In naive mice that received rabbit ABM Ab, these bound to the glomerular basement membrane but not to the ABM and their lungs were normal. In the lungs of mice injected with human recombinant IL-2 and IFN-alpha specific binding of ABM IgG, C3, and fibrinogen to the ABM, diffuse and severe erythrocyte extravasation, and accumulation of mononuclear and polymorphonuclear leukocytes were constantly observed. ABM Ab and IL-2 or ABM Ab and IFN-alpha did not produce comparable effects. Mice injected only with IL-2 and IFN-alpha had enlarged, edematous lungs without pulmonary hemorrhages. The results show that the synergism of IL-2 and IFN-alpha convert the lung into a preferential target for AMB Ab, suggesting that cytokines may have a role in the pathogenesis of human Goodpasture's pneumonitis.
T H Queluz, I Pawlowski, M J Brunda, J R Brentjens, A O Vladutiu, G Andres
We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product.
M L Baeza, S R Reddigari, D Kornfeld, N Ramani, E M Smith, P A Hossler, T Fischer, C W Castor, P G Gorevic, A P Kaplan
Mutations of the androgen receptor that impair the action of 5 alpha-dihydrotestosterone and testosterone result in abnormal male sexual development. The definition of the organization of the androgen receptor gene has permitted us to examine its structure in nine patients with androgen resistance that exhibit absent 5 alpha-dihydrotestosterone binding in cultured fibroblasts (receptor-negative androgen resistance). Using labeled probes specific for each individual coding exon, we find no gross rearrangements, insertions, or deletions of the androgen receptor gene in these patients. To analyze the genetic defect in these receptor-negative patients, we used the polymerase chain reaction to amplify each individual exon of the androgen receptor gene in nine affected patients. In all patients, the size of each amplified exon segment was identical to that in normal individuals. The nucleotide sequence of the entire coding region of the androgen receptor was determined in one of these patients. A single nucleotide substitution was identified that results in a premature termination codon in exon 6 at amino acid 794. S1 nuclease protection assays demonstrated that normal levels of androgen receptor mRNA are present in skin fibroblasts of this patient. Transfection of a mutated androgen receptor cDNA containing a termination codon at position 794 into eukaryotic cells resulted in formation of a normal amount of receptor protein, as indicated by immunoblotting, but the expressed protein does not bind 5 alpha-dihydrotestosterone. These findings suggest that the presence of a premature termination codon at amino acid 794 of the androgen receptor is the cause of androgen resistance in this patient.
M Marcelli, W D Tilley, C M Wilson, J D Wilson, J E Griffin, M J McPhaul
In vitro ultraviolet B (UVB) irradiation of human blood monocytes inhibits their accessory cell function for antigen- and mitogen-induced T cell responses. These studies were designed to characterize the nature of the UVB-induced defect in human monocyte accessory cell function. Irradiated monocytes were deficient in their ability to serve as accessory cells for OKT3-induced T cell activation. In vitro exposure of monocytes to 100 J/m2 UVB completely inhibited the T cell proliferative response (51502 cpm, non-UVB-irradiated; 302 cpm, UVB-irradiated). Analysis of the accessory signals altered by UVB indicated that irradiated monocytes were incapable of binding to OKT3 molecules attached to the CD3 antigen on T cells. Provision of an alternative mechanism for binding of OKT3 molecules by attaching anti-mouse IgG to the bottom of microtiter wells completely restored accessory cell function. Further characterization of the defect demonstrated that UVB radiation did not deplete p72 Fc receptors from the surface of irradiated monocytes. However, UVB exposure did produce a dose-dependent decrease in monocyte membrane expression of ICAM-1. It is proposed that UVB radiation leads to changes within the cell membrane that inhibit the ability of monocytes to express selected molecules necessary for binding of T cells.
J Krutmann, I U Khan, R S Wallis, F Zhang, E A Rich, J J Ellner, C A Elmets
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide isolated from porcine endothelial cells. We have previously demonstrated widespread binding sites for ET-1 in blood vessels, heart, kidney, adrenal, lung, and brain in a distribution that paralleled that of endothelial cells. To determine whether these cells are capable of synthesizing ET-1 in close proximity to its binding sites, amplification of cDNA using the polymerase chain reaction and in situ hybridization were used to study the distribution of ET-1 mRNA. We have found widespread transcription of ET-1 mRNA in human and porcine tissues. The identity of the transcripts was confirmed by prediction of restriction fragment lengths or sequencing. In situ hybridization in the kidney showed that the regional expression of these transcripts is localized, probably to small blood vessels, but the failure to visualize ET-1 mRNA in the capillaries may reflect absence of expression or insufficient sensitivity of the technique. These results should permit investigation of the role of ET-1 as a local factor in vascular pathophysiology.
D J Nunez, M J Brown, A P Davenport, C B Neylon, J P Schofield, R K Wyse
Lp(a) lipoprotein purified from human plasma bound with high affinity to isolated bovine LDL receptors on nitrocellulose blots and in a solid-phase assay. Lp(a) also competed with 125I-LDL for binding to human LDL receptors in intact fibroblasts. Binding led to cellular uptake of Lp(a) with subsequent stimulation of cholesterol esterification. After intravenous injection, human Lp(a) was cleared slowly from the plasma of normal mice. The clearance was markedly accelerated in transgenic mice that expressed large amounts of LDL receptors. We conclude that the covalent attachment of apo(a) to apo B-100 in Lp(a) does not interfere markedly with the ability of apo B-100 to bind to the LDL receptor and that this receptor has the potential to play a major role in clearance of Lp(a) from the circulation of intact humans.
S L Hofmann, D L Eaton, M S Brown, W J McConathy, J L Goldstein, R E Hammer
Over 80% of infants with severe combined immunodeficiency (SCID) of unknown genetic etiology are males, yet less than a third of these affected males have a family history of X-linked disease. To help identify new mutations of the X-linked SCID gene and to provide genetic counseling, X chromosome inactivation patterns in T cells from 16 women who had sons with sporadic SCID were examined. Between 9 and 35 human/hamster hybrids that selectively retained the active human X chromosome were produced from the T cells of each woman and analyzed with an X-linked restriction fragment length polymorphism for which the woman in question was heterozygous. Exclusive use of a single X as the active X was seen in the T cell hybrids from 7 of the 16 women, identifying these women as carriers of X-linked SCID. Studies on additional family members confirmed the mutant nature of the inactive X and revealed the source of the new mutation in three families. To determine whether there were any laboratory characteristics that might differentiate the boys whose mothers were identified as carriers of X-linked SCID from those whose mothers were not, the clinical records of both groups were compared to each other and to a group of 14 boys with a family history of X-linked SCID. The most consistent finding in the 21 patients with X-linked SCID was an elevated proportion of B cells. These data demonstrate the high incidence of spontaneous mutation for the X-linked SCID gene and help clarify the characteristic presenting features of this disorder.
M E Conley, R H Buckley, R Hong, C Guerra-Hanson, C M Roifman, J A Brochstein, S Pahwa, J M Puck
Chromogranin A is an acidic protein costored and coreleased with catecholamines from storage vesicles. Its serum concentration is elevated in patients with peptide-producing endocrine neoplasia. We measured serum chromogranin A at the time of diagnosis in 34 children with all stages of neuroblastoma. With a sensitivity of 91% and specificity of 100%, serum chromogranin A emerged as a useful diagnostic tool for neuroblastoma, comparable to or better than other measurements such as neuron-specific enolase, ferritin, or dopamine-beta-hydroxylase. Mean serum chromogranin A correlated with disease stage (r = 0.76, P less than 0.01). The relationship of prognosis (progression-free survival) to baseline serum chromogranin A, age, and disease stage was determined in 34 patients at risk for relapse, with a median followup period of 18 mo (range, 1-48 mo). The survival rate for patients with lower serum chromogranin A levels (less than 190 ng/ml at the time of diagnosis) was 69%, whereas it was 30% for those with higher chromogranin A levels (P less than 0.05). Furthermore, when subjects were additionally stratified by either age or stage, chromogranin A was an effective prognostic tool in patients who either were older than 1 yr (P less than 0.005) or had more advanced disease (stage III or IV; P less than 0.05). We conclude that serum chromogranin A in neuroblastoma is (a) a valuable (sensitive and specific) diagnostic tool, (b) a correlate of tumor burden, and (c) a useful predictor of survival.
R J Hsiao, R C Seeger, A L Yu, D T O'Connor
Interleukin-3 (IL-3) is a hematopoietic growth factor that supports the growth of early hematopoietic progenitors in vitro. In vivo administration of recombinant human interleukin-3 (rhIL-3) to normal primates results in a modest and delayed leukocytosis secondary to increases in neutrophils, basophils, and eosinophils. We postulated that the effects of rhIL-3 might be more pronounced in hematologically stressed primates, and therefore administered rhIL-3 to primates after intensive myelosuppressive therapy. Primates received either cyclophosphamide (CPM) at 60 mg/kg or 5-fluorouracil (5-FU) at 75 mg/kg i.v. on two consecutive days. Subsequently, rhIL-3 was administered intravenously or subcutaneously at 20 micrograms/kg per d for 14 d. Compared to controls, all rhIL-3 treated primates experienced higher absolute neutrophil count (ANC) nadirs and dramatic decreases in the period of severe neutropenia (ANC less than 500) after myelosuppressive therapy. RhIL-3 administration resulted in a significant basophilia and eosinophilia, which resolved after discontinuation of the drug. RhIL-3 did not enhance erythroid recovery. Platelet recovery was earlier in rhIL-3-treated animals. However, variations in the platelet recovery observed in control animals, precluded accurate estimation of this effect or its significance. Our results indicate that the administration of rhIL-3 following intensive myelosuppressive therapy dramatically enhances myeloid recovery and ablates the predicted period of prolonged severe neutropenia.
A P Gillio, C Gasparetto, J Laver, M Abboud, M A Bonilla, M B Garnick, R J O'Reilly
To analyze the autoepitopes on the SS-B/La protein, a cDNA covering the entire region coding the protein was isolated from a human cDNA library. The cDNA was subcloned into an expression plasmid vector, pEX, to express its protein product as a fusion protein with cro-beta-galactosidase in Escherichia coli. A recombinant pEX plasmid expressing three-fourths of the protein (amino acid 112-408) was also constructed. The antigenicities of these recombinant proteins were confirmed with a patient's serum. Their various deletion mutants were produced with exonuclease III treatment from the 3' ends of the cDNAs without changing the proper translational frame. Immunoblot analysis and enzyme-linked immunosorbent assay were used to evaluate the reactivities of the recombinant proteins with patients' sera to determine the autoepitopes. A narrow segment (amino acid 88-101) and the region where several epitopes were located (amino acid 283-338) on the SS-B/La protein were universally recognized by all the sera with anti-SS-B/La antibodies examined. An additional epitope region (amino acid 179-220) was recognized by some patients' sera. Computer analysis revealed that the most distinct autoepitope, amino acid 88-101, had a striking homology to a retroviral gag polyprotein. These findings indicate that exogenous or endogenous retroviruses may play a role in initiation of the anti-SS-B/La autoimmunity.
H Kohsaka, K Yamamoto, H Fujii, H Miura, N Miyasaka, K Nishioka, T Miyamoto
Two distinct mutant alleles of the precursor (p) short chain acyl-CoA dehydrogenase (SCAD) gene were identified in a SCAD-deficient patient (YH2065) using the polymerase chain reaction to amplify cDNA synthesized from total RNA from her fibroblasts. Cells from this patient had previously been shown to synthesize a labile variant SCAD in contrast to the normal stability of variant SCADs in two other SCAD-deficient cell lines (Naito, E., Y. Indo, and K. Tanaka. 1989. J. Clin. Invest. 84:1671-1674). In the present study, both mutant alleles of YH2065 were found to contain a C----T transition, one at position 136 and the other at position 319 of the coding region of pSCAD cDNA. Clones of cDNA amplified from this region showed only one of the C----T transitions, indicating that each mutation was derived from different pSCAD alleles. Each of these mutations altered a known restriction endonuclease site, and restriction analysis of additional cDNA clones from amplified mutant cDNA and Southern blotting of mutant genomic DNA confirmed the presence of two unique mutant alleles in YH2065, indicating YH2065 is a compound heterozygote. These C----T transitions result in the substitution of Arg-22 and Arg-83 of the mature SCAD with Trp and Cys, respectively.
E Naito, Y Indo, K Tanaka
Most cases of cytosol-defective chronic granulomatous disease are due to the deficiency of a 47-kD protein (p47-phox) whose phosphorylation normally accompanies the activation of the respiratory burst oxidase. Recently, a form of chronic granulomatous disease was described in which the failure of O2- production was associated with the absence of a 67-kD polypeptide (p67-phox) from the cytosol of affected neutrophils. Using neutrophils obtained from a patient with this form of the disease, we examined the function of p67-phox in the activation of the oxidase. Our studies showed that in whole p67-phox-deficient neutrophils, p47-phox was phosphorylated in a normal fashion. In the cell-free oxidase-activating system, the ability of the p67-phox-deficient cytosol to support oxidase activation was partly restored by the addition of p47-phox-deficient cytosol; the p67-phox-deficient cytosol, however, was not complemented by cytosol inactivated with NADPH dialdehyde, an affinity label previously found to block the NADPH-binding component of the oxidase. Despite these differences, the kinetic properties of the p67-phox-deficient cytosol closely resembled those of the p47-phox-deficient cytosol. Taken together with earlier findings, these results suggest that (a) in the neutrophil cytosol, p67-phox is at least partly complexed to p47-phox; (b) it is in the form of this complex that p67-phox participates in oxidase activation; and (c) p47-phox appears to be translocated from the cytosol to the plasma membrane during oxidase activation, but complexation to p67-phox is not necessary for this translocation, nor for the accompanying extra protein phosphorylation.
N Okamura, B M Babior, L A Mayo, P Peveri, R M Smith, J T Curnutte
To compare extra-renal 1,25(OH)2D3 production in different types of granulomatous disease, and to identify the cell types responsible, we have evaluated the conversion of 25(OH)D3 in 1,25(OH)2D3 by uncultured cells recovered by bronchoalveolar lavage and blood mononuclear cells from normocalcemic patients with sarcoidosis and tuberculosis. 1,25(OH)2D3 was produced both by lavage cells (12/12 tuberculosis patients, 2/6 sarcoidosis patients) and blood mononuclear cells (3/5 tuberculosis patients, 0/3 sarcoidosis patients) from patients but not controls, but significantly greater amounts were produced by lavage cells from tuberculosis patients than those of sarcoidosis patients (P less than 0.001). 1,25(OH)2D3 production by lavage cells from tuberculosis patients correlated with the number of CD8+ T lymphocytes present but not other cell types. T lymphocytes appeared to be an important source of 1,25(OH)2D3 production, since purified T lymphocytes from all patients with tuberculosis produced 1,25(OH)2D3, and 1,25(OH)2D3 production by these cells correlated closely with that produced by unseparated lavage cells. Because 1,25(OH)2D3 can improve the capacity of macrophages to kill mycobacteria, our results support the conclusion that macrophage-lymphocyte interactions, mediated at least in part by 1,25(OH)2D3, may be an important component of a successful antituberculous immune response.
J Cadranel, M Garabedian, B Milleron, H Guillozo, G Akoun, A J Hance
Effective T cell vaccination against experimental autoimmune diseases involves treatment with activated, autoimmune T lymphocytes. The present study was undertaken to learn whether antigen-specific T cells present in low frequency could be selected in vitro without using the specific antigen. The rat models of adjuvant arthritis and experimental autoimmune encephalomyelitis were investigated using proliferation assays and limiting dilution techniques to quantify the changes in reactivity of a heterogenous population of lymphocytes to the relevant antigen. Stimulation with concanavalin A for 2 d and then culture in IL-2-containing medium led to a substantial increase in the activity and frequency of the specific autoimmune T cells. Enrichment of antigen-specific T cells could be demonstrated using lymph node, spleen, or peripheral blood lymphocytes, from rats late in the course of disease. The effect was not evident in lymphocytes from the thymus. These results are relevant to the clinical application of T cell vaccination and to investigation of self-antigens in autoimmune disease.
F Mor, A W Lohse, N Karin, I R Cohen
Experiments were performed in human working myocardium to investigate the relationship of intracellular calcium handling and availability to alterations in the strength of contraction produced by changes in stimulation rate and pattern. Both control and myopathic muscles exhibited potentiation of peak isometric force during the postextrasystolic contraction which was associated with an increase in the peak intracellular calcium transient. Frequency-related force potentiation was attenuated in myopathic muscles compared to controls. This occurred despite an increase in resting intracellular calcium and in the peak amplitude of the calcium transient as detected with aequorin. Therefore, abnormalities in contractile function of myopathic muscles during frequency-related force potentiation are not due to decreased availability of intracellular calcium, but more likely reflect differences in myofibrillar calcium responsiveness. Sarcolemmal calcium influx may also contribute to frequency-related changes in contractile force in myopathic muscles as suggested by a decrease in action potential duration with increasing stimulation frequency which is associated with fluctuations in peak calcium transient amplitude.
J K Gwathmey, M T Slawsky, R J Hajjar, G M Briggs, J P Morgan
Age-dependent alterations in the effects of catecholamines on lipolysis were investigated in 25 young (21-35 yr) and 10 elderly (58-72 yr) healthy, nonobese subjects using isolated adipocytes obtained from abdominal subcutaneous tissue. Basal lipolysis was not affected by aging, while the rate of catecholamine-stimulated lipolysis was reduced by 50% in the elderly subjects (P less than 0.005). To elucidate the mechanisms behind this phenomenon lipolysis was stimulated with agents that act at well-defined steps in the lipolytic cascade, from the receptor down to the final step: the activation of the protein kinase/hormone-sensitive lipase complex. All agents stimulated lipolysis at a 50% lower rate in elderly as compared with young subjects (P less than 0.05 or less). However, half-maximum effective concentrations of the lipolytic agents were similar in both groups. The antilipolytic effects of alpha 2-adrenoceptor agonists were also the same in young and old subjects. Moreover, the stoichiometric properties of the beta- and alpha 2-receptors did not change with increasing age. In vivo studies performed on the same individuals likewise demonstrated an impaired lipolytic responsiveness, with 50% lower plasma glycerol concentrations during exercise in the elderly subjects (P less than 0.05), in spite of a normal rise in plasma norepinephrine. The plasma glycerol levels correlated strongly to the glycerol release caused by catecholamine-stimulated lipolysis in vitro in both young and elderly subjects (r = 0.8-0.9, P less than 0.001). In conclusion, a decreased activation of the hormone-sensitive lipase complex appears to be the mechanism underlying a blunted lipolytic response of fat cells to catecholamine stimulation in elderly subjects. This finding may, explain the age-dependent decreased lipolytic response to exercise in vivo.
F Lönnqvist, B Nyberg, H Wahrenberg, P Arner
Adenosine has been proposed to act within the juxtaglomerular apparatus (JGA) as a mediator of the inhibition of renin secretion produced by a high NaCl concentration at the macula densa. To test this hypothesis, we studied the effects of the adenosine1 (A1)-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (CPX) on renin release from single isolated rabbit JGAs with macula densa perfused. The A1-receptor agonist, N6-cyclohexyladenosine (CHA), applied in the bathing solution at 10(-7) M, was found to inhibit renin secretion, an effect that was completely blocked by adding CPX (10(-5) M) to the bath. Applied to the lumen, 10(-5) M CPX produced a modest stimulation of renin secretion rates suppressed by a high NaCl concentration at the macula densa (P less than 0.05). The effect of changing luminal NaCl concentration on renin secretion rate was examined in the presence of CPX (10(-7) and 10(-5) M) in the bathing solution and in vehicle control experiments. The control response to increasing luminal NaCl concentration was a marked suppression of renin secretion, that was maintained as long as luminal NaCl concentration was high and was promptly reversible when concentration was lowered. CPX did not alter renin release when luminal NaCl was low, but diminished the reduction caused by high NaCl (P less than 0.01). It is concluded that A1-receptors are located within the JGA, and that A1-receptor activation inhibits renin release. A high NaCl concentration at the macula densa appears to influence A1-receptor activation, but a low NaCl concentration does not. The findings support participation of adenosine in macula densa control of renin secretion.
H Weihprecht, J N Lorenz, J Schnermann, O Skøtt, J P Briggs
In the in vitro perfused rectal gland of the dogfish shark (Squalus acanthias), the adenosine analogue 2-chloroadenosine (2Clado) completely and reversibly inhibited forskolin-stimulated chloride secretion with an IC50 of 5 nM. Other A1 receptor agonists including cyclohexyladenosine (CHA), N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyl-adenosine (R-PIA) also completely inhibited forskolin stimulated chloride secretion. The "S" stereoisomer of PIA (S-PIA) was a less potent inhibitor of forskolin stimulated chloride secretion, consistent with the affinity profile of PIA stereoisomers for an A1 receptor. The adenosine receptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline completely blocked the effect of 2Clado to inhibit forskolin-stimulated chloride secretion. When chloride secretion and tissue cyclic (c)AMP content were determined simultaneously in perfused glands, 2Clado completely inhibited secretion but only inhibited forskolin stimulated cAMP accumulation by 34-40%, indicating that the mechanism of inhibition of secretion by 2Clado is at least partially cAMP independent. Consistent with these results, A1 receptor agonists only modestly inhibited (9-15%) forskolin stimulated adenylate cyclase activity and 2Clado markedly inhibited chloride secretion stimulated by a permeant cAMP analogue, 8-chlorophenylthio cAMP (8CPT cAMP). These findings provide the first evidence for a high affinity A1 adenosine receptor that inhibits hormone stimulated ion transport in a model epithelia. A major portion of this inhibition occurs by a mechanism that is independent of the cAMP messenger system.
G G Kelley, E M Poeschla, H V Barron, J N Forrest Jr
During the pathogenesis of Lyme disease, Borrelia burgdorferi spreads hematogenously from the site of a tick bite to several tissues throughout the body. The specific mechanism of spirochete emigration is presently unknown. Using cultured human umbilical vein endothelial cells, we found that Borrelia burgdorferi bound to the endothelial cells and to the subendothelial matrix. Low passage isolates adhered 22-30-fold greater than a strain maintained in culture continuously. Spirochete binding to subendothelial matrix was inhibited 48-63% by pretreatment of the matrix with anti-fibronectin antiserum. Spirochete migration across endothelial monolayers cultured on amniotic membrane was increased when the monolayers were damaged by chemical or physical means. Electron microscopic examination of spirochete-endothelial interactions demonstrated the presence of spirochetes in the intercellular junctions between endothelial cells as well as beneath the monolayers. Scanning electron microscopy identified a mechanism of transendothelial migration whereby spirochetes pass between cells into the amniotic membrane at areas where subendothelium is exposed.
A Szczepanski, M B Furie, J L Benach, B P Lane, H B Fleit
The effects of hyperglycemia on myocardial glucose metabolism were investigated in seven healthy male subjects (age 24 +/- 4 yr). [6-14C]Glucose and [U-13C]lactate were infused as tracers. Circulating glucose was elevated to two hyperglycemic levels using a clamp technique for 1 h at each level. The mean arterial glucose concentration was 4.95 +/- 0.29 (control), 8.33 +/- 0.31 and 10.84 +/- 0.60 mumols/ml, respectively. Glucose extraction increased significantly from control (0.15 +/- 0.13 mumols/ml) during each level of the glucose clamp (0.28 +/- 0.12, P less than 0.02, and 0.54 +/- 0.14 mumols/ml, P less than 0.005, respectively). Myocardial production of 14CO2 showed that during control 9 +/- 10% of exogenous glucose was oxidized immediately upon extraction. Despite a significant increase in the amount of exogenous glucose oxidized with level II hyperglycemia, it represented only 32 +/- 10% of the glucose extracted. [13C]Lactate analysis showed that the myocardium was releasing lactate; during control 40 +/- 30% of this lactate was derived from exogenous glucose and during hyperglycemia this value increased to 97 +/- 37% (P less than 0.005). Thus, these data show that during short-term hyperglycemia, myocardial glucose extraction is enhanced. However, despite increases in exogenous glucose oxidation and the contribution of exogenous glucose to lactate release, the majority of the extracted glucose (i.e., 57%) is probably stored as glycogen.
J A Wisneski, W C Stanley, R A Neese, E W Gertz
A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake.
D A Greene, S A Lattimer, P B Carroll, J D Fernstrom, D N Finegold
The hyper-IgE (HIE) syndrome is characterized by high IgE serum levels, chronic dermatitis, and recurrent infections. The mechanisms responsible for hyperproduction of IgE in HIE patients are presently unknown. We investigated whether spontaneous in vitro IgE synthesis by PBMC from seven HIE patients was sensitive to signals (cell adhesion, T/B cell cognate interaction and lymphokines: IL-4, IL-6, and IFN-gamma) known to regulate IgE induction in normals. Our results show that, unlike IL-4 dependent IgE synthesis induced in normals, spontaneous IgE production by PBMC from HIE patients was not blocked by monoclonal antibodies to CD2, CD4, CD3, and MHC class II antigens. Furthermore, antibodies to IL-4 and IL-6 did not significantly suppress IgE production. IFN-gamma had no significant effects on spontaneous in vitro IgE synthesis. To test whether an imbalance in lymphokine production might underlie hyperproduction of IgE in HIE patients, mitogen-induced secretion of IL-4 and IFN-gamma by PBMC was assessed. No significant difference was detected between HIE patients and normal controls. Thus, ongoing IgE synthesis in the HIE syndrome is largely independent of cell-cell interactions and endogenous lymphokines, and is due to a terminally differentiated B cell population, no longer sensitive to regulatory signals.
D Vercelli, H H Jabara, C Cunningham-Rundles, J S Abrams, D B Lewis, J Meyer, L C Schneider, D Y Leung, R S Geha
The mechanism of tumor-associated hypoglycemia was examined in 11 patients with hepatocellular carcinoma, 6 of whom presented with severe hypoglycemia and 5 in whom plasma glucose was persistently normal. Serum insulin levels in the hypoglycemic patients were low. Although total serum insulin-like growth factor II (IGF-II) levels in both groups of tumor patients were lower than normal, tumor tissue from hypoglycemic patients contained levels of IGF-II mRNA that were 10-20-fold higher than those present in normal liver. IGF-II immunoreactivity consisted in all cases of a mixture of both higher molecular weight forms and material having the character of IGF-II itself. The former comprised a greater proportion of total IGF-II, in patients with hypoglycemia. Studies to characterize the interactions of IGF-II with serum proteins showed that (a) the radiolabeled peptide bound to an approximately 40,000-D protein in sera from both hypoglycemic patients and normal subjects, (b) sera from hypoglycemic patients and normal subjects had similar capacity to bind the radiolabeled peptide, and (c) the apparent affinities of serum binding proteins for IGF-II were the same for both hypoglycemic patients and normal subjects. Whereas, acid extracted, tumor-derived IGF-II immunoreactive peptides with low or intermediate molecular weights bound to serum proteins in a manner indistinguishable from that of IGF-II itself, the highest molecular weight IGF-II immunoreactive peptide exhibited negligible ability to compete for radiolabeled ligand binding to serum proteins. The low affinity of serum binding proteins for this component suggests that high molecular weight IGF-II immunoreactivity might circulate free and be available for interaction with cell-surface receptors.
E T Shapiro, G I Bell, K S Polonsky, A H Rubenstein, M C Kew, H S Tager
The control of gene transcription is usually mediated by transacting transcriptional factors that bind to upstream regulatory elements. As insulin regulates transcription of the growth hormone (GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated Chinese hamster ovarian (CHO) cells for binding to four human GH (hGH) gene promoter oligonucleotide fragments identified as target-binding sequences by DNAse I footprinting. Using a mobility shift assay, an insulin-induced DNA-binding protein was identified. This protein binds to two upstream overlapping oligonucleotide sequences. Binding activity is present at low levels in unstimulated CHO cells and is stimulated by insulin treatment with a time course suggesting that protein synthesis is required. Incubation of the cells with cycloheximide and puromycin confirmed that de novo protein synthesis is necessary for the increased binding activity. Competition with excess unlabeled specific competitor oligonucleotides prevented binding, while unrelated similar-sized oligonucleotides failed to compete for binding, indicating that the observed DNA-protein complex formation is specific. A protein of approximately 70-80 kD was detected by gradient gel electrophoresis. In conclusion, insulin-mediated DNA-protein binding has been identified on the upstream hGH promoter, suggesting a trans-active role for insulin in mediating polypeptide hormone gene expression.
D Prager, S Gebremedhin, S Melmed
Pulmonary infections with Pneumocystis carinii are an important cause of morbidity and mortality in patients with AIDS. P. carinii infections are seen in patients with decreased numbers of helper T lymphocytes, suggesting that these cells are important in preventing infection. To test this hypothesis, we sought to establish experimental infection with P. carinii in mice selectively depleted of helper T lymphocytes. Weekly injections of a monoclonal anti-CD4 antibody produced sustained depletion of helper T lymphocytes from blood and lymphoid organs. To establish pulmonary infection, lymphocyte-depleted mice were then given intratracheal inoculations of P. carinii organisms derived from the lungs of chronically infected athymic mice. Pulmonary infection with P. carinii was demonstrable in the antibody-treated mice and was centered around the conducting airways. Infection was persistent for up to 3 mo with continued antibody treatments, and yet could be cleared from the lungs if antibody treatments were discontinued. This experimental model of P. carinii infection permits the study of infection associated with a specific immune defect and implicates the helper T lymphocyte as a critical cell in host defense against this pathogen.
J Shellito, V V Suzara, W Blumenfeld, J M Beck, H J Steger, T H Ermak
Human monocytes cultured on adherent IgG produce a specific IL-1 inhibitor that functions as a receptor antagonist (IL-1ra). This molecular has been purified, sequenced, cloned as a cDNA, and expressed in Escherichia coli. Recombinant IL-1ra has 17,000 mol wt and binds to IL-1 receptors on T lymphocytes, synovial cells, and chondrocytes with an affinity nearly equal to that of IL-1. These studies have examined some biological properties of purified recombinant human IL-1ra. This protein exhibits a dose-responsive inhibition of Il-1 alpha and Il-1 beta augmentation of PHA-induced murine thymocyte proliferation. The recombinant IL-1ra also blocks IL-1 alpha and IL-1 beta stimulation of PGE2 production in human synovial cells and rabbit articular chondrocytes, and of collagenase production by the synovial cells. A 50% inhibition of these IL-1-induced biological responses requires amounts of IL-1ra up to 100-fold in excess of the amounts of IL-1 alpha or IL-1 beta present. IL-1ra may play an important role in normal physiology or in pathophysiological states by functioning as a natural IL-1 receptor antagonist in the cell microenvironment.
W P Arend, H G Welgus, R C Thompson, S P Eisenberg
Studies in animal models have suggested that alterations affecting phospholamban-mediated stimulation of Ca2+ uptake by sarcoplasmic reticulum are involved in the pathophysiology of heart disease. A monoclonal antibody that binds to phospholamban and stimulates Ca2+ uptake was used to characterize phospholamban-mediated effects in human cardiac sarcoplasmic reticulum and to compare these effects in tissue from normal and failing hearts. Stimulation of Ca2+ uptake by anti-phospholamban monoclonal antibody simulated the effect of phosphorylation of phospholamban by cAMP-dependent protein kinase. Binding of anti-phospholamban antibody reduced the K0.5 of the Ca2(+)-transporting ATPase from 0.53 microM [( Ca2+]) to 0.29 microM [( Ca2+]), without affecting Vmax or nHill. At 0.2 microM Ca2+, stimulation was 1.93-fold in sarcoplasmic reticulum prepared from normal human left ventricular myocardium and 1.94-fold in sarcoplasmic reticulum prepared from the left ventricular myocardium of patients with heart failure resulting from idiopathic dilated cardiomyopathy. Stimulation of Ca2+ uptake in canine cardiac sarcoplasmic reticulum under identical conditions was 1.89-fold. Phospholamban-mediated stimulation of Ca2+ uptake in human cardiac sarcoplasmic reticulum is thus comparable in magnitude to that observed in other species and results from an increase in the apparent affinity of the Ca2(+)-transporting ATPase for Ca2+. The pathogenesis of heart failure in idiopathic dilated cardiomyopathy does not, however, appear to involve intrinsic alterations of this mechanism.
M A Movsesian, J Colyer, J H Wang, J Krall
Several inherited disorders of fatty acid beta-oxidation have been described that relate mainly to saturated precursors. This study is the first report of an enzyme defect related only to unsaturated fatty acid oxidation and provides the first in vivo evidence that fat oxidation in humans proceeds by the reductase-dependent pathway. The patient was a black female, presenting in the neonatal period with persistent hypotonia. Biochemical studies revealed hyperlysinemia, hypocarnitinemia, normal organic acid profile, and an unusual acylcarnitine species in both urine and blood. The new metabolite was positively identified by mass spectrometry as 2-trans,4-cis-decadienoylcarnitine, derived from incomplete oxidation of linoleic acid. In spite of dietary therapy, the patient died of respiratory acidosis at four months of age. Samples of liver and muscle from the autopsy were assayed for 2,4-dienoyl-coenzyme A reductase activity. Using the substrate 2-trans,4-cis-decadienoylcoenzyme A, the reductase activity was 40% of the control value in liver and only 17% of that found in normal muscle. It is suggested that unsaturated substrates should be used for in vitro testing to cover the full range of potential beta-oxidation defects and that acylcarnitine species identification be used for in vivo detection of this disorder.
C R Roe, D S Millington, D L Norwood, N Kodo, H Sprecher, B S Mohammed, M Nada, H Schulz, R McVie