J A Berzofsky
H F McFarland
The metabolic and functional alterations which occur during the acute phase of nephrotoxic nephritis (NTN) in rats, a model of immune-mediated glomerulonephritis, result from a cooperative interaction between PMNs and platelets (PLTs). In consequence, we hypothesized that fibrinogen (Fg) might play a critical role in this process and, accordingly, we found that defibrination of animals decreased both the acute phase proteinuria in NTN (approximately 70%) as well as the influx of PLTs and PMNs into the glomerulus (approximately 40-50%). In contrast, blockade of the PLT Fg receptor, alpha IIb beta 3, with the RGD peptidomimetic SC-49992 decreased proteinuria (approximately 90%) without substantially altering the influx of PMNs or PLTs. Immunocytochemistry showed a marked increase in beta 3 integrin expression in inflamed glomeruli which was prevented either by PMN or PLT depletion before disease induction. FACS and immunocytochemical analysis of glomerular cell dissociates demonstrated that beta 3 integrin expression was predominantly on intraglomerular PLTs. In vitro, activated PLTs stimulated the PMN respiratory burst, an interaction which could be inhibited by Fg receptor blockade. In sum, acute NTN is accompanied by a marked increase in glomerular beta 3 integrin expression predominantly due to the influx of PLTs which localize to the glomerulus in a PMN-dependent fashion. Fg appears to serve a major role as a coactivating stimulus for PLT-PMNs in situ via alpha IIb beta 3, potentially mediating the PMN respiratory burst which contributes to proteinuria. Fg may also play a subsidiary role in PMN/PLT comigration.
X Wu, M H Helfrich, M A Horton, L P Feigen, J B Lefkowith
With the aim of establishing whether a genetically reduced capability of producing apolipoprotein E (apo E) can affect atherogenesis, we have compared the consequences of dietary stress on normal mice and on mice heterozygous or homozygous for a disrupted apo E gene. A dramatically accelerated development of lesions occurred in the vasculature of the homozygous mutants as a result of feeding an atherogenic diet for 12 wk, and extensive deposition of lipid-filled macrophages was found outside the cardiovascular system. In nine heterozygotes fed the atherogenic diet for 12 wk, the amount of apo E in their total plasma lipoproteins increased to a level comparable to normal, but all nine developed much larger foam cell lesions in their proximal aorta than those found in 3 of 9 normal mice fed the same diet. The other six normals had no lesions. Our study demonstrates that heterozygous mice with only one functional apo E gene are more susceptible to diet-induced atherosclerosis than are normal, two-copy mice. Genetically determined quantitative limitations of apo E could, therefore, have similar effects in humans when they are stressed by an atherogenic diet.
S H Zhang, R L Reddick, B Burkey, N Maeda
Matrix metalloproteinases are a highly regulated family of enzymes, that together can degrade most components of the extracellular matrix. These proteins are active in normal and pathological processes involving tissue remodeling; however, their sites of synthesis and specific roles are poorly understood. Using in situ hybridization, we determined cellular distributions of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, an inhibitor of matrix metalloproteinases, in endometrium during the reproductive cycle. The mRNAs for all the metalloproteinases were detected in menstrual endometrium, but with different tissue distributions. The mRNA for matrilysin was localized to epithelium, while the others were detected in stromal cells. Only the transcripts for the 72-kD gelatinase and tissue inhibitor of metalloproteinases-1 were detected throughout the cycle. Transcripts for stromelysin-2 and the 92-kD gelatinase were only detected in late secretory and menstrual endometrium, while those for matrilysin, the 72-kD gelatinase, and stromelysin-3 were also consistently detected in proliferative endometrium. These data indicate that matrix metalloproteinases are expressed in cell-type, tissue, and reproductive cycle-specific patterns, consistent with regulation by steroid hormones, and with specific roles in the complex tissue growth and remodeling processes occurring in the endometrium during the reproductive cycle.
W H Rodgers, L M Matrisian, L C Giudice, B Dsupin, P Cannon, C Svitek, F Gorstein, K G Osteen
Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma.
M Weller, K Frei, P Groscurth, P H Krammer, Y Yonekawa, A Fontana
The pathogenic gram-positive bacterium Streptococcus pyogenes (group A streptococcus) causes numerous diseases of cutaneous tissue, each of which is initiated after the interaction of the bacterium with the cells of the epidermis. In this study, we show that different surface proteins of S. pyogenes play important roles in determining the cell-specific tropism of the bacterium in skin. Using streptococcal strains with defined mutations in the genes which encode surface proteins in combination with primary cultures of human skin and an in situ adherence assay which uses histological sections of human skin, we show that the M protein of S. pyogenes mediates the binding of the bacterium to keratinocytes, while a second streptococcal surface protein, protein F, directs the adherence of the organism to Langerhans' cells. Characterization of binding revealed that adherence was inhibited by purified streptococcal proteins and pretreatment of both host cells with the protease trypsin. Adherence was only slightly affected by the state of keratinocyte differentiation in vitro, but was considerably modulated in response to environmental conditions known to regulate expression of M protein and protein F, suggesting that the interaction between these bacterial cell-surface structures/adhesins and keratinocytes and Langerhans' cells may play an important role in streptococcal skin disease.
N Okada, A P Pentland, P Falk, M G Caparon
In vitro studies have demonstrated that angiotensin (Ang) II directly stimulates vascular smooth muscle cell (VSMC) growth. However, it is still unclear if Ang II exerts a direct effect on vascular hypertrophy in vivo independent of its effect on blood pressure. In vivo gene transfer provides the opportunity to assess the effects of increased activity of the vascular angiotensin system in the intact animal while avoiding an increase in circulating angiotensin or in blood pressure. Accordingly, we transfected the human angiotensin converting enzyme (ACE) vector into intact rat carotid arteries by the hemagglutinating virus of Japan-liposome method. 3 d after transfection, we detected increased ACE activity in the transfected artery. Immunohistochemistry localized immunoreactive ACE in the medial VSMC as well as in the intimal endothelial cells. The increase in vascular ACE activity was associated with a parallel increase in DNA synthesis as assessed by BrdU (bromo-deoxyuridine) index and vascular DNA content. This increase in DNA synthesis was abolished by the in vivo administration of an Ang II receptor-specific antagonist (DuP 753). Morphometry at 2 wk after transfection revealed an increase in the wall to lumen ratio of the ACE-transfected blood vessel as compared with control vector transfected vessels. This was accompanied by increases in protein and DNA contents without an increase in cell number. Local transfection of ACE vector did not result in systemic effects such as increased blood pressure, heart rate, or serum ACE activity. These morphological changes were abolished by the administration of the Ang II receptor antagonist. In this study, we used in vivo gene transfer to increase local expression of vascular angiotensin converting enzyme and provided proof that increased autocrine/paracrine angiotensin can directly cause vascular hypertrophy independent of systemic factors and hemodynamic effects. This approach has important potentials for defining the role of autocrine/paracrine substances in vascular biology and hypertension.
R Morishita, G H Gibbons, K E Ellison, W Lee, L Zhang, H Yu, Y Kaneda, T Ogihara, V J Dzau
The human erythrocyte chemokine receptor has recently been shown to be identical to the Duffy blood group antigen and is expressed in multiple organs, including kidney. Here we have examined the molecular properties of the renal isoform. Immunoblot analysis of erythrocyte and kidney detergent lysates, with a monoclonal antibody (Fy6) to the Duffy antigen, revealed that the renal isoform had a molecular mass of 43-45 kD, which could be distinguished from that observed in erythroid cells (38-47 kD). Chemical cross-linking of kidney membranes to 125I-melanoma growth stimulatory activity (MGSA) indicated that the renal chemokine receptor had a molecular mass of 38-45 kD. Binding of 125I-labeled MGSA to kidney membranes was competitively inhibited by the addition of unlabeled MGSA, IL-8, regulated on activation, normal T expressed and secrted, and monocyte chemotactic protein-1. Scatchard analysis of MGSA binding showed that the chemokine receptor from renal tissues had a binding affinity of 3.5 nM similar to that observed for the erythroid isoform (5-10 nM). The primary structure of the renal chemokine receptor predicted from the nucleotide sequence of cDNA from renal tissues is identical to that reported for the erythroid isoform. Immunocytochemical staining of kidney with Fy6 localized expression to endothelial cells present in postcapillary venules. These studies implicate the Duffy antigen/chemokine receptor in the complex interactions between postcapillary endothelial cells and granulocytes, which are modulated by pro-inflammatory chemokines.
T J Hadley, Z H Lu, K Wasniowska, A W Martin, S C Peiper, J Hesselgesser, R Horuk
Accelerated coronary atherosclerosis in cardiac transplants (cardiac allograft vasculopathy, CAV) is characterized by coronary intimal hyperplasia. Acidic fibroblast growth factor (aFGF) is a potent mitogen for vascular smooth muscle cells and endothelial cells, and its expression is increased in cardiac allografts, suggesting it may play a role in the pathogenesis of CAV. The activity of aFGF is dependent on binding to transmembrane receptors. To investigate whether receptors for aFGF are also induced after transplantation, polymerase chain reaction, in situ hybridization, and immunohistochemistry were used to analyze expression of four receptors for aFGF (FGFR1-FGFR4). Expression of mRNA encoding extracellular immunoglobulin-like domains of FGFR1 was increased 35-fold in cardiac allografts compared with normal hearts and was predominantly present in cardiac myocytes and vascular structures. Alternatively spliced mRNA that encodes transmembrane forms of FGFR1, which contain the signal-transducing tyrosine kinase domains, was induced in allografts during rejection, in infiltrating cells, vascular structures, and myocytes. In vitro experiments showed that differential expression of FGF receptor isoforms was induced by aFGF, and also by IL-6 and TGF-beta, which are expressed in cardiac allografts during rejection. The results show that expression of both aFGF and its receptors is altered in cardiac allografts and suggest that these events are important in the pathogenesis of CAV.
X M Zhao, W H Frist, T K Yeoh, G G Miller
We evaluated G-proteins that are components of adenylyl cyclase (AC) signal transduction in erythrocyte and lymphocyte membranes from 26 family history positive (FHP) non-alcoholic and 26 family history negative (FHN) nonalcoholic subjects. Subjects were classified as FHP if their father met criteria for alcohol dependence; as FHN, if there was no history of alcoholism in any first or second degree relatives. Immunoblot analysis indicated that levels of erythrocyte membrane Gs alpha from FHP subjects were greater than levels in FHN subjects (171 +/- 11 vs 100 +/- 6, P < 0.001). To confirm the results of the immunoblot analysis, Gs alpha was quantitated by cholera toxin-dependent [32P]ADP-ribosylation. Levels of erythrocyte [32P]ADP-ribose-Gs alpha from FHP subjects were greater than levels in FHN subjects (236 +/- 28 vs 100 +/- 14, P < 0.001). Gs alpha levels did not correlate with age or alcohol consumption. By contrast to differences in Gs alpha, immunoblot analysis showed similar levels of Gi(2)alpha and Gi(3)alpha in erythrocyte membranes of FHP and FHN subjects. Pertussis toxin-catalyzed [32P]ADP-ribosylation of Gi-like G-proteins confirmed the immunoblot observations. Lastly, compared to FHN subjects, FHP subjects had enhanced Gs alpha expression in lymphocyte membranes as well (138 +/- 11 vs 100 +/- 5.5; P < 0.02). In summary, compared to FHN nonalcoholic men, FHP nonalcoholic men had greater levels of the stimulatory G-protein, Gs alpha, in erythrocyte and lymphocyte membranes. Enhanced expression of Gs alpha may be a marker of increased risk for the future development of alcoholism.
G S Wand, C Waltman, C S Martin, M E McCaul, M A Levine, D Wolfgang
We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.
A E Koch, S L Kunkel, L A Harlow, D D Mazarakis, G K Haines, M D Burdick, R M Pope, A Walz, R M Strieter
At 4 wk after intraperitoneal inoculation of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable only in the salivary glands. When T cells of these mice were activated by a single injection of anti-CD3 epsilon monoclonal antibody, mice died of interstitial pneumonitis at 24-48 h after injection, accompanied by elevation of serum levels of TNF-alpha and IFN-gamma. However, MCMV remained undetectable in the lungs during the period. Simultaneous injection of cyclosporin A reduced such effects of anti-CD3. In conclusion, although the presence of MCMV in the host may be required, MCMV-associated pneumonitis is not mediated by virus in the lung but probably by the cytokines released from T cells, of which responsiveness to stimulation via CD3 molecule has been presumably modified by MCMV infection.
K Tanaka, Y Koga, Y Y Lu, X Y Zhang, Y Wang, G Kimura, K Nomoto
Autoimmune myocarditis is considered to play a major role in the pathogenesis of dilated cardiomyopathy. A new autoimmune myocarditis model was attained by repeated immunization using murine cardiac C-protein with the immunological adjuvant, Klebsiella pneumoniae O3 lipopolysaccharide. For further analysis of a pathological epitope, the cDNA encoding C-protein was isolated; a fusion protein encoded by part of this cDNA induced myocarditis in SMA mice as well as in three other strains: DBA/1J (H-2q), O20/A (H-2pz1), and SJL (H-2s). The nucleotide sequence and its deduced amino acid analysis revealed that this protein had immunoglobulin-like and fibronectin-like repeats. This study provides a new animal model of autoimmune myocarditis which may shed light on the pathogenesis of dilated cardiomyopathy.
H Kasahara, M Itoh, T Sugiyama, N Kido, H Hayashi, H Saito, S Tsukita, N Kato
The dystrophin gene, which is mutated in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously unidentified alternative promoter. The case study presented is that of a patient with Duchenne muscular dystrophy who had a deletion extending from the 5' end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5' part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5' end of the new transcript indicated that the 5' end of exon 3 was extended by 9 codons, only the last (most 3') of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5' of the previously identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.
H Nishio, Y Takeshima, N Narita, H Yanagawa, Y Suzuki, Y Ishikawa, Y Ishikawa, R Minami, H Nakamura, M Matsuo
Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.
B L Smith, G M Preston, F A Spring, D J Anstee, P Agre
Channel-forming integral protein (CHIP) is the archetypal member of the Aquaporin family of water channels. Delayed CHIP expression was shown recently in perinatal rat (Smith, B. L., R. Baumgarten, S. Nielsen, D. Raben, M. L. Zeidel, and P. Agre. 1993. J. Clin. Invest. 92:2035-2041); here we delineate the human patterns. Compared with adult, second and third trimester human fetal red cells had lower CHIP/spectrin ratios (0.72 +/- 0.12, 0.94 +/- 0.22 vs 1.18 +/- 0.11) and reduced osmotic water permeability (0.029, 0.026 vs 0.037 cm/s); CHIP was already present in human renal tubules by the second trimester. A patient with a novel form of congenital dyserythropoietic anemia (CDA) with persistent embryonic and fetal globins and absent red cell CD44 protein was studied because of reduced CHIP-associated Colton antigens. Novel CDA red cells contained < 10% of the normal level of CHIP and had remarkably low osmotic water permeability (< 0.01 cm/s), but no mutation was identified in Aquaporin-1, the gene encoding CHIP. These studies demonstrate: (a) unlike rat, human CHIP expression occurs early in fetal development; (b) red cell water channels are greatly reduced in a rare phenotype; and (c) disrupted expression of red cell CHIP and CD44 suggests an approach to the molecular defect in a novel form of CDA.
P Agre, B L Smith, R Baumgarten, G M Preston, E Pressman, P Wilson, N Illum, D J Anstee, M B Lande, M L Zeidel
Brain and atrial natriuretic peptides (BNP and ANP) are cardiac hormones with diuretic, natriuretic, and vasodilatory activities. Cardiomyopathic hamsters are widely used animal models of heart failure. Due to the structural divergence of BNP among species, examination on pathophysiological roles of BNP using cardiomyopathic hamsters is so far impossible. We therefore isolated hamster BNP and ANP cDNAs, and investigated synthesis and secretion of these peptides in normal and cardiomyopathic hamsters. The COOH-terminal 32-residue peptide of cloned hamster preproBNP with 122 amino acids, preceded by a single arginine residue, supposedly represents hamster BNP showing < 50% homology to rat BNP. Alpha-hamster ANP, 28-residue peptide, is identical to alpha-rat ANP. In hamsters, BNP and ANP occur mainly in the ventricle and the atrium, respectively. The 32-wk-old hypertrophic cardiomyopathic BIO14.6 strain exhibited ventricular hypertrophy. The 32-wk-old dilated cardiomyopathic BIO53.58 strain remained at the stage without apparent heart failure. In BIO14.6 and BIO53.58 strains at this age, ventricular BNP and ANP gene expressions are augmented, and the plasma BNP concentration is elevated to 136 and 108 fmol/ml, respectively, three times greater than the elevated plasma ANP concentration, which well mimics changes of the plasma BNP and ANP concentrations in human heart failure. Cardiomyopathic hamsters, therefore, are useful models to investigate the implication of BNP in human cardiovascular diseases.
N Tamura, Y Ogawa, H Itoh, H Arai, S Suga, O Nakagawa, Y Komatsu, I Kishimoto, K Takaya, T Yoshimasa
Human radiocontrast nephrotoxicity is predicted by the presence of multiple risk factors, often associated with compromised renal circulation. To produce a simple model of radiocontrast nephropathy, rats were pretreated with indomethacin and N omega-nitro-L-arginine methyl ester (L-NAME, to inhibit nitric oxide synthesis) before the administration of iothalamate. Acute renal failure consistently developed, with a decline in creatinine clearance from 1.05 +/- 0.10 to 0.27 +/- 0.05 ml/min (P < 0.001) associated with selective necrosis of 49 +/- 9% of medullary thick ascending limbs. Hemodynamic studies using laser-Doppler probes revealed that when injected alone, iothalamate increased outer medullary blood flow to 196 +/- 25% of baseline (P < 0.001). Pretreatment by L-NAME or indomethacin both reduced basal medullary blood flow and transformed the medullary vasodilator response to radiocontrast into vasoconstriction, with a prolonged reduction of medullary blood flow to less then half of baseline. Combined administration of indomethacin, L-NAME, and iothalamate lowered medullary blood flow to 12 +/- 4% of baseline. We conclude that prostanoids and nitric oxide have an important protective role in the renal response to radiocontrast material. Reduced synthesis of these vasoactive substances in renal/vascular diseases may predispose patients to radiocontrast nephropathy.
Y Agmon, H Peleg, Z Greenfeld, S Rosen, M Brezis
Advanced human malignancies cannot be permanently cured by adoptive transfer of autologous lymphokine-activated killer (LAK) cells. Moreover, administration of high doses of IL-2 to maintain LAK activity in vivo is associated with severe toxicity. In this study, we tested the anti-tumor efficacy of a uniquely potent MHC non-restricted human killer T cell clone (TALL-104) in humanized severe combined immunodeficient (SCID) mice bearing acute myelogenous leukemia (AML). We show that, in appropriate experimental conditions, TALL-104 cells could effectively reverse the aggressive growth of the myelomonocytic leukemia cell line U937 in SCID mouse tissues, leading to complete abrogation of AML. A single transfer of TALL-104 cells at the time of tumor challenge in combination with recombinant human (rh) IL-12 (1 microgram/d) prolonged significantly the life of the mice. However, eradication of leukemia, as monitored both clinically and by PCR, was achieved by repeated injection of the effectors at close intervals. Complete cure was obtained also upon transfer of lethally irradiated (non-proliferating) TALL-104 cells together with low doses of rh IL-2 (100 U/d). Most notably, of the mice that received multiple transfers of TALL-104 cells without cytokines in an advanced disease stage, 50% were clinically cured, and 50% survived significantly longer. The potential of TALL-104 cells as an effective and safe leukemia purging agent is discussed.
A Cesano, S Visonneau, L Cioé, S C Clark, G Rovera, D Santoli
The frequency of the D allele of an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene has been reported to be elevated in myocardial infarction and other patients. We therefore hypothesized that death rate of DD individuals should be increased in the population as a whole and this should be evident as a decrease in DD frequency with age. This hypothesis was tested in 118 Caucasian subjects who were already at high risk of cardiovascular events by having severe, early onset, familial hypertension (HT). A group of 196 age-, sex- and body mass index-matched normotensives (NTs) was used as a control. In the NT group II, ID, and DD genotype frequencies were similar for different age groups. DD frequency was 0.42 in NTs, but in HTs was 0.28, 0.26, and 0.10 for the age groups < 50, 50-59, and > or = 60 yr, respectively. Corresponding D allele frequencies were 0.52, 0.46, and 0.40 in the respective age groups of HTs, compared with 0.61 in NTs (by chi 2-analysis, P = 0.1, 0.047, and 0.0006, respectively). In HTs aged > or = 60, DD frequency was only 14% of expected. Plasma ACE activity tracked similarly with I/D genotype in HTs (P = 0.027; n = 35) as in NTs (P = 0.0001; n = 94) and Michaelis constant was identical for DD and II. Neither blood pressure, body mass index, nor sex bore any relationship with I/D genotype. In conclusion, in a group of severely HT patients not selected for cardiac pathology, there appeared to be a marked, selective decrease, in subgroups of increasing age, in frequency of the ACE DD genotype. One possibility suggested by this data might be that DD increases risk of premature death, at least in HTs who have two HT parents.
B J Morris, R Y Zee, A P Schrader
We have found that an estrogen deficiency causes a marked increase in bone marrow cells. To examine the effect of estrogen on hemopoiesis, we characterized the increased population of bone marrow cells after ovariectomy (OVX). In OVX mice, the percentage of myeloid cells and granulocytes was decreased, whereas that of B220-positive B lymphocytes was selectively increased 2-4 wk after surgery. The total number of myeloid cells and granulocytes did not change appreciably, but that of B220-positive cells was greatly increased by OVX. When OVX mice were treated with estrogen, the increased B lymphopoiesis returned to normal. B220-positive cells were classified into two subpopulations, B220low and B220high. The majority of the B220low cells were negative for the IgM mu chain, whereas most of the B220high cells were mu-positive. OVX selectively increased the precursors of B lymphocytes identified by B220low. mu-negative phenotype, suggesting that an estrogen deficiency stimulates accumulation of B lymphocyte precursors. When bone marrow-derived stromal cells (ST2) were pretreated with estrogen then co-cultured with bone marrow cells in the presence of estrogen, the stromal cell-dependent B lymphopoiesis was greatly inhibited. The present study suggests that estrogen plays an important role in the regulation of B lymphocyte development in mouse bone marrow.
T Masuzawa, C Miyaura, Y Onoe, K Kusano, H Ohta, S Nozawa, T Suda
We examined the distribution of glycoprotein IIb-IIIa (GPIIb-IIIa) and its ligands fibrinogen and von Willebrand factor (vWf) on platelets which had adhered under flow conditions. Immunoelectron microscopy was performed on whole mounts and frozen thin sections of adhering platelets. GPIIb-IIIa was homogeneously distributed on dendritic platelets and on interplatelet membranes of formed thrombi. Fibrinogen and vWf were predominantly associated with interplatelet membranes and membranes facing the substrate. On whole mounts, vWf appeared in clumps and linear arrays, representing the tangled or extended forms of the multimeric molecule. From semiquantitative analysis, it appeared that fibrinogen and vWf were, respectively, nine- and fourfold higher on interplatelet membranes than on surface membranes facing the blood stream, while GPIIb-IIIa was evenly distributed over all platelet plasma membranes. Ligand-induced binding sites (LIBS) of GPIIb-IIIa, as measured with conformation specific monoclonal antibodies RUU 2.41 and LIBS-1, were present on the surface of adhered platelets and thrombi. A redistribution of LIBS-positive forms of GPIIb-IIIa towards interplatelet membranes was not observed. Our data support the hypothesis that, under flow conditions, ligands have first bound to activated GPIIb-IIIa but this binding is reversed on the upper surface of adhering platelets. This relative absence of ligands on the exposed surface of thrombi may play a role in limiting their size.
H F Heynen, M Lozano Molero, P G de Groot, H K Nieuwenhuis, J J Sixma
Upon activation of human neutrophils by chemoattractants, functionally important proteins are rapidly transported from intracellular granules and storage vesicles to the plasma membrane. This is accompanied by a marked increase in the rate of endocytosis and by ligand-independent internalization of type 1 complement receptors (CR1). To define the pathway of endocytosis, we used gold-conjugated BSA in a pulse-chase protocol. This tracer was initially internalized into small endocytic vesicles which rapidly traversed the cytoplasm and coalesced to form large, conspicuous multivesicular bodies. Within 5 min after addition of the chemoattractant, multivesicular bodies contained > 60% of the cell-associated BSA-gold. CR1 colocalized with the endocytic tracer in both the early endosomes and multivesicular bodies. In unstimulated cells, there was much less uptake of BSA-gold and multivesicular bodies were rarely seen. Using the acidotropic amine, DAMP, and anti-DNP antibodies, we found that the multivesicular bodies were acidified but the early endosomes did not concentrate DAMP. Neither the early endosomes nor the multivesicular bodies initially contained the lysosomal membrane antigens hLAMP 1 or 2, but hLAMP-positive structures subsequently joined the multivesicular bodies. The rapid activation of the endocytic pathway upon stimulation of neutrophils allowed us to visualize the de novo formation and maturation of multivesicular bodies. Our observations suggest that vesicles containing ion pumps and acid hydrolases fuse with multivesicular bodies, giving them characteristics of lysosomes, and that these are the probable sites of degradation of CR1. The observations do not support models which would require transport of CR1 from multivesicular bodies to defined, pre-existing lysosomes for degradation.
M Berger, E Wetzler, J T August, A M Tartakoff
Insulin-like growth factor-I (IGF-I) is considered to be the mediator of the growth-promoting effects of growth hormone (GH). The metabolic effects of these two hormones, however, are different. Whereas GH treatment leads to elevated insulin and glucose levels, reduced insulin sensitivity, and impaired glucose tolerance, IGF-I treatment leads to reduced insulin and GH levels and enhanced insulin sensitivity. IGF-I may, therefore, not only be the mediator of the growth-promoting effects of GH but also a modulator of the effects of GH on insulin action and glucose metabolism. To study the influence of GH and IGF-I on substrate metabolism and insulin sensitivity (assessed by euglycemic, hyperinsulinemic clamping combined with indirect calorimetry and glucose tracer infusion), we have treated eight GH-deficient adults with GH (2 IU/m2 daily subcutaneously [s.c.]), IGF-I (10 micrograms/kg.h s.c.), or both hormones together for 7 d, respectively, and compared the effects of these treatment regimens with a control phase. Our findings suggest that (a) both GH and IGF-I promote lipolysis and lipid oxidation, albeit by different mechanisms; (b) treatment with either hormone is followed by enhanced energy expenditure and reduced protein oxidation; and (c) IGF-I reverses the insulin resistance induced by GH.
M A Hussain, O Schmitz, A Mengel, Y Glatz, J S Christiansen, J Zapf, E R Froesch
Macrophages represent a critical component in the inflammatory lesions of giant cell arteritis. By combining immunohistochemistry and in situ hybridization, we have analyzed the functional heterogeneity of tissue-infiltrating macrophages in patients with untreated vasculitis. 20% of macrophages in temporal artery tissue synthesized IL-6-specific mRNA and produced IL-6 and IL-1 beta proteins. IL-6 and IL-1 beta production was not limited to CD68+ cells in the lymphoid aggregates but was a feature of CD68+ cells dispersed throughout the tissue. 50% of tissue-infiltrating CD68+ cells synthesized 72-kD type IV collagenase. Only a small subset of CD68+ cells produced cytokines as well as collagenase, indicating functional specialization or distinct differentiation stages of CD68+ cells in the inflamed tissue. Activation of CD68+ cells was not restricted to tissue-infiltrating cells. Expression of IL-6 and IL-1 beta was found in 60-80% of circulating monocytes of patients with untreated giant cell arteritis, whereas collagenase production was restricted to tissue macrophages. IL-6 and IL-1 beta production by the majority of circulating monocytes was a shared feature of patients with giant cell arteritis and polymyalgia rheumatica but was not found in rheumatoid arthritis. These data suggest that giant cell arteritis has two components of disease, an inflammatory reaction in vessel walls and a systemic activation of monocytes. Systemic monocyte activation can manifest independently without vasculitis as exemplified in patients with polymyalgia rheumatica.
A D Wagner, J J Goronzy, C M Weyand
Insulin receptor substrate-1 (IRS-1) plays an important role in insulin-stimulated signaling mechanisms. Therefore, we investigated the frequency and clinical significance of variants in the coding region of this gene in patients with non-insulin-dependent diabetes (NIDDM). Initial screening included a population-based sample of 40 Finnish patients with typical NIDDM. Applying single strand conformation polymorphism analysis the following amino acid substitutions were found among the 40 NIDDM patients: Gly818-Arg, Ser892Gly, and Gly971Arg. The first two variants have not been previously reported. Additional samples of 72 patients with NIDDM and 104 healthy control subjects with completely normal oral glucose tolerance test and a negative family history of diabetes were screened. The most common polymorphism was the Gly971Arg substitution which was found in 11 (9.8%) of 112 NIDDM patients and in 9 (8.7%) of 104 control subjects. The Gly818Arg substitution was found in 2 (1.8%) of NIDDM patients and in 2 (1.9%) of control subjects, and the Ser892Gly substitution was found in 3 (2.7%) NIDDM patients and in 1 (1.0%) control subject. The Gly971Arg substitution was not associated with an impairment in insulin secretion capacity (estimated by insulin responses in an oral glucose tolerance test or by the hyperglycemic clamp) or insulin action (estimated by the euglycemic clamp). Of the three amino acid substitutions observed Ser892Gly is the most interesting one since it abolishes one of the potential serine phosphorylation sites (SPGE) which is located immediately NH2-terminal to the only SH2 binding site of growth factor receptor-bound protein (GRB2), and thus could potentially influence some aspects of signal transduction and metabolic response to insulin.
M Laakso, M Malkki, P Kekäläinen, J Kuusisto, S S Deeb
Human umbilical vein endothelial cells have recently been shown to respond to C5a with increases in intracellular Ca2+, production of D-myo-inositol 1,4,5-triphosphate and superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and endothelial cells in the acute inflammatory response.
K E Foreman, A A Vaporciyan, B K Bonish, M L Jones, K J Johnson, M M Glovsky, S M Eddy, P A Ward
Since we have previously shown a direct inhibitory effect of platelet-activating factor (PAF) on Cl reabsorption in the medullary thick ascending limb of Henle's loop (TAL), the aim of this study was to extend this effect to the whole TAL and to further investigate the signaling pathway involved. In microperfused cortical TALs, PAF significantly decreased Cl reabsorption by 50.3 +/- 6.5%. On the one hand, this effect was not modified in the presence of staurosporine and was not mimicked by phorbol ester; chelating cytosolic Ca by BAPTA/AM failed to suppress the inhibitory effect of PAF on Cl reabsorption; moreover, no significant increase in intracellular Ca concentration could be observed in the presence of PAF on isolated tubules. On the other hand, 8-bromo cyclic GMP mimicked the PAF effect on Cl reabsorption and prevented a further effect of this agent; the PAF effect was significantly reduced by H-8, a cyclic GMP-dependent protein kinase inhibitor; in medullary TALs, PAF significantly increased by twofold cyclic GMP content, an effect inhibited by the PAF antagonist BN 50730, whereas PAF did not significantly modify cAMP content in basal or stimulated conditions. Finally, inhibition of nitric oxide production by NAME or NMMA failed to prevent the effect of PAF on Cl reabsorption. It is concluded that the PAF-induced inhibition of Cl reabsorption in the TAL was mediated by cyclic GMP, likely independent of a nitric oxide synthesis.
F Néant, M Imbert-Teboul, C Bailly
L Zhu, D Wigle, A Hinek, J Kobayashi, C Ye, M Zuker, H Dodo, F W Keeley, M Rabinovitch
The purpose of this study was to examine whether insulin's effect to vasodilate skeletal muscle vasculature is mediated by endothelium-derived nitric oxide (EDNO). N-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO synthase, was administered directly into the femoral artery of normal subjects at a dose of 16 mg/min and leg blood flow (LBF) was measured during an infusion of saline (NS) or during a euglycemic hyperinsulinemic clamp (HIC) designed to approximately double LBF. In response to the intrafemoral artery infusion of L-NMMA, LBF decreased from 0.296 +/- 0.032 to 0.235 +/- 0.022 liters/min during NS and from 0.479 +/- 0.118 to 0.266 +/- 0.052 liters/min during HIC, P < 0.03. The proportion of NO-dependent LBF during NS and HIC was approximately 20% and approximately 40%, respectively, P < 0.003 (NS vs. HIC). To elucidate whether insulin increases EDNO synthesis/release or EDNO action, vasodilative responses to graded intrafemoral artery infusions of the endothelium-dependent vasodilator methacholine chloride (MCh) or the endothelium-independent vasodilator sodium nitroprusside (SNP) were studied in normal subjects during either NS or HIC. LBF increments in response to intrafemoral artery infusions of MCh but not SNP were augmented during HIC versus NS, P < 0.03. In summary, insulin-mediated vasodilation is EDNO dependent. Insulin vasodilation of skeletal muscle vasculature most likely occurs via increasing EDNO synthesis/release. Thus, insulin appears to be a novel modulator of the EDNO system.
H O Steinberg, G Brechtel, A Johnson, N Fineberg, A D Baron
The gusmps/gusmps mouse is a model of the human lysosomal storage disease mucopolysaccharidosis type VII caused by deficient beta-glucuronidase activity. Bone marrow transplantation has been shown to correct some of their biochemical and pathological abnormalities but its efficacy in correcting their neurological functional deficits is unknown. We transplanted the neonatal gusmps/gusmps mice and their normal controls and evaluated their central nervous system function with two behavioral tests: the grooming test, a developmentally regulated and genetically based activity, and a Morris water maze test which assessed spatial learning abilities. The two transplanted groups groomed less than the normals, were unable to remember the location of an invisible platform from day to day, and were severely impaired at developing strategies to locate the platform in unfamiliar locations. The performance of both normal and mutant transplanted groups was clearly inferior to the untreated normals and, in some instances, close to or worse than the untreated mutants, even though the enzyme abnormalities of the mutants have been partially corrected. Hence, the behavioral deficits in the mutant mice were not restored to normal while similarly treated normal mice showed significant functional deterioration, indicating the detrimental consequence of this therapy in the neonatal period.
L Bastedo, M S Sands, D T Lambert, M A Pisa, E Birkenmeier, P L Chang
Previous studies have established that in a variety of human glomerulopathies the reduced glomerular filtration rate (GFR) is due to a marked lowering of the ultrafiltration coefficient (Kf). To identify the factors which lower Kf, we measured the filtering surface area per glomerulus, filtration slit frequency, basement membrane thickness, and GFR and its determinants in patients with minimal change and membraneous nephropathies and in age-matched healthy controls. Overall values of Kf for the two kidneys were calculated from GFR, renal plasma flow rate, systemic colloid osmotic pressure, and three assumed values for the transcapillary pressure difference. "Experimental" values of the glomerular hydraulic permeability (kexp) were then calculated from Kf, glomerular filtering surface area, and estimates of the total number of nephrons of the two kidneys. Independent estimates of the glomerular hydraulic permeability (kmodel) were obtained using a recent mathematical model that is based on analyses of viscous flow through the various structural components of the glomerular capillary wall. Individual values of basement membrane thickness and filtration slit frequency were used as inputs in this model. The results indicate that the reductions of Kf in both nephropathies can be attributed entirely to reduced glomerular hydraulic permeability. The mean values of kexp and kmodel were very similar in both disorders and much smaller in the nephrotic groups than in healthy controls. There was good agreement between kexp and kmodel for any given group of subjects. It was shown that, in both groups of nephrotics, filtration slit frequency was a more important determinant of the water flow resistance than was basement membrane thickness. The decrease in filtration slit frequency observed in both disorders caused the average path length for the filtrate to increase, thereby explaining the decreased hydraulic permeability.
M C Drumond, B Kristal, B D Myers, W M Deen
20 normoglycemic first degree relatives of non-insulin-dependent diabetes mellitus (NIDDM) patients were compared with 20 matched subjects without any family history of diabetes using the intravenous glucose tolerance test with minimal model analysis of glucose disappearance and insulin kinetics. Intravenous glucose tolerance index (Kg) was similar in both groups (1.60 +/- 0.14 vs 1.59 +/- 0.18, x 10(-2) min-1, NS). However, insulin sensitivity (Si) was reduced (3.49 +/- 0.43 vs 4.80 +/- 0.61, x 10(-4) min-1 per mU/liter, P = 0.05), whereas glucose effectiveness (Sg) was increased (1.93 +/- 0.14 vs 1.52 +/- 0.16, x 10(-2) min-1, P < 0.05) in the relatives. Despite insulin resistance neither fasting plasma insulin concentration (7.63 +/- 0.48 vs 6.88 +/- 0.45, mU/liter, NS) nor first phase insulin responsiveness (Phi1) (3.56 +/- 0.53 vs 4.13 +/- 0.62, mU/liter min-1 per mg/dl, NS) were increased in the relatives. Phi1 was reduced for the degree of insulin resistance in the relatives so that the Phi1 x Si index was lower in the relatives (11.5 +/- 2.2 vs 16.7 +/- 2.0, x 10(-4) min-2 per mg/dl, P < 0.05). Importantly, glucose effectiveness correlated with Kg and with basal glucose oxidation but not with total glucose transporter 4 (GLUT4) content in a basal muscle biopsy. In conclusion we confirm the presence of insulin resistance in first degree relatives of NIDDM patients. However, insulin secretion was altered and reduced for the degree of insulin resistance in the relatives, whereas glucose effectiveness was increased. We hypothesize that increased glucose effectiveness maintains glucose tolerance within normal limits in these "normoinsulinemic" relatives of NIDDM patients.
J E Henriksen, F Alford, A Handberg, A Vaag, G M Ward, A Kalfas, H Beck-Nielsen
We examined the molecular defect in two kindreds with "variant" X-linked chronic granulomatous disease (CGD). Western blots of neutrophil extracts showed decreased immunoreactive cytochrome b558 components gp91-phox and p22-phox. Analysis of mRNA demonstrated reduced gp91-phox transcripts, with relative preservation of an alternative mRNA species created by transcription initiation in the third exon of the gene. Single strand conformation polymorphism analysis of the 5' flanking region of the patients' gp91-phox genes revealed an electrophoretic abnormality not detected in 40 other gp91-phox genes. Genomic sequencing demonstrated a single base change associated with CGD in each kindred: in one, adenine to cytosine at base pair-57 and in the other, thymidine to cytosine at -55. These mutations are located between the "CCAAT" and "TATA" box consensus sequences involved in eukaryotic gene transcription. Gel shift assays revealed two specific DNA-protein complexes formed between phagocyte nuclear extracts and an oligonucleotide probe representing bases -31 to -68 of the gp91-phox promoter region; the faster-migrating complex could not be formed with oligonucleotides containing either of the promoter mutations. Thus, these promoter region mutations appear to be causally related to the loss of association of a DNA-binding protein and lead to diminished gp91-phox expression, abnormal transcription initiation, and the development of CGD.
P E Newburger, D G Skalnik, P J Hopkins, E A Eklund, J T Curnutte
Controversy still exists concerning the therapy for viral myocarditis which manifests a wide variety of clinical symptoms. Vesnarinone, a quinolinone derivative that was developed as a positive inotropic agent with complex actions, including phosphodiesterase inhibition and cation channel modification, has recently been confirmed to improve the prognosis of patients with chronic heart failure. However, the precise mechanism of this beneficial effect is not yet clearly understood. In this study, using a murine model of acute viral myocarditis resulting from encephalomyocarditis virus infection, survival and myocardial damage were markedly improved by treatment with vesnarinone. In contrast, survival was not improved by treatment with amrinone, a phosphodiesterase inhibitor. Although vesnarinone did not inhibit viral replication or protect myocytes from viral direct cell injury, it did inhibit the increase in natural killer cell activity after viral infection. On the other hand, amrinone failed to inhibit natural killer cell activity. Both vesnarinone and amrinone suppressed the production of tumor necrosis factor-alpha. Therefore, we postulate that vesnarinone exerted its beneficial effects through an inhibition of natural killer cell activity, and that it serves as an immunomodulator providing new therapeutic possibilities for the treatment of viral myocarditis and/or immunological disorders.
S Matsui, A Matsumori, Y Matoba, A Uchida, S Sasayama
We previously found that exogenous GSH enhances mucosal GSH and promotes lipid hydroperoxide metabolism by rat small intestine (AW, T. Y., and M. W. WIlliams, 1992. Am. J. Physiol. 263:G665-G672). In this study, we have developed an in vivo bile and lymph fistula rat model to test the hypothesis that biliary GSH is an important luminal source of GSH. Peroxidized fish oil was infused into the proximal intestine, and hydroperoxide accumulation in lumen, mucosa, and lymph was determined. Diversion of bile decreased mucosal GSH and increased hydroperoxide accumulation in all fractions. Supplementation with GSH, but not with GSSG, increased tissue GSH and attenuated hydroperoxide accumulation (50-60%), consistent with enhancement of hydroperoxide removal by exogenous GSH. Addition of native bile deficient in GSH, but not cysteine, cystine, or GSSG, decreased luminal and lymph hydroperoxide levels by 20-30%. Amino acid supplementation concurrently attenuated hydroperoxide recoveries in these fractions by 30-40% and increased mucosal GSH by 40%, indicating a role for biliary amino acids in hydroperoxide elimination. The effect of amino acids was abolished by buthionine sulfoximine, confirming their role in GSH biosynthesis. Collectively, the results demonstrate that bile is a rich source of reductant for maintaining mucosal GSH to promote intestinal metabolism of luminal peroxidized lipids.
T Y Aw
Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 125I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (Bmax) of the ETA and ETB subtype was 2.6 +/- 0.5 pmol/mg protein (m +/- SEM) and 1.19 +/- 0.6, respectively. The dissociation constant (Kd) of ET-1, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells.
G Rossi, G Albertin, A Belloni, L Zanin, M A Biasolo, T Prayer-Galetti, M Bader, G G Nussdorfer, G Palù, A C Pessina
The expression of the insulin-like growth factors (IGFs) and their receptors has been linked to cellular proliferation and tumorigenicity in a number of model systems. Since rhabdomyosarcoma cells express IGF-I receptors, an autocrine or paracrine loop involving this receptor and its ligands could be responsible in part for the growth characteristics of this tumor. To assess directly the role of the IGF-I receptor in rhabdomyosarcoma cell growth and tumorigenicity, a human alveolar rhabdomyosarcoma cell line with high IGF-I receptor expression was transfected with an amplifiable IGF-I receptor antisense expression vector. Four unique, transfected clones were analyzed and found to have reduced IGF-I receptor expression relative to the parental line. Integration of the antisense sequence was demonstrated by Southern blot analysis, and expression of antisense message in these clones was shown by S1 nuclease protection assay. Reduced IGF-I receptor surface expression in the transfectants was shown by decreased immunofluorescence with an IGF-I receptor monoclonal antibody and by decreased IGF-I binding as measured by Scatchard analysis. These clones had markedly reduced growth rates in vitro, impaired colony formation in soft agar, and failed to form tumors in immunodeficient mice when compared with vector-transfected clones. These results demonstrate that reduction of IGF-I receptor expression can inhibit both the in vitro and in vivo growth of a human rhabdomyosarcoma cell line and suggest a role for the IGF-I receptor in mediating neoplastic growth in this mesenchymally derived tumor.
D N Shapiro, B G Jones, L H Shapiro, P Dias, P J Houghton
Activated platelets express P-selectin and release leukocyte chemoattractants; however, they have not been known to express integrin ligands important in the stabilization of leukocyte interactions with the vasculature. We now demonstrate the presence of intercellular adhesion molecular-2 (ICAM-2) (CD102), and lack of expression of other beta 2-integrin ligands, ICAM-1 (CD54) and ICAM-3 (CD50), on the surface of resting and stimulated platelets. ICAM-2 isolated from platelets migrates as a band of 59,000 M(r) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of bone marrow aspirates with anti-ICAM-2 mAb demonstrates strong reactivity to megakaryocytes. Using frozen thin sections and immunogold labeling, the antigen was shown to be present on the plasma membrane and surface-connected canalicular system of resting platelets. The average number of ICAM-2 molecules per platelet is 3,000 +/- 230 and does not change after activation. In adhesion assays, resting and stimulated platelets were capable of binding through ICAM-2 to purified leukocyte function-associated antigen-1. Activation of T lymphocytes with PMA stimulated binding to platelets that was Mg2+ dependent and could be specifically inhibited by mAbs to either ICAM-2 or leukocyte function-associated antigen-1. ICAM-2 is the only known beta 2-integrin ligand present on platelets, suggesting that it may play an important role in leukocyte-platelet interactions in inflammation and thrombosis.
T G Diacovo, A R deFougerolles, D F Bainton, T A Springer
Genetic and biochemical studies were carried out in 96 relatives of six independently ascertained probands with familial dysbetalipoproteinemia (FD) carrying the APOE*2 (Lys146-->Gln) allele. Compared to noncarriers, the 40 heterozygous APOE*2 (Lys146-->Gln) allele carriers exhibited markedly increased mean levels of cholesterol and triglyceride in the very low density lipoproteins (VLDL) (1.89 +/- 0.37 vs 0.30 +/- 0.27 and 1.86 +/- 0.37 vs 0.68 +/- 0.27 mmol/liter, respectively) and plasma apolipoprotein (apo) E levels (28.1 +/- 1.6 vs 4.6 +/- 1.1 mg/dl), which is characteristic for FD. By means of a pedigree-based maximum likelihood method we calculated that carrier-status accounted for 57% and 71%, respectively, of the total variance of the ratio (VLDL + IDL)-cholesterol/plasma triglyceride and plasma apoE levels. APOE*2 (Lys146-->Gln) and APOE*3-Leiden allele carriers were found to differ significantly in: (a) plasma apoE levels, (b) in the amounts of triglycerides in the VLDL and VLDL + IDL fraction, and (c) in the amount of cholesterol in the VLDL and VLDL + IDL fraction relative to the amount of triglyceride in these fractions. In the APOE*2 (Lys146-->Gln) allele carriers the VLDL and VLDL + IDL fraction is relatively rich in triglycerides as compared with that in APOE*3-Leiden carriers. We hypothesize that these two rare mutations of apoE both lead to dominantly inherited forms of FD along different underlying metabolic defects.
P de Knijff, A M van den Maagdenberg, D I Boomsma, A F Stalenhoef, A H Smelt, J J Kastelein, A D Marais, R R Frants, L M Havekes
Electroconvulsive shock (ECS) is a highly effective therapy for the treatment of major depression, but its mechanisms of action are not known. We report that repeated ECS in rats produces enduring changes in two clinically relevant stress-responsive brain systems: (a) the hypothalamic-pituitary-adrenal axis regulated by corticotropin-releasing hormone (CRH) in the paraventricular nucleus; and (b) the NE system in the locus coeruleus regulated by tyrosine hydroxylase (TH). CRH and TH mRNA levels in these brain regions were assessed by in situ hybridization histochemistry. A single interaural ECS elevated TH but not CRH mRNA measured 24 h later. Repeated daily treatments (3, 7, or 14) elevated both mRNAs, maximally with 7, correlating with the time course of clinical efficacy. The elevations persisted for 3 (CRH) or 8 wk (TH) after the ECS. No other therapeutic treatment is known to produce such long-lasting changes in central nervous system gene expression. The time course of events (delayed onset, long duration) implicate CRH as a principal mediator of the antidepressant effects of ECS. The locus coeruleus-NE system may be important in initiating the central nervous system response.
L S Brady, A B Lynn, J R Glowa, D Q Le, M Herkenham
To study the ionic basis of salt sensitivity in hypertension, 19F-, 13P-, and 23Na-nuclear magnetic resonance techniques were used to measure cytosolic free calcium (Cai), pH (pHi), free magnesium (Mgi), and sodium (Nai) in erythrocytes of essential hypertensive subjects (n = 19). Individuals were studied for 2 mo each on low- (UNaV < 50 meq/d) and high- (UNaV > 200 meq/d) salt diets, with the concomitant administration of nifedipine (10 mg t.i.d.) or placebo tablets for 1 mo of each diet. Salt loading elevated Cai and Nai while suppressing Mgi and pHi; these changes occurred predominantly in salt-sensitive subjects (n = 9). Nifedipine blunted the pressor response to salt loading > 50% (delta diastolic BP [high-low salt vs placebo] = 5 +/- 2 vs 14 +/- 2 mmHg, P < 0.05) and reversed salt-induced ionic changes, lowering Cai and elevating Mgi and pHi. Regardless of the definition of salt sensitivity, continuous relationships were observed between the pressure response to salt loading, the levels of Cai (r = 0.726, P < 0.001), Nai (r = 0.747, P < 0.001), and pHi (r = -0.754, P < 0.001), and the salt-induced change in Mgi (r = -0.757, P < 0.001). Altogether, these results emphasize the reciprocal and coordinate nature of intracellular ionic changes in response to dietary salt loading and calcium channel blockade in essential hypertension. They suggest that salt sensitivity is mediated by cellular calcium accumulation from the extracellular space, in association with magnesium depletion and acidification. Lastly, interpretation of intracellular ion measurements in the future will require concurrent assessment of dietary salt intake.
L M Resnick, R K Gupta, B DiFabio, M Barbagallo, S Mann, R Marion, J H Laragh
Pulses of growth hormone (GH) release in acromegaly may arise from hypothalamic regulation or from random events intrinsic to adenomatous tissue. To distinguish between these possibilities, serum GH concentrations were measured at 5-min intervals for 24 h in acromegalic men and women with active (n = 19) and inactive (n = 9) disease and in normal young adults in the fed (n = 20) and fasted (n = 16) states. Daily GH secretion rates, calculated by deconvolution analysis, were greater in patients with active acromegaly than in fed (P < 0.05) but not fasted normal subjects. Significant basal (nonpulsatile) GH secretion was present in virtually all active acromegalics but not those in remission or in fed and fasted normal subjects. A recently introduced scale- and model-independent statistic, approximate entropy (ApEn), was used to test for regularity (orderliness) in the GH data. All but one acromegalic had ApEn values greater than the absolute range in normal subjects, indicating reduced orderliness of GH release; ApEn distinguished acromegalic from normal GH secretion (fed, P < 10(-12); fasted, P < 10(-7)) with high sensitivity (95%) and specificity (100%). Acromegalics in remission had ApEn scores larger than those of normal subjects (P < 0.0001) but smaller than those of active acromegalics (P < 0.001). The coefficient of variation of successive incremental changes in GH concentrations was significantly lower in acromegalics than in normal subjects (P < 0.001). Fourier analysis in acromegalics revealed reduced fractional amplitudes compared to normal subjects (P < 0.05). We conclude that GH secretion in acromegaly is highly irregular with disorderly release accompanying significant basal secretion.
M L Hartman, S M Pincus, M L Johnson, D H Matthews, L M Faunt, M L Vance, M O Thorner, J D Veldhuis
The human insulin receptor has two isoforms derived from alternative splicing of exon 11 of the insulin receptor gene. The type B (containing exon 11, or exon 11+) isoform binds insulin with twofold lower affinity than the type A (lacking exon 11, or exon 11-) isoform. In efforts to resolve the controversy over whether altered splicing is involved in the development of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM), the spontaneously obese diabetic rhesus monkey, a unique model that is extraordinarily similar to human NIDDM, was used. Cross-sectional studies of insulin receptor mRNA splicing variants in vastus lateralis muscle were performed on 19 rhesus monkeys. When monkeys were divided into four groups based upon the known stages of progression to NIDDM: normal (normoglycemic/normoinsulinemic), prediabetic (normoglycemic/hyperinsulinemic), early NIDDM (hyperglycemic/hyperinsulinemic), and late NIDDM (hyperglycemic/hypoinsulinemic), both hyperinsulinemic groups had significantly higher percentages of the exon 11- mRNA splicing variant compared to the normal (74.8 +/- 1.7 vs 59.0 +/- 2.3%; P < 0.005) and late NIDDM groups (74.8 +/- 1.7 vs 64.2 +/- 3.9%; P < 0.05). Our findings provide the first direct evidence linking hyperinsulinemia to alterations in insulin receptor mRNA splicing, and suggest that alterations of insulin receptor mRNA splicing in muscle is an early molecular marker that may play an important role in NIDDM.
Z Huang, N L Bodkin, H K Ortmeyer, B C Hansen, A R Shuldiner
These studies examined the effect of acidosis on immediate early (IE) gene expression in renal tubule cells. In MCT cells, an SV40 transformed mouse proximal tubule cell line, incubation in acid media led to transient increases in c-fos, c-jun, junB, and egr-1 mRNA abundance, peaking at 30 min to 1 h. In vivo metabolic acidosis caused more prolonged increases in these mRNA species in renal cortex. Nuclear runon studies demonstrated increased rates of transcription for these IE genes. In addition, pretreatment of cells with cycloheximide caused superinduction of these mRNA by acid incubation. These responses are similar to those elicited by growth factors. Inhibition of tyrosine kinase pathways prevented IE gene activation by acid, while inhibition of protein kinase C and/or increases in cell calcium had no effect. In 3T3 cells, acid activated IE genes by a different mechanism in that the increase in mRNA did not include c-jun, was more prolonged, and was blocked by cycloheximide. In summary, incubation of renal cells in acid media leads to activation of IE genes that is similar to growth factor-induced IE gene activation, and is likely mediated by tyrosine kinase pathways.
Y Yamaji, O W Moe, R T Miller, R J Alpern
Infection and inflammation induce alterations in hepatic synthesis and plasma concentrations of the acute phase proteins. Our results show that apolipoprotein (apo) J is a positive acute phase protein. Endotoxin (LPS), tumor necrosis factor (TNF), and interleukin (IL)-1 increased hepatic mRNA and serum protein levels of apo J in Syrian hamsters. Hepatic apo J mRNA levels increased 10- to 15-fold with doses of LPS from 0.1 to 100 micrograms/100 g body weight within 4 h and were elevated for > or = 24 h. Serum apo J concentrations were significantly increased by 16 h and further elevated to 3.3 times that of control, 24 h after LPS administration. Serum apo J was associated with high density lipoprotein and increased fivefold in this fraction, after LPS administration. Hepatic apo J mRNA levels increased 3.5- and 4.6-fold, with TNF and IL-1, respectively, and 8.2-fold with a combination of TNF and IL-1. Serum apo J concentrations were increased 2.3-fold by TNF, 79% by IL-1, and 2.9-fold with a combination of TNF and IL-1. These results demonstrate that apo J is a positive acute phase protein.
I Hardardóttir, S T Kunitake, A H Moser, W T Doerrler, J H Rapp, C Grünfeld, K R Feingold
The proinflammatory chemokine interleukin-8 (IL-8/NAP-1) has been implicated in recruiting neutrophils to sites of acute and chronic tissue inflammation. In transgenic mice, elevated serum IL-8 levels ranging from 1 to 118 ng/ml were correlated with proportional increases in circulating neutrophils and proportional decreases in L-selectin expression on the surface of blood neutrophils. No change in the expression of the beta 2-integrins Mac-1 and LFA-1 was apparent on peripheral blood neutrophils of the IL-8 transgenic mice. Additionally, L-selectin expression on bone marrow neutrophils and neutrophil precursors was normal in all transgenic lines. IL-8 transgenic mice demonstrated an accumulation of neutrophils in the microcirculation of the lung, liver and spleen. Moreover, there was no evidence of neutrophil extravasation, plasma exudation or tissue damage in any IL-8 transgenic mice. Neutrophil migration into the inflamed peritoneal cavity was severely inhibited in IL-8 transgenic mice, but not in nontransgenic littermates. The IL-8 transgenic mice should serve as useful models for studying the putative role of neutrophils in mediating tissue damage in models of inflammation, such as hepatic ischemia and reperfusion injury, cecal puncture and ligation, and glomerulonephritis.
W S Simonet, T M Hughes, H Q Nguyen, L D Trebasky, D M Danilenko, E S Medlock
Meth-A sarcoma cells were stable transfected to overexpress (sense construct) or underexpress (antisense construct) tissue factor. In vitro, there was no difference in plating efficiency or growth between these cell lines. In vivo, tumor cells transfected to overexpress tissue factor grew more rapidly, and established larger and more vascularized tumors than control transfectants. Antisense transfectants grew the slowest and were the least vascularized. Anticoagulation of mice with warfarin did not alter the difference between these tumor lines. Tumor cells over-expressing tissue factor released more (compared with control transfectants) mitogenic activity for endothelial cells in parallel with enhanced transcription of vascular permeability factor/vascular endothelial cell growth factor (VEGF/VPF), and diminished transcription of thrombospondin (TSP2), a molecule with anti-angiogenic properties. Antisense tissue factor transfectants, while releasing the lowest amount of mitogenic activity, had increased thrombospondin and decreased VEGF/VPF transcription compared with control transfectants or wild-type cells. Experiments with these sense, antisense, truncated sense, or vector tumor lines gave comparable results in complete medium, serum free medium or in the presence of hirudin, indicating that the activation of the coagulation mechanism was not likely to be responsible for changes in tumor cell properties. These results suggest that tissue factor regulates angiogenic properties of tumor cells by altering the production of growth regulatory molecules of endothelium by a mechanism distinct from tissue factor activation of the coagulation mechanism.
Y Zhang, Y Deng, T Luther, M Müller, R Ziegler, R Waldherr, D M Stern, P P Nawroth
Peritoneal macrophages undergoing apoptosis induced by Shigella flexneri infection release the inflammatory cytokine interleukin 1 (IL-1), but not IL-6 or tumor necrosis factor alpha (TNF alpha). Wild type shigella causes a very fast and significant release of IL-1 from prestimulated peritoneal macrophages, before the cell's integrity is compromised. Both IL-1 alpha and IL-1 beta are released, IL-1 beta in its mature processed form. IL-1 is released from presynthesized cytoplasmic pools. These results demonstrate that bacteria-induced apoptosis of macrophages may play an active role in vivo by releasing IL-1, which in turn mediates an early inflammatory response in epithelial tissues.
A Zychlinsky, C Fitting, J M Cavaillon, P J Sansonetti
Chronic and acute graft-versus-host disease (cGVHD and aGVHD) result from donor cells responding to host disparate MHC alleles. In cGVHD (H-2d-->H-2bd), heightened polyclonal immunoglobulin production is due to the interaction of donor allospecific helper T cells (Th) and the host B cells. In vivo administration of antibody to the ligand for CD40, gp39, blocked cGVHD-induced serum anti-DNA autoantibodies, IgE production, spontaneous immunoglobulin production in vitro, and associated splenomegaly. Antibody production remained inhibited for extended periods of time after termination of anti-gp39 administration. Antiallogeneic CTL responses induced in a GVHD were also prevented by the in vivo administration of anti-gp39 as was the associated splenomegaly. These data suggest that CD40-gp39 interactions are critical in GVHD and that CD40-gp39 may be a valuable ligand-receptor pair for targeting immunotherapeutic agents to control GVHD.
F H Durie, A Aruffo, J Ledbetter, K M Crassi, W R Green, L D Fast, R J Noelle
C P Genain, D Lee-Parritz, M H Nguyen, L Massacesi, N Joshi, R Ferrante, K Hoffman, M Moseley, N L Letvin, S L Hauser