Toxin A, a 308,000-Mr enterotoxin from Clostridium difficile, mediates antibiotic-associated diarrhea and colitis in humans. Injection of toxin A into animal intestine triggers an acute inflammatory response characterized by activation of sensory neurons and immune cells of the intestinal lamina propria, including mast cells and macrophages, and migration of circulating neutrophils in the involved intestinal segment. In this study we show that mice genetically deficient in the neurokinin-1 receptor are protected from the secretory and inflammatory changes as well as from epithelial cell damage induced by toxin A. The protective effect of neurokinin-1R deletion correlates with diminished intestinal levels of the cytokine TNF-alpha and its mRNA and the leukocyte enzyme myeloperoxidase. These results demonstrate a major requirement for substance P receptors in the pathogenesis of acute inflammatory diarrhea.
I Castagliuolo, M Riegler, A Pasha, S Nikulasson, B Lu, C Gerard, N P Gerard, C Pothoulakis
Coronary arteries contain a network of vasa vasorum in the adventitia. The three-dimensional anatomy of the vasa vasorum in early coronary atherosclerosis is unknown. This study was designed to visualize and quantitate the three-dimensional spatial pattern of vasa vasorum in normal and experimental hypercholesterolemic porcine coronary arteries, using a novel computed tomography technique. Animals were killed after being fed either a high cholesterol diet (n = 4) or a control diet (n = 4) for 12 wk. The proximal left anterior descending coronary artery was removed from the heart, scanned, and reconstructed, and quantitation of vasa vasorum density was performed. Two different types of vasa vasorum were defined: first-order vasa vasorum ran longitudinally parallel to the vessel and second-order originated from first-order vasa circumferentially around the vessel wall. Compared with controls in hypercholesterolemic coronary arteries, there was a significant increase in the area of the vessel wall (3.86+/-0.22 vs. 8.07+/-0.45 mm2, respectively, P < 0.01) and in the density of vasa vasorum (1. 84+/-0.05/mm2 vs. 4.73+/-0.24/mm2; respectively, P = 0.0001). This occurred especially by an increase of second-order vasa vasorum and disorientation of normal vasa vasorum spatial pattern. This study suggests that adventitial neovascularization of vasa vasorum occurs in experimental hypercholesterolemic coronary arteries and may be a part of the early atherosclerotic remodeling process.
H M Kwon, G Sangiorgi, E L Ritman, C McKenna, D R Holmes Jr, R S Schwartz, A Lerman
Adipose tissue growth results from de novo adipocyte recruitment (hyperplasia) and increased size of preexisting adipocytes. Adipocyte hyperplasia accounts for the severalfold increase in adipose tissue mass that occurs throughout life, yet the mechanism of adipocyte hyperplasia is unknown. We studied the potential of macrophage colony-stimulating factor (MCSF) to mediate adipocyte hyperplasia because of the profound effects MCSF exerts on pluripotent cell recruitment and differentiation in other tissues. We found that MCSF mRNA and protein were expressed by human adipocytes and that adipocyte MCSF expression was upregulated in rapidly growing adipose tissue that encircled acutely inflamed bowel and in adipose tissue from humans gaining weight (4-7 kg) with overfeeding. Localized overexpression of adipocyte MCSF was then induced in rabbit subcutaneous adipose tissue in vivo using adenoviral-mediated gene transfer. Successful overexpression of MCSF was associated with 16-fold increases in adipose tissue growth compared with a control adenovirus expressing beta-galactosidase. This occurred in the absence of increased cell size and in the presence of increased nuclear staining for MIB-1, a marker of proliferation. We conclude that MCSF participates in adipocyte hyperplasia and the physiological regulation of adipose tissue growth.
J A Levine, M D Jensen, N L Eberhardt, T O'Brien
The delivery of biologically active factors to the developing mammalian embryo by in utero gene transfer has generated considerable interest but limited success. The chorioallantoic placenta is a potential alternative target for providing therapeutic transgenes to the fetus during gestation. We demonstrate that somatic gene transfer to the midgestation rat placenta may be efficiently accomplished in situ through the implantation of a variety of genetically modified cells with different antigenic and growth properties. Ex vivo-modified cells survived and retained transgene expression until term. Proteins secreted from the transplanted cells were detectable within the fetal trunk blood. These studies suggest that gene transfer to the placenta may be a useful tool for answering questions of both embryonic and placental development and providing therapeutic proteins during gestation for amelioration of diseases with onset during embryonic life.
M C Senut, S T Suhr, F H Gage
Distinct genetic abnormalities (loss-of-function mutations of APC and p53 and oncogenic activation of Ki-ras) are associated with specific stages of the sporadic, most common types of colorectal tumors. However, the inability to maintain primary colon epithelial cells in culture has hindered the analysis of the pathogenetic role of these abnormalities in colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of p53-deficient mice; these cells are nontumorigenic as indicated by their failure to form colonies in soft agar and to grow as tumors in immunodeficient SCID mice and in immunocompetent syngeneic hosts. Upon ectopic expression of an activated Ki-ras gene, p53-deficient colon epithelial cells form colonies in soft agar and highly invasive subcutaneous tumors in both immunodeficient and immunocompetent mice. Ectopic expression of wild-type p53, but not of a DNA-binding-deficient mutant, markedly suppressed the colony-forming ability of the Ki-ras-transformed p53-deficient epithelial cells. Together, these findings establish a functional synergism in colorectal tumorigenesis dependent on the effects of an oncogenic Ki-ras in a p53-deficient background. This model of tumorigenic conversion of colon epithelial cells might be useful to identify genetic changes associated with disease progression and to evaluate the therapeutic response to conventional and novel anticancer drugs.
C Sevignani, P Wlodarski, J Kirillova, W E Mercer, K G Danielson, R V Iozzo, B Calabretta
HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.
M Aviram, M Rosenblat, C L Bisgaier, R S Newton, S L Primo-Parmo, B N La Du
Previously we presented suggestive evidence from an F2 segregating population for an interaction on blood pressure (BP) between quantitative trait loci (QTL) on rat chromosomes (Chr) 2 and 10. To prove the existence of such an interaction, we developed congenic strains for Chr 2 and 10 by introgressing the low BP QTL alleles into the Dahl salt-sensitive (S) strain. A double congenic strain was also constructed with both the Chr 2 and 10 low BP QTL alleles on the S background. The four strains (S, Chr 2 congenic, Chr 10 congenic, and Chr 2/10 double congenic) were studied for BP response to increased salt intake. An analysis of variance showed significant main effects of Chr 2, Chr 10, and a significant interaction between Chr 2 and 10 on BP and heart weight (all P < 0.0001). The interaction accounted for 24 mmHg of BP and 79 mg of heart weight. Thus, the discovery and proof of epistatic interactions are clearly critical to understanding the genetics of blood pressure.
J P Rapp, M R Garrett, A Y Deng
Biochemical investigations into the pathogenesis of osteoarthritis have, for the last two decades, concentrated on the mechanisms involved in the destruction of the articular cartilage. Although bone changes are known to occur, the biochemistry of the collagenous matrix within osteoarthritic bone has received scant attention. We report that bone collagen metabolism is increased within osteoarthritic femoral heads, with the greatest changes occurring within the subchondral zone. Collagen synthesis and its potential to mineralize were determined by the carboxy-terminal propeptide content and alkaline phosphatase activity, respectively. These data supported elevated new matrix formation. Our finding of a three- to fourfold increase in TGF-beta in osteoarthritic bone indicates that this might represent a stimulus for the increased collagen synthesis observed. Of additional significance is the hypomineralization of deposited collagen in the subchondral zone of osteoarthritic femoral heads, supporting a greater proportion of osteoid in the diseased tissue. The cross-linking of collagen was similar to that observed for controls. In addition, the degradative potential of osteoarthritic bone was considerably higher as demonstrated by increased matrix metalloproteinase 2 activity, and again the greater activity was associated with the subchondral bone tissue. The polarization exhibited in the metabolism of bone collagen from osteoarthritic hips might exacerbate the processes involved in joint deterioration by altering joint morphology. This in turn may alter the distribution of mechanical forces to the various tissues, to which bone is a sensitive responder. Bone collagen metabolism is clearly an important factor in the pathogenesis of osteoarthritis and certainly warrants further biochemical study.
J P Mansell, A J Bailey
Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.
M Romano, V Ricci, A Di Popolo, P Sommi, C Del Vecchio Blanco, C B Bruni, U Ventura, T L Cover, M J Blaser, R J Coffey, R Zarrilli
Our understanding of the pathogenesis of atopic dermatitis (AD) and its relationship to asthma remains incomplete. Herein, we describe a murine model of epicutaneous (EC) sensitization to the protein allergen, chicken egg albumin, ovalbumin (OVA), which results in a rise in total and OVA-specific serum IgE and leads to the development of a dermatitis characterized by infiltration of CD3(+) T cells, eosinophils, and neutrophils and by local expression of mRNA for the cytokines IL-4, IL-5, and interferon-gamma. A single exposure of the EC sensitized mice to aerosolized OVA induced eosinophilia in the bronchoalveolar lavage fluid and airway hyperresponsiveness to intravenous methacholine as assessed by measurement of pulmonary dynamic compliance (Cdyn). These results suggest a possible role for EC exposure to antigen in atopic dermatitis and in the development of allergic asthma.
J M Spergel, E Mizoguchi, J P Brewer, T R Martin, A K Bhan, R S Geha
This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.
Y P Zhou, D Teng, F Dralyuk, D Ostrega, M W Roe, L Philipson, K S Polonsky
Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.
E V Valore, C H Park, A J Quayle, K R Wiles, P B McCray Jr, T Ganz
The activation of platelets and the formation of neutrophil- platelet conjugates may lead to the development of thromboemboli. We studied whether blockade of adenosine receptors during coronary hypoperfusion may cause thromboemboli via P-selectin-dependent mechanisms in 30 open-chest dogs. When coronary blood flow was reduced to 20% of the control, it was stable at low levels with increases in adenosine levels. When 8-p-sulfophenyltheophylline, an adenosine receptor antagonist, was infused during coronary hypoperfusion, coronary blood flow decreased gradually and approached almost zero 20 min after its administration. Histological examination revealed thromboemboli in the small coronary vessels. During hypoperfusion in the presence of 8-p-sulfophenyltheophylline, the mAb against P-selectin attenuated both the reduction in coronary blood flow and the formation of thromboemboli, and improved contractile and metabolic dysfunction of the myocardium. Flow cytometric analysis indicated that the expression of P-selectin on platelet and neutrophil-platelet adhesion were increased during coronary hypoperfusion, and that both were further augmented by 8-p-sulfophenyltheophylline. Immunohistochemical examination showed no staining of P-selectin in the ischemic myocardium. Adenosine inhibited the thrombin-induced expression of P-selectin on platelet and neutrophil- platelet adhesion via adenosine A2 receptors. Adenosine appears to inhibit the formation of thromboemboli during coronary hypoperfusion by suppressing the expression of P-selectin on platelets and neutrophil-platelet adhesion.
T Minamino, M Kitakaze, H Asanuma, Y Tomiyama, M Shiraga, H Sato, Y Ueda, H Funaya, T Kuzuya, Y Matsuzawa, M Hori
Muscle acidosis has been implicated as a major determinant of reflex sympathetic activation during exercise. To test this hypothesis we studied sympathetic exercise responses in metabolic myopathies in which muscle acidosis is impaired or augmented during exercise. As an index of reflex sympathetic activation to muscle, microneurographic measurements of muscle sympathetic nerve activity (MSNA) were obtained from the peroneal nerve. MSNA was measured during static handgrip exercise at 30% of maximal voluntary contraction force to exhaustion in patients in whom exercise-induced muscle acidosis is absent (seven myophosphorylase deficient patients; MD [McArdle's disease], and one patient with muscle phosphofructokinase deficiency [PFKD]), augmented (one patient with mitochondrial myopathy [MM]), or normal (five healthy controls). Muscle pH was monitored by 31P-magnetic resonance spectroscopy during handgrip exercise in the five control subjects, four MD patients, and the MM and PFKD patients. With handgrip to exhaustion, the increase in MSNA over baseline (bursts per minute [bpm] and total activity [%]) was not impaired in patients with MD (17+/-2 bpm, 124+/-42%) or PFKD (65 bpm, 307%), and was not enhanced in the MM patient (24 bpm, 131%) compared with controls (17+/-4 bpm, 115+/-17%). Post-handgrip ischemia studied in one McArdle patient, caused sustained elevation of MSNA above basal suggesting a chemoreflex activation of MSNA. Handgrip exercise elicited an enhanced drop in muscle pH of 0.51 U in the MM patient compared with the decrease in controls of 0.13+/-0.02 U. In contrast, muscle pH increased with exercise in MD by 0.12+/-0.05 U and in PFKD by 0.01 U. In conclusion, patients with glycogenolytic, glycolytic, and oxidative phosphorylation defects show normal muscle sympathetic nerve responses to static exercise. These findings indicate that muscle acidosis is not a prerequisite for sympathetic activation in exercise.
J Vissing, S F Vissing, D A MacLean, B Saltin, B Quistorff, R G Haller
Recent data suggest that sex hormones affect the thiazide-sensitive NaCl cotransporter (TSC) density or binding capacity (Chen, Z., D.A. Vaughn, and D.D. Fanestil. 1994. J. Am. Soc. Nephrol. 5:1112-1119). Thus, we determined the effect of ovariectomy (OVX) and estrogen replacement on the ultrastructural localization of TSC in rat kidney using immunocytochemistry. Kidneys of intact female (CON) and OVX rats fed ad libitum for 6 and 9 wk or pair-fed for 9 wk were processed for transmission electron microscopy. Immunogold localization of rat TSC (rTSC1) demonstrated intense label in the apical plasma membrane of CON distal convoluted tubule (DCT). In OVX DCT, rTSC1 label and apical plasma membrane microprojections were decreased. Western blots of renal membrane protein from pair-fed CON and OVX revealed bands at 129-135 kD, but the OVX signal was reduced. Morphometric analyses demonstrated that injecting 10 microg/ kg body weight 17beta-estradiol subcutaneously 4x/wk in OVX rats restored DCT apical microprojections and label density for rTSC1. Thus, in OVX rats (a) rTSC1 immunoreactive renal membrane protein is reduced; (b) apical plasma membrane complexity and immunogold label for rTSC1 in DCT is decreased; and (c) estradiol replacement restores DCT ultrastructure and rTSC1 label to normal. We conclude that estrogen enhances the density of rTSC1 in the DCT, and may alter renal Na transport by this mechanism.
J W Verlander, T M Tran, L Zhang, M R Kaplan, S C Hebert
In this study, we investigated the impact of the common apoE polymorphism on apoE metabolism and cholesterol homeostasis in monocyte-derived macrophages isolated from E2/2, E3/3, and E4/4 subjects. Unloaded cells of all genotypes contained similar amounts of free cholesterol, cholesteryl ester, and apoE mRNA. E3/3 cells secreted 77 and 30% more apoE than E2/2 or E4/4 cells, respectively. Pulse-chase studies confirmed that the apoE secretion rate was greatest in E3/3 and least in E2/2 cells and showed that a portion of apoE2, but not apoE3 or apoE4, was degraded intracellularly. Surface binding of apoE was greatest in E4/4 cells, as revealed by heparinase treatment. On cholesterol loading with acetylated LDL, apoE mRNA levels and protein secretion rose most in E4/4 and least in E2/2 cells. Cholesterol and cholesteryl ester content, however, rose most in E2/2 and least in E3/3 cells. Incubations with 3H-cholesterol-labeled acetylated LDL revealed that E2/2 cells were most efficient at secreting cholesterol. The greatest reuptake of 3H-cholesterol-rich particles was from E4/4 macrophage- conditioned media. Thus, E2/2 macrophages, despite a low apoE secretion rate, are protected from cholesterol storage by apoE-mediated cholesterol efflux. In E3/3 macrophages, cholesterol accumulation is lessened by a high basal apoE secretion rate. E4/4 macrophages secrete the most apoE but lack effective net cholesterol efflux due to enhanced surface binding and reuptake of cholesterol-rich particles.
P Cullen, A Cignarella, B Brennhausen, S Mohr, G Assmann, A von Eckardstein
Our previous work showed that the proteoglycan aggrecan can induce erosive polyarthritis and spondylitis in BALB/c mice, and that the G1 domain of the proteoglycan aggrecan (G1) is the arthritogenic region. In this study, two T cell epitopes residing on G1 within residues 70-84 (peptide G5) and 150-169 (peptide G9) were identified using synthetic peptides and aggrecan-specific T cell lines. Two G1-specific T cell hybridomas exclusively responded to peptide G5. When the G5-specific T cell line was injected intraperitoneally into BALB/c mice, it induced acute inflammatory arthritis in joints, but only in those that had been injected with the epitope recognized by these T cells. Furthermore, we also demonstrate that the keratan sulfate chain(s) (KS) on G1 possess immunosuppressive properties with respect to T and B cell epitope recognition. T cell lines that recognize both G1 and peptide G5 show an increased response to G1 after KS is removed. Antibodies in hyperimmune sera of mice immunized with G1 show increased epitope recognition (quantitative and qualitative) after KS removal before immunization. These studies reveal that a T cell line specific to an epitope on the G1 domain of aggrecan, also recognizing a corresponding mouse G1 epitope, can induce arthritis by adoptive transfer and homing to the intraarticular epitope, thereby implicating T cells in arthritis development caused by immunity to the G1 domain of aggrecan. Moreover, the presence of KS on G1 can inhibit arthritis development by suppressing T and B cell epitope recognition.
Y Zhang, A Guerassimov, J Y Leroux, A Cartman, C Webber, R Lalic, E de Miguel, L C Rosenberg, A R Poole
The cervical cellular transcription factors Brn-3a and Brn-3b have antagonistic effects on transcription of the human papilloma virus types 16 and 18 E6 and E7 oncogenes, with Brn-3a activating expression and Brn-3b repressing it. We therefore measured expression of Brn-3a and Brn-3b mRNAs in biopsies from 16 women with no detectable cervical abnormality, and in 14 women with cervical intraepithelial neoplasia grade 3 (CIN3) lesions. Although the mean level of Brn-3b expression was similar in both groups, the mean level of Brn-3a expression was over 300-fold higher in the CIN3 samples when compared with normals. Elevated expression of Brn-3a was also detected in 16 histologically normal regions of the cervix adjacent to the CIN3 lesions, indicating that elevation of Brn-3a levels is not confined to the lesion in women with CIN3, and is thus not a consequence of the oncogenic process. The elevated levels of Brn-3a in the CIN3 patient samples, together with the activating effect of Brn-3a on HPV-16 and -18 oncogene expression, suggest that induction of this factor is involved in activating HPV-16 and -18 oncogene expression in the cervix, and hence in the production of cervical cancers induced by HPV.
D Ndisdang, P J Morris, C Chapman, L Ho, A Singer, D S Latchman
At sites of injury, macrophages secrete growth factors and proteins that promote tissue repair. While this central role of the macrophage has been well studied, the specific stimuli that recruit macrophages into sites of injury are not well understood. This study examines the role of macrophage inflammatory protein 1alpha (MIP-1alpha), a C-C chemokine with monocyte chemoattractant capability, in excisional wound repair. Both MIP-1alpha mRNA and protein were detectable in murine wounds from 12 h through 5 d after injury. MIP-1alpha protein levels peaked 3 d after injury, coinciding with maximum macrophage infiltration. The contribution of MIP-1alpha to monocyte recruitment into wounds was assessed by treating mice with neutralizing anti-MIP-1alpha antiserum before injury. Wounds of mice treated with anti-MIP-1alpha antiserum had significantly fewer macrophages than control (41% decrease, P < 0. 01). This decrease in wound macrophages was paralleled by decreased angiogenic activity and collagen synthesis. When tested in the corneal micropocket assay, wound homogenates from mice treated with anti-MIP-1alpha contained significantly less angiogenic activity than control wound homogenates (27% positive for angiogenic activity versus 91% positive in the control group, P < 0.01). Collagen production was also significantly reduced in the wounds from anti-MIP-1alpha treated animals (29% decrease, P < 0.05). The results demonstrate that MIP-1alpha plays a critical role in macrophage recruitment into wounds, and suggest that appropriate tissue repair is dependent upon this recruitment.
L A DiPietro, M Burdick, Q E Low, S L Kunkel, R M Strieter
To identify molecular factors regulating apo A-I production in vivo, we induced in transgenic mice the experimental nephrotic syndrome, which results in elevated levels of HDL cholesterol (HDL-C), plasma apo A-I, and hepatic apo A-I mRNA. Human (h) apo A-I transgenic mice with different length 5' flanking sequences (5.5 or 0.256 kb, the core promoter for hepatic-specific basal expression) were injected with nephrotoxic (NTS) or control serum. With nephrosis, there were comparable (greater than twofold) increases in both lines of HDL-C, h-apo A-I, and hepatic h-apo A-I mRNA, suggesting that cis-acting elements regulating induced apo A-I gene expression were within its core promoter. Hepatic nuclear extracts from control and nephrotic mice footprinted the core promoter similarly, implying that the same elements regulated basal and induced expression. Hepatic mRNA levels for hepatocyte nuclear factor (HNF) 4 and early growth response factor (EGR) 1, trans-acting factors that bind to the core promoter, were measured: HNF4 mRNA was not affected, but that of EGR-1 was elevated approximately fivefold in the nephrotic group. EGR-1 knockout (EGR1-KO) mice or mice expressing EGR-1 were injected with either NTS or control serum. Levels of HDL-C, apo A-I, and hepatic apo A-I mRNA were lowest in nonnephrotic EGR1-KO mice and highest in nephrotic mice expressing EGR-1. Although in EGR1-KO mice HDL-C, apo A-I, and apo A-I mRNA levels also increased after NTS injection, they were approximately half of those in the nephrotic EGR-1-expressing mice. We conclude that in this model, basal and induced apo A-I gene expression in vivo are regulated by the trans-acting factor EGR-1 and require the same cis-acting elements in the core promoter.
M Zaiou, N Azrolan, T Hayek, H Wang, L Wu, M Haghpassand, B Cizman, M P Madaio, J Milbrandt, J B Marsh, J L Breslow, E A Fisher
We used wild-type and isogenic mutant strains of group A Streptococcus (GAS) that expressed M protein, capsule, or both to study the function of M protein and the hyaluronic acid capsular polysaccharide in attachment of GAS to human keratinocytes. Types 6 and 24, but not type 18, M protein were found to mediate attachment of GAS to soft palate or skin keratinocytes, but this interaction was prevented by the hyaluronic acid capsule on highly encapsulated, or mucoid, strains. Monoclonal antibody to CD44, the principal hyaluronic acid-binding receptor on keratinocytes, inhibited attachment of both highly encapsulated and poorly encapsulated wild type strains of GAS, but not the attachment of acapsular mutants. Transfection of K562 cells with cDNA encoding human CD44 conferred the capacity to bind each of six wild-type strains of GAS, but not to bind acapsular mutants. Because, in contrast to other potential adhesins, the group A streptococcal capsule is both highly conserved and surface-exposed, it may serve as a universal adhesin for attachment of diverse strains of GAS to keratinocytes of the pharyngeal mucosa and the skin.
H M Schrager, S Albertí, C Cywes, G J Dougherty, M R Wessels
Atherosclerosis is an inflammatory-fibrotic response to accumulation of cholesterol in the artery wall. In hypercholesterolemia, low density lipoproteins (LDL) accumulate and are oxidized to proinflammatory compounds in the arterial intima, leading to activation of endothelial cells, macrophages, and T lymphocytes. We have studied immune cell activation and the autoimmune response to oxidized LDL in atherosclerotic apo E-knockout mice. Autoantibodies to oxidized LDL exhibited subclass specificities indicative of T cell help, and the increase in antibody titers in peripheral blood was associated with increased numbers of cytokine-expressing T cells in the spleen. In addition to T cell-dependent antibodies, IgM antibodies to oxidized LDL were also increased in apo E-knockout mice. This suggests that both T cell-dependent and T cell-independent epitopes may be present on oxidized LDL. In moderate hypercholesterolemia, IgG antibodies were largely of the IgG2a isotype, suggesting that T cell help was provided by proinflammatory T helper (Th) 1 cells, which are prominent components of atherosclerotic lesions. In severe hypercholesterolemia induced by cholesterol feeding of apo E-knockout mice, a switch to Th2-dependent help was evident. It was associated with a loss of IFN-gamma-producing Th1 cells in the spleen, whereas IL-4-producing Th2 cells were more resistant to hypercholesterolemia. IFN-gamma but not IL-4 mRNA was detected in atherosclerotic lesions of moderately hypercholesterolemic apo E-knockout mice, but IL-4 mRNA appeared in the lesions when mice were made severely hypercholesterolemic by cholesterol feeding. These data show that IFN-gamma-producing Th1 cells infiltrate atherosclerotic lesions and provide T cell help for autoimmune responses to oxidized LDL in apo E-knockout mice. However, severe hypercholesterolemia is associated with a switch from Th1 to Th2, which results not only in the formation of IgG1 autoantibodies to oxidized LDL, but also in the appearance of Th2-type cytokines in the atherosclerotic lesions. Since the two subsets of T cells counteract each other, this switch may have important consequences for the inflammatory/immune process in atherosclerosis.
X Zhou, G Paulsson, S Stemme, G K Hansson
According to the secretion-capture model of remnant lipoprotein clearance, apo E secreted by hepatocytes into the space of Disse serves to enrich the remnants with a ligand for receptor-mediated lipoprotein endocytosis. Current evidence supports a two-receptor model of lipoprotein removal, in which apo E-containing remnants bind either the low density lipoprotein receptor (LDLR) or the LDLR-related protein (LRP). Recently, we demonstrated that reconstitution of apo E(-/-) mice with apo E(+/+) marrow results in normalization of plasma lipoprotein levels, indicating that hepatic expression of apo E is not required for remnant clearance and calling into question the relevance of the secretion-capture mechanism. To dissect the relative contributions of LDLR and LRP to the cellular catabolism of remnant lipoproteins by the hepatocyte, bone marrow transplantation (BMT) was used to reconstitute macrophage expression of apo E in mice that were null for expression of both apo E and the LDLR. Reconstitution of macrophage apo E in apo E(-/-)/LDLR(-/-) mice had no effect on serum lipid and lipoprotein concentrations, although it produced plasma apo E levels up to 16-fold higher than in C57BL/6 controls. Immunocytochemistry of hepatic sections revealed abundant staining for apo E in the space of Disse, but no evidence of receptor-mediated endocytosis of remnant lipoproteins. Transient expression of human LDLR in the livers of apo E(+/+)--> apo E(-/-)/LDLR(-/-) mice by adenoviral gene transfer resulted in normalization of serum lipid levels and in the clearance of apo E-containing lipoproteins from the space of Disse. We conclude that whereas the LDLR efficiently clears remnant lipoproteins irrespective of the site of origin of apo E, endocytosis by the chylomicron remnant receptor (LRP) is absolutely dependent on hepatic expression of apo E. These data demonstrate in vivo the physiologic relevance of the apo E secretion-capture mechanism in the liver.
M F Linton, A H Hasty, V R Babaev, S Fazio
We have isolated progenitor cells from the stromal system of the fibrous dysplastic marrow of patients with McCune-Albright Syndrome. Analysis of the Gsalpha gene from individual colonies provided direct evidence for the presence of two different genotypes within single fibrous dysplastic lesions: marrow stromal cells containing two normal Gsalpha alleles, and those containing one normal allele and an allele with an activating mutation. Transplantation of clonal populations of normal cells into the subcutis of immunocompromised mice resulted in normal ossicle formation. In contrast, transplantation of clonal populations of mutant cells always led to the loss of transplanted cells from the transplantation site and no ossicle formation. However, transplantation of a mixture of normal and mutant cells reproduced an abnormal ectopic ossicle recapitulating human fibrous dysplasia and providing an in vivo cellular model of this disease. These results provide experimental evidence for the necessity of both normal and mutant cells in the development of McCune-Albright Syndrome fibrous dysplastic lesions in bone.
P Bianco, S A Kuznetsov, M Riminucci, L W Fisher, A M Spiegel, P G Robey
Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.
S S Lin, B C Weidner, G W Byrne, L E Diamond, J H Lawson, C W Hoopes, L J Daniels, C W Daggett, W Parker, R C Harland, R D Davis, R R Bollinger, J S Logan, J L Platt
Type 2 diabetes mellitus is a common chronic disease that is thought to have a substantial genetic basis. Identification of the genes responsible has been hampered by the complex nature of the syndrome. Abnormalities in insulin secretion and insulin action predict the development of type 2 diabetes and are, themselves, highly heritable traits. Since fewer genes may contribute to these precursors of type 2 diabetes than to the overall syndrome, such genes may be easier to identify. We, therefore, undertook an autosomal genomic scan to identify loci linked to prediabetic traits in Pima Indians, a population with a high prevalence of type 2 diabetes. 363 nondiabetic Pima Indians were genotyped at 516 polymorphic microsatellite markers on all 22 autosomes. Linkage analyses were performed using three methods (single-marker, nonparametric multipoint [MAPMAKER/SIBS], and variance components multipoint). These analyses provided evidence for linkage at several chromosomal regions, including 3q21-24 linked to fasting plasma insulin concentration and in vivo insulin action, 4p15-q12 linked to fasting plasma insulin concentration, 9q21 linked to 2-h insulin concentration during oral glucose tolerance testing, and 22q12-13 linked to fasting plasma glucose concentration. These results suggest loci that may harbor genes contributing to type 2 diabetes in Pima Indians. None of the linkages exceeded a LOD score of 3.6 (a 5% probability of occurring in a genome-wide scan). These findings must, therefore, be considered tentative until extended in this population or replicated in others.
R E Pratley, D B Thompson, M Prochazka, L Baier, D Mott, E Ravussin, H Sakul, M G Ehm, D K Burns, T Foroud, W T Garvey, R L Hanson, W C Knowler, P H Bennett, C Bogardus
The role of various MHC genes in determining the progression of multiple sclerosis (MS) remains controversial. The HLA-DR3 gene has been associated with benign relapsing MS in some genetic epidemiologic studies, but with disease progression in others. We induced demyelination in highly susceptible B10.M and B10.Q mice expressing the DR3 (HLA-DRB1*0301) transgene to determine directly the effects of a human transgene by infecting them with Theiler's murine encephalomyelitis virus (TMEV). DR3+ mice experienced a dramatic reduction in the extent and severity of demyelination compared with DR3- littermate controls, whereas anti-TMEV antibody titers, delayed-type hypersensitivity responses, and levels of infectious virus, virus antigen, and virus RNA were similar in both groups. To address a possible mechanism of how the human transgene is reducing virus-induced demyelination, we analyzed cytokine expression in the lesions and also determined whether B10.M mice can respond to peptides derived from the DR3 molecule. Intense staining for IFN-gamma and IL-4, T helper (TH) 1 and TH2 cytokines, respectively, was found in the lesions of TMEV-infected DR3- mice but not in the DR3+ transgenic mice at day 21 after infection. DR3 peptides elicited strong proliferative responses in B10.M mice but not in B10.M (DR3+) mice. These experiments are the first to demonstrate that a human class II DR gene can alter the severity of demyelination in an animal model of MS without influencing viral load. These experiments are consistent with a mechanism by which DR3 reduces demyelination by altering the cytokine expression in the lesions, possibly by deleting T cells involved in virus-induced pathology.
K M Drescher, L T Nguyen, V Taneja, M J Coenen, J L Leibowitz, G Strauss, G J Hammerling, C S David, M Rodriguez
An arginine to glutamine missense mutation at position 403 of the beta-cardiac myosin heavy chain causes familial hypertrophic cardiomyopathy. Here we study mice which have this same missense mutation (alphaMHC403/+) using an isolated, isovolumic heart preparation where cardiac performance is measured simultaneously with cardiac energetics using 31P nuclear magnetic resonance spectroscopy. We observed three major alterations in the physiology and bioenergetics of the alphaMHC403/+ mouse hearts. First, while there was no evidence of systolic dysfunction, diastolic function was impaired during inotropic stimulation. Diastolic dysfunction was manifest as both a decreased rate of left ventricular relaxation and an increase in end-diastolic pressure. Second, under baseline conditions alphaMHC403/+ hearts had lower phosphocreatine and increased inorganic phosphate contents resulting in a decrease in the calculated value for the free energy released from ATP hydrolysis. Third, hearts from alphaMHC403/+ hearts that were studied unpaced responded to increased perfusate calcium by decreasing heart rate approximately twice as much as wild types. We conclude that hearts from alphaMHC403/+ mice demonstrate work load-dependent diastolic dysfunction resembling the human form of familial hypertrophic cardiomyopathy. Changes in high-energy phosphate content suggest that an energy-requiring process may contribute to the observed diastolic dysfunction.
M Spindler, K W Saupe, M E Christe, H L Sweeney, C E Seidman, J G Seidman, J S Ingwall
Insulin resistance is often associated with atherosclerotic diseases in subjects with obesity and impaired glucose tolerance. This study examined the effects of insulin resistance on coronary risk factors in IRS-1 deficient mice, a nonobese animal model of insulin resistance. Blood pressure and plasma triglyceride levels were significantly higher in IRS-1 deficient mice than in normal mice. Impaired endothelium-dependent vascular relaxation was also observed in IRS-1 deficient mice. Furthermore, lipoprotein lipase activity was lower than in normal mice, suggesting impaired lipolysis to be involved in the increase in plasma triglyceride levels under insulin-resistant conditions. Thus, insulin resistance plays an important role in the clustering of coronary risk factors which may accelerate the progression of atherosclerosis in subjects with insulin resistance.
H Abe, N Yamada, K Kamata, T Kuwaki, M Shimada, J Osuga, F Shionoiri, N Yahagi, T Kadowaki, H Tamemoto, S Ishibashi, Y Yazaki, M Makuuchi
To evaluate the concept that in vivo transfer of the human carboxylesterase gene will confer sensitivity of a solid tumor to the prodrug CPT-11 (irinotecan), we constructed an adenovirus vector (AdCMV.CE) carrying the human carboxylesterase gene driven by the cytomegalovirus (CMV) promoter, infected A549 human lung adenocarcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CE produced a functional carboxylesterase protein in A549 cells in vitro and in vivo as evidenced by ability of lysates from the infected cells to convert CPT-11 to its active metabolite SN-38. The AdCMV.CE vector effectively suppressed A549 cell growth in vitro in the presence of CPT-11. Cell mixing studies demonstrated that when as few as 10% of cells expressed the human carboxylesterase gene, there was bystander growth suppression in the presence of CPT-11. Consistent with these in vitro observations, when AdCMV.CE was directly injected into established subcutaneous A549 tumors in nude mice receiving CPT-11, there was 35% reduction in tumor size at day 27 compared to controls, and a 41% reduction at day 34 (P < 0.01, both comparisons to controls). Similar observations were made with the cell line H157 and HeLa. These observations suggest that local gene transfer of the human carboxylesterase gene and concomitant local administration of CPT-11 may have potential as a strategy for control of the growth of solid tumors.
A Kojima, N R Hackett, A Ohwada, R G Crystal
Insulin-like growth factor (IGF)-1 is a cytokine that promotes oligodendrocyte development and myelin production. This study investigated whether treatment of chronic, relapsing murine experimental autoimmune encephalomyelitis (EAE) with IGF-1 or IGF-1 associated with its binding protein, IGFBP3, altered the course of disease. Administration of IGF-1/IGFBP3 (1-100 mg/kg per day) delayed the onset of disease in a dose-dependent manner and histologic examination showed a delay in inflammatory cells entering the central nervous system. However, once signs of EAE developed, disease was enhanced in the mice that had been given the highest dose of IGF-1/IGFBP3. Treatment with IGF-1/IGFBP3 after the onset of signs resulted in a severe relapse. Administration of free IGF-1 (10 mg/kg per day) provided mild protection when given before disease onset, but did not significantly alter the course of disease if given after disease onset. Possible mechanisms that could explain the altered disease in IGF-1/IGFBP3-treated mice included (a) IGF-1/IGFBP3 administration delayed the onset of EAE by downregulating ICAM-1 gene expression in the central nervous system, and (b) IGF-1/IGFBP3 treatment of EAE resulted in more severe disease due to enhanced expansion of encephalitogenic T cells. Although IGF-1 may enhance remyelination, these results indicate that administration of IGF-1 associated with IGFBP3 may also accentuate autoimmune demyelinating disease.
A E Lovett-Racke, P Bittner, A H Cross, J A Carlino, M K Racke