Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.
C Barazzone, D Belin, P F Piguet, J D Vassalli, A P Sappino
Polycystin, the product of autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) is the cardinal member of a novel class of proteins. As a first step towards elucidating the function of polycystin and the pathogenesis of ADPKD, three types of information were collected in the current study: the subcellular localization of polycystin, the spatial and temporal distribution of the protein within normal tissues and the effects of ADPKD mutations on the pattern of expression in affected tissues. Antisera directed against a synthetic peptide and two recombinant proteins of different domains of polycystin revealed the presence of an approximately 400-kD protein (polycystin) in the membrane fractions of normal fetal, adult, and ADPKD kidneys. Immunohistological studies localized polycystin to renal tubular epithelia, hepatic bile ductules, and pancreatic ducts, all sites of cystic changes in ADPKD, as well as in tissues such as skin that are not known to be affected in ADPKD. By electron microscopy, polycystin was predominantly associated with plasma membranes. Polycystin was significantly less abundant in adult than in fetal epithelia. In contrast, polycystin was overexpressed in most, but not all, cysts in ADPKD kidneys.
L Geng, Y Segal, B Peissel, N Deng, Y Pei, F Carone, H G Rennke, A M Glücksmann-Kuis, M C Schneider, M Ericsson, S T Reeders, J Zhou
Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.
A Bosch, P B McCray Jr, S M Chang, T R Ulich, W S Simonet, D J Jolly, B L Davidson
Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.
B J Hicke, S R Watson, A Koenig, C K Lynott, R F Bargatze, Y F Chang, S Ringquist, L Moon-McDermott, S Jennings, T Fitzwater, H L Han, N Varki, I Albinana, M C Willis, A Varki, D Parma
Activation of naive T cells requires at least two signals. In addition to the well characterized interaction of the T cell antigen receptor with the antigen/MHC expressed on an antigen-presenting cell, T cell activation also requires costimulation by a second set of signals. The best characterized costimulatory receptor is CD28, which binds to a family of B7 ligands expressed on antigen-presenting cells. In asthma, although activated T cells play a role in the initiation and maintenance of airway inflammation, the importance of T cell costimulation in bronchial hyperresponsiveness had not been characterized. Therefore, we tested the hypothesis that inhibition of the CD28:B7 costimulatory pathway would abrogate airway hyperresponsiveness. Our results show that blockade of costimulation with CTLA4-Ig, a fusion protein known to prevent costimulation by blocking CD28:B7 interactions, inhibits airway hyperresponsiveness, inflammatory infiltration, expansion of thoracic lymphocytes, and allergen-specific responsiveness of thoracic T cells in this murine model of allergic asthma.
S J Krinzman, G T De Sanctis, M Cernadas, D Mark, Y Wang, J Listman, L Kobzik, C Donovan, K Nassr, I Katona, D C Christiani, D L Perkins, P W Finn
Plasmid DNA vaccines capable of preventing viral, bacterial, and parasitic infections are currently under development. Our labs have shown that a plasmid DNA vaccine encoding the circumsporozoite protein of the malaria parasite elicits protective immunity against live sporozoite challenge in adult BALB/c mice. We now find that the same DNA vaccine induces tolerance rather than immunity when administered to 2-5 d-old mice. Neonatally tolerized animals were unable to mount antibody, cytokine or cytotoxic responses when rechallenged with DNA vaccine in vitro or in vivo. Tolerance was specific for immunogenic epitopes expressed by the vaccine-encoded, endogenously produced antigen. Mice challenged with exogenous circumsporozoite protein produced antibodies against a different set of epitopes, and were not tolerized. These findings demonstrate important differences in the nature and specificity of the immune response elicited by DNA vaccines versus conventional protein immunogens.
G Mor, G Yamshchikov, M Sedegah, M Takeno, R Wang, R A Houghten, S Hoffman, D M Klinman
We studied the effects of recombinant growth hormone on systemic nitric oxide (NO) formation and hemodynamics in a double-blind, placebo-controlled trial in adult patients with acquired growth hormone deficiency. 30 patients were randomly allocated to either recombinant human growth hormone (r-hGH; 2.0 IU/d) or placebo for 12 mo. In the subsequent 12 mo, the study was continued with both groups of patients receiving r-hGH. In months 1, 3, 6, 9, and 12 of each year, urine and plasma samples were collected for the determination of urinary nitrate and cyclic GMP as indices of systemic NO production, and of plasma IGF-1 levels. Cardiac output was measured in months 1, 12, and 24 by echocardiography. r-hGH induced a fourfold increase in plasma IGF-1 concentrations within the first month of treatment. Urinary nitrate and cyclic GMP excretion rates were low at baseline in growth hormone-deficient patients (nitrate, 96.8+/-7.4 micromol/mmol creatinine; cyclic GMP, 63.6+/-7.1 nmol/mmol creatinine) as compared with healthy controls (nitrate, 167.3+/-7.5 micromol/mmol creatinine; cyclic GMP, 155.2+/-6.9 nmol/mmol creatinine). These indices of NO production were significantly increased by r-hGH, within the first 12 mo in the GH group, and within the second 12 mo in the placebo group. While systolic and diastolic blood pressure were not significantly altered by r-hGH, cardiac output significantly increased by 30-40%, and total peripheral resistance decreased by approximately 30% in both groups when they were assigned to r-hGH treatment. In the second study year, when both groups were given r-hGH, there were no significant differences in plasma IGF-1, urinary nitrate, or cyclic GMP excretion, or hemodynamic parameters between both groups. In conclusion, systemic NO formation is decreased in untreated growth hormone-deficient patients. Treatment with recombinant human growth hormone normalizes urinary nitrate and cyclic GMP excretion, possibly via IGF-1 stimulation of endothelial NO formation, and concomitantly decreases peripheral arterial resistance. Increased NO formation may be one reason for improved cardiovascular performance of patients with acquired hypopituitarism during growth hormone therapy.
R H Böger, C Skamira, S M Bode-Böger, G Brabant, A von zur Muhlen, J C Frolich
Neutrophil (PMNL) function defects occur as a consequence of HIV infection. This study examined PMNL apoptosis in patients with the acquired immunodeficiency syndrome (AIDS) to determine if accelerated apoptosis contributes to impaired function. PMNL were isolated from 10 HIV-infected patients with CD4+ lymphocyte counts < 200/mm3 without signs of active infection and 7 healthy volunteers. PMNL were stained with acridine orange and ethidium bromide after 0, 3, 6, and 18 h in culture, and examined for the morphologic changes of apoptosis and viability by fluorescent microscopy. Apoptosis was also demonstrated by electron microscopy, flow cytometry, and DNA gel electrophoresis. Apoptosis was minimal at 0 h, but PMNL from AIDS patients exhibited significantly greater apoptosis than controls at 3 h (22.5+/-11.5 vs. 8.9+/-6.9%, P = 0.015), 6 h (38.1+/-14.2 vs. 18.1+/-4.5%, P = 0.003), and 18 h (71.3+/-19.0 vs. 38.8+/-16.7%, P = 0.002). Viabilities were > or = 88.0% for both groups from 0-6 h, but by 18 h viability was significantly decreased for the HIV group (58.8+/-12.4 vs. 83.5+/-10.4%, P = 0.001) due to an increase in non-viable apoptotic cells. Incubation with serum from AIDS patients had no effect on control PMNL, and incubation with control serum did not reduce the rate of apoptosis of PMNL from AIDS patients. Incubation with granulocyte colony-stimulating factor (G-CSF) in vitro significantly decreased apoptosis for PMNL from AIDS patients. PMNL from patients with AIDS exhibit markedly accelerated apoptosis ex vivo. In vivo, apoptosis and functional impairment of PMNL may contribute to the risk of secondary infections, and cytokine therapy may be of potential clinical benefit in this circumstance.
D L Pitrak, H C Tsai, K M Mullane, S H Sutton, P Stevens
To determine the effects of hammerhead ribozymes against hepatitis C virus (HCV) RNA on viral protein translation, a luciferase reporter gene vector, pCMV/T7-NCRCdelta-luc, was constructed containing the 5'-noncoding region (5'-NCR) and part of the core region of HCV. Four ribozymes, Rz1-Rz4, were designed to cleave at nucleotide positions 136-160, 313-337, 496-520, and 373-388, respectively. Each ribozyme cleaved the target RNA at expected positions under cell-free conditions. Rz2 and Rz4 significantly suppressed translation of NCRCdelta-luc RNA by 71 and 49%, respectively. Translation of control luciferase mRNA lacking viral elements was not affected by the ribozymes. Furthermore, when NCRCdelta-luc RNA and ribozymes were cotransfected into cells, Rz2 and Rz4 significantly suppressed expression by 73 and 56%, respectively. In contrast, cleavage-deficient ribozymes with a point mutation in the hammerhead domain had no significant effect. To determine the effects of endogenously produced ribozymes, eukaryotic expression vectors for Rz2 and Rz4 were constructed. Cotransfection of the vectors with CMV/T7-NCRCdelta-luc showed suppression of luciferase activities to 50 and 61%, respectively. Moreover, transfection of pCMV/T7-NCRCdelta-luc into stable Rz2 and Rz4 producer cells also showed substantial inhibition of luciferase activity. Ribozymes directed against the HCV genome can substantially and specifically inhibit viral gene expression under intracellular conditions.
N Sakamoto, C H Wu, G Y Wu
SR 121463A, a potent and selective, orally active, nonpeptide vasopressin V2 receptor antagonist, has been characterized in several in vitro and in vivo models. This compound displayed highly competitive and selective affinity for V2 receptors in rat, bovine and human kidney (0.6 < or = Ki [nM] < or = 4.1). In this latter preparation, SR 121463A potently antagonized arginine vasopressin (AVP)-stimulated adenylyl cyclase activity (Ki = 0.26+/-0.04 nM) without any intrinsic agonistic effect. In autoradiographic experiments performed in rat kidney sections, SR 121463A displaced [3H]AVP labeling especially in the medullo-papillary region and confirmed that it is a suitable tool for mapping V2 receptors. In comparison, the nonpeptide V2 antagonist, OPC-31260, showed much lower affinity for animal and human renal V2 receptors and lower efficacy to inhibit vasopressin-stimulated adenylyl cyclase (Ki in the 10 nanomolar range). Moreover, OPC-31260 exhibited a poor V2 selectivity profile and can be considered as a V2/V1a ligand. In normally hydrated conscious rats, SR 121463A induced powerful aquaresis after intravenous (0.003-0.3 mg/kg) or oral (0.03-10 mg/kg) administration. The effect was dose-dependent and lasted about 6 hours at the dose of 3 mg/kg p.o. OPC-31260 had a similar aquaretic profile but with markedly lower oral efficacy. The action of SR 121463A was purely aquaretic with no changes in urine Na+ and K+ excretions unlike that of known diuretic agents such as furosemide or hydrochlorothiazide. In addition, no antidiuretic properties have been detected with SR 121463A in vasopressin-deficient Brattleboro rats. Thus, SR 121463A is the most potent and selective, orally active V2 antagonist yet described and could be a powerful tool for exploring V2 receptors and the therapeutical usefulness of V2 blocker aquaretic agents in water-retaining diseases.
C Serradeil-Le Gal, C Lacour, G Valette, G Garcia, L Foulon, G Galindo, L Bankir, B Pouzet, G Guillon, C Barberis, D Chicot, S Jard, P Vilain, C Garcia, E Marty, D Raufaste, G Brossard, D Nisato, J P Maffrand, G Le Fur
Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis.
E N Unemori, L B Pickford, A L Salles, C E Piercy, B H Grove, M E Erikson, E P Amento
MHC class I allele, HLA-B27, is strongly associated with a group of human diseases called spondyloarthropathies. Some of these diseases have an onset after an enteric or genitourinary infection. In the present study, we describe spontaneous disease in HLA-B27 transgenic mice where endogenous beta2-microglobulin (beta2m) gene was replaced with transgenic human beta2m gene. These mice showed cell surface expression of HLA-B27 similar to that of human peripheral blood mononuclear cells. In addition, free heavy chains (HCs) of HLA-B27 were also expressed on thymic epithelium and on a subpopulation of B27-expressing PBLs. These mice developed spontaneous arthritis and nail changes in the rear paws. Arthritis occurred primarily in male animals and only when mice were transferred from the pathogen-free barrier facility to the conventional area. Transgenic mice expressing HLA-B27 with mouse beta2m have undetectable levels of free HCs on the cell surface and do not develop arthritis. In vivo treatment with anti-HC-specific antibody delayed the onset of disease. Our data demonstrate specific involvement of HLA-B27 'free' HCs in the disease process.
S D Khare, J Hansen, H S Luthra, C S David
The objective of this study was to evaluate the effect of muscarinic receptor modulation on basal and beta-adrenergic stimulated left ventricular function in patients with heart failure. 21 heart failure patients and 14 subjects with normal ventricular function were studied. In Protocol 1 intracoronary acetylcholine resulted in a 60+/-8% inhibition of the left ventricular +dP/dt response to intracoronary dobutamine in the normal group, and a similar 70+/-13% inhibition in the heart failure group. Acetylcholine also attenuated the dobutamine-mediated acceleration of isovolumic relaxation (Tau) in both groups. Acetylcholine alone had no effect on Tau in the normal group, while it prolonged Tau in the heart failure group. In Protocol 2 intracoronary atropine resulted in a 35+/-10% augmentation of the inotropic response to dobutamine in the normal group, versus a non-significant 12+/-15% augmentation of the dobutamine response in the heart failure group. In Protocol 3, in 6 heart failure patients, both effects of acetylcholine, the slowing of ventricular relaxation and the inhibition of beta-adrenergic responses, were reversed by the addition of atropine. Therefore, in the failing human left ventricle muscarinic stimulation has an independent negative lusitropic effect and antagonizes the effects of beta-adrenergic stimulation.
G E Newton, A B Parker, J S Landzberg, W S Colucci, J D Parker
The association between HLA-B27 and spondylarthropathies is currently being reinvestigated in the light of HLA-B27 subtyping. At least 11 different subtypes have been described among which B*2703, B*2706, and B*2709 could be less closely associated with disease at the population level. Differences in the presentation of antigenic peptides by these subtypes could be related to differences in disease susceptibility. We focused our work on the comparison of B*2705 and B*2703 which differ at a single position at residue 59 in pocket A of the peptide binding groove. Endogenous peptides from the human C1R line transfected by B*2705 or B*2703 were acid-eluted and separated by HPLC. Major individual fractions were sequenced by Edman NH2-terminal degradation. Differences observed between B*2705 versus B*2703 individual ligands were confirmed in an in vitro stabilization assay with T2-B*2705 or B*2703 transfected cells in the presence of synthetic peptides. One B*2705 associated peptide is derived from the sequence 169-179 in the second extracellular domain of several HLA class I molecules including HLA-B27. This sequence (RRYLENGKETL) is highly homologous to a previously reported sequence (LRRYLENGK) sharing similarities with proteins from enteric bacteria. We show here that it is naturally presented as a major endogenous peptide by B*2705 and B*2702 disease-associated subtypes and not by B*2703.
F Boisgérault, V Tieng, M C Stolzenberg, N Dulphy, I Khalil, R Tamouza, D Charron, A Toubert
During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.
A Mallat, A M Préaux, C Serradeil-Le Gal, D Raufaste, C Gallois, D A Brenner, C Bradham, J Maclouf, V Iourgenko, L Fouassier, D Dhumeaux, P Mavier, S Lotersztajn
The effect of spinal adenosine receptor ligation on peripheral leukocyte accumulation was studied in two rat models of inflammation. Neutrophil infiltration into dermal inflammatory sites was signficantly reduced by adenosine A1 receptor agonists injected through intrathecal catheters. These effects were reversed by N-methyl-D-aspartate (NMDA), and were mimicked by (+/-)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate NMDA receptor antagonist. Peripheral adenosine levels, as measured in air pouch exudates, decreased markedly in inflamed pouches but remained near normal after intrathecal treatment with AP-5. Moreover, the antiinflammatory effects of intrathecal A1 receptor agonists and AP-5 were reversed by an adenosine A2 receptor antagonist administered intraperitoneally. Hence, central NMDA receptor activity can regulate neutrophil accumulation in peripheral inflammatory sites by reducing local levels of adenosine, an antiinflammatory autacoid which inhibits neutrophil function through A2 receptor activation. This represents a previously unknown pathway by which the central nervous system influences inflammatory responses.
G W Bong, S Rosengren, G S Firestein
FK506 (tacrolimus) is an immunosuppressive drug which interrupts Ca2+-calmodulin-calcineurin signaling pathways in T lymphocytes, thereby blocking antigen activation of T cell early activation genes. Regulation of insulin gene expression in the beta cell may also involve Ca2+-signaling pathways and FK506 has been associated with insulin-requiring diabetes mellitus during clinical use. The purpose of this study was to characterize the effects of FK506 on human insulin gene transcription, insulin mRNA levels, and insulin secretion using as a model the HIT-T15 beta cell line. FK506 had no acute effect on insulin secretion in the HIT cell, but caused a reversible time- and dose-dependent (10(-9)-10(-6) M) decrease in HIT cell insulin secretion. Decreased insulin secretion in the presence of FK506 was also accompanied by a dose-dependent decrease in HIT cell insulin content, insulin mRNA levels, and expression of a human insulin promoter-chloramphenicol acetyl transferase (CAT) reporter gene. FK506 decreased HIT cell expression of the human insulin promoter-CAT reporter gene by 40% in the presence of both low (0.4 mM) at high (20 mM) glucose concentrations. Western blot analysis of HIT cell proteins gave evidence for the presence of calcineurin in the HIT cell. These findings suggest that FK506 may have direct effects to reversibly inhibit insulin gene transcription, leading to a decline in insulin mRNA levels, insulin synthesis, and ultimately insulin secretion.
J B Redmon, L K Olson, M B Armstrong, M J Greene, R P Robertson
The factors controlling immunoglobulin (Ig) gene repertoire formation are poorly understood. Studies on monozygotic twins have helped discern the contributions of genetic versus environmental factors on expressed traits. In the present experiments, we applied a novel anchored PCR-ELISA system to compare the heavy chain V gene (V(H)) subgroup repertoires of mu and gamma expressing B lymphocytes from ten pairs of adult monozygotic twins, including eight pairs who are concordant or discordant for rheumatoid arthritis. The results disclosed that the relative expression of each Ig V(H) gene subgroup is not precisely proportional to its relative genomic size. The monozygotic twins had more similar IgM V(H) gene repertoires than did unrelated subjects. Moreover, monozygotic twins who are discordant for RA also use highly similar IgM V(H) gene-subgroup repertoires. Finally, the V(H) gene repertoire remained stable over time. Collectively, these data reveal that genetic factors predominantly control V(H) gene repertoire formation.
H Kohsaka, D A Carson, L Z Rassenti, W E Ollier, P P Chen, T J Kipps, N Miyasaka
A major goal of tumor immunotherapy is the induction of tumor-specific T cell responses that are effective in eradicating disseminated tumor, as well as mounting a persistent tumor-protective immunity. We demonstrate here that a genetically engineered fusion protein consisting of human/mouse chimeric anti-ganglioside GD2 antibody and human interleukin-2 is able to induce eradication of established B78-D14 melanoma metastases in immunocompetent syngeneic C57BL/6J mice. This therapeutic effect is mediated by host immune cells, particularly CD8+ T cells and is associated with the induction of a long-lived immunity preventing tumor growth in the majority of animals when challenged up to four months later with B78-D14 cells. This effect was tumor-specific, since no cross-protection against syngeneic, ganglioside GD2+ EL-4 thymoma cells was observed. Furthermore, this tumor-specific protection can be transmitted horizontally to naive, syngeneic SCID mice by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of cytokines provides a means to elicit effective immune responses against established tumors in the immunotherapy of neoplastic disease.
J C Becker, N Varki, S D Gillies, K Furukawa, R A Reisfeld
Human beta cells can be maintained in serum-free culture at 6 mmol/liter glucose, with 80% cell recovery and preserved glucose-inducible functions after 1 wk. Between 0 and 10 mmol/liter, glucose dose-dependently increases the number of beta cells in active protein synthesis (15% at 0 mmol/liter glucose, 60% at 5 mmol/liter, and 82% at 10 mmol/liter), while lacking such an effect in islet non-beta cells (> 75% activated irrespective of glucose concentrations). As in rat beta cells, this intercellular difference in glucose sensitivity determines the dose-response curves during acute glucose stimulation of human beta cells. During 2-h incubations, human beta cells synthesize 7 fmol insulin/10(3) cells at 0 mmol/liter glucose, 20 fmol at 5 mmol/liter, and 31 fmol at 10 mmol/liter. Culture at higher (10 or 20 mmol/liter) glucose does not affect beta cell recovery but decreases by 50-85% the net effect of glucose upon insulin synthesis and release. These reduced responses to glucose are not caused by diminished cellular activities but are the consequence of a shift of beta cells to a state of sustained activation. The presence of more activated cells at low glucose eliminates glucose-dependent cell recruitment as a mechanism for adjusting beta cell responses to acute variations in glucose concentration. It leads to elevated basal biosynthetic (3-fold) and secretory (10-fold) activities, and, hence, to a 4-fold reduction in the beta cell insulin content and the amount of insulin released at maximal glucose stimulation. Prolonged exposure of human beta cells to high glucose can thus lead to a loss of their glucose regulation as a consequence of sustained cellular activation, without signs of glucose-induced toxicity or desensitization.
Z Ling, D G Pipeleers
The structure of the carbohydrate of the 40-kD major outer membrane component of Chlamydia trachomatis and its role in defining infectivity of the organism were investigated. The oligosaccharides were released from the glycoprotein by N-glycanase digestion, coupled to a 2-aminopyridyl residue, and subjected to two-dimensional sugar mapping technique. The major fractions consisted of "high-mannose type" oligosaccharides containing 8-9 mannose residues. Bi- and tri-antennary "complex type" oligosaccharides having terminal galactose were detected as minor components. These oligosaccharides were N-linked and contained no sialic acid. This structural profile is consistent with our previous characterization based on lectin-binding and glycosidase digestion. Functional specificity of identified chlamydial oligosaccharides was analyzed using glycopeptides fractionated from ovalbumin and structurally defined oligosaccharides from other sources. The glycopeptide fraction having high-mannose type oligosaccharide, as compared to those having complex or hybrid-type, showed a stronger inhibitory effect on attachment and infectivity of chlamydial organisms to HeLa cells. Among high-mannose type oligosaccharides, the strongest inhibition was observed with mannose 8 as compared with mannose 6, 7, or 9. These results indicate that a specific high-mannose type oligosaccharide linked to the major outer membrane protein of C. trachomatis mediates attachment and infectivity of the organism to HeLa cells.
C Kuo, N Takahashi, A F Swanson, Y Ozeki, S Hakomori
The administration of the immunosuppressive humanized monoclonal antibody CAMPATH 1-H, which recognizes CD52 on lymphocytes and monocytes, is associated with a first-dose cytokine-release syndrome involving TNFalpha, IFNgamma, and IL-6 clinically. In vitro models have been used to establish the cellular source and mechanism responsible for cytokine release, demonstrating that cytokine release is isotype dependent, with the rat IgG2b and human IgG1 isotype inducing the highest levels of cytokine release, which was inhibited with antibody to CD16, the low affinity Fc-receptor for IgG (FcgammaR). Cross-linking antibody opsonized CD4 T lymphocytes failed to stimulate TNFalpha release, which together with the observation that TNFalpha release by purified natural killer (NK) cells stimulated by fixed autologous CAMPATH 1-H-opsonized targets was inhibited with anti-CD16, indicates that cytokine release results from ligation of CD16 on the NK cells, rather than Fc-receptor (FcR)-dependent cross-linking of CD52 on the targeted cell. Since the hierarchy of isotypes inducing cytokine release in these cultures matches that seen clinically, we conclude that ligation of CD16 on NK cells is also responsible for cytokine release after injection of CAMPATH 1-H in vivo.
M G Wing, T Moreau, J Greenwood, R M Smith, G Hale, J Isaacs, H Waldmann, P J Lachmann, A Compston
To investigate the autoantibody repertoire associated with SLE, we have created phage display IgG Fab libraries from two clinically active SLE patients and from the healthy identical twin of one of these patients. The libraries from the lupus discordant twins were found to both include unusually large representations of the V(H)5 gene family. By panning with DNA, the SLE libraries each yielded IgG anti-double-stranded (ds) DNA autoantibodies, which are characteristic of lupus disease. These included a V(H)5 autoantibody from the affected twin, that has a targeted cluster of mutations that potentially improves binding affinity. The recovered IgG anti-dsDNA autoantibodies expressed the same idiotypes associated with the in vivo IgG anti-dsDNA response of the respective SLE donor. Heavy-light chain shuffling experiments demonstrated a case in which the in vitro creation of anti-dsDNA binding activity required restrictive pairing of a heavy chain with Vlambda light chains similar to those in circulating anti-dsDNA autoantibodies. By contrast, IgG anti-ds autoantibodies could not be recovered from the library from the healthy twin, or from shuffled libraries with heavy chains from the healthy twin. These repertoire analyses illustrate how inheritance and somatic processes interplay to produce lupus-associated IgG autoantibodies.
P Roben, S M Barbas, L Sandoval, J M Lecerf, B D Stollar, A Solomon, G J Silverman
Recent advances in understanding the molecular pathogenesis of congenital hypothyroid goiter in cog/cog mice, have raised important questions concerning the maturation of thyroglobulin (the thyroid prohormone) in certain human kindreds with congenital goiter. We have now examined affected siblings from two unrelated families that synthesize an apparently normally glycosylated, > 300 kD immunoreactive thyroglobulin, yet have a reduced quantity of intraglandular thyroglobulin and that secreted into the circulation. From thyroid tissues of the four patients, light microscopic approaches demonstrated presence of intracellular thyroglobulin despite its absence in thyroid follicle lumina, while electron microscopy indicated abnormal distention of the endoplasmic reticulum (ER). We have confirmed biochemically that most intrathyroidal thyroglobulin fails to reach the (Golgi) compartment where complex carbohydrate modification takes place. Moreover, the disease in the affected patients is associated with massive induction of specific ER molecular chaperones including the hsp90 homolog, GRP94, and the hsp70 homolog, BiP. The data suggest that these patients synthesize a mutant thyroglobulin which is defective for folding/assembly, leading to a markedly reduced ability to export the protein from the ER. Thus, these kindreds suffer from a thyroid ER storage disease, a cell biological defect phenotypically indistinguishable from that found in cog/cog mice.
G Medeiros-Neto, P S Kim, S E Yoo, J Vono, H M Targovnik, R Camargo, S A Hossain, P Arvan
Interleukin-11 is a pleotropic cytokine produced by lung stromal cells in response to respiratory viruses, cytokines, and histamine. To further define its potential effector functions, the Clara cell 10-kD protein promoter was used to express IL-11 and the airways of the resulting transgene mice were characterized. In contrast to transgene (-) littermates, the airways of IL-11 transgene (+) animals manifest nodular peribronchiolar mononuclear cell infiltrates and impressive airways remodeling with subepithelial fibrosis. The inflammatory foci contained large numbers of B220(+) and MHC Class II(+) cells and lesser numbers of CD3(+), CD4(+), and CD8(+) cells. The fibrotic response contained increased amounts of types III and I collagen, increased numbers of alpha smooth muscle actin and desmin-containing cells and a spectrum of stromal elements including fibroblasts, myofibroblasts, and smooth muscle cells. Physiologic evaluation also demonstrated that 2-mo-old transgene (+) mice had increased airways resistance and non-specific airways hyperresponsiveness to methacholine when compared with their transgene (-) littermates. These studies demonstrate that the targeted expression of IL-11 in the mouse airway causes a B and T cell-predominant inflammatory response, airway remodeling with increased types III and I collagen, the local accumulation of fibroblasts, myofibroblasts, and myocytes, and obstructive physiologic dysregulation. IL-11 may play an important role in the inflammatory and fibrotic responses in viral and/or nonviral human airway disorders.
W Tang, G P Geba, T Zheng, P Ray, R J Homer, C Kuhn 3rd, R A Flavell, J A Elias
In the present study, it was shown that physiologically relevant levels of the proinflammatory cytokine TNFalpha induced apoptosis in rat cardiomyocytes in vitro, as quantified by single cell microgel electrophoresis of nuclei ("cardiac comets") as well as by morphological and biochemical criteria. It was also shown that TNFalpha stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNFalpha-mediated cardiomyocyte apoptosis. Consistent with this hypothesis, sphingosine strongly induced cardiomyocyte apoptosis. The ability of the appropriate stimulus to drive cardiomyocytes into apoptosis indicated that these cells were primed for apoptosis and were susceptible to clinically relevant apoptotic triggers, such as TNFalpha. These findings suggest that the elevated TNFalpha levels seen in a variety of clinical conditions, including sepsis and ischemic myocardial disorders, may contribute to TNFalpha-induced cardiac cell death. Cardiomyocyte apoptosis is also discussed in terms of its potential beneficial role in limiting the area of cardiac cell involvement as a consequence of myocardial infarction, viral infection, and primary cardiac tumors.
K A Krown, M T Page, C Nguyen, D Zechner, V Gutierrez, K L Comstock, C C Glembotski, P J Quintana, R A Sabbadini
More than 30 missense mutations in the beta-cardiac myosin heavy chain gene have been shown to be responsible for familial hypertrophic cardiomyopathy. To clarify the effects of these point mutations on myosin motor function, we expressed wild-type and mutant human beta-cardiac myosin heavy chains in insect cells with human cardiac light chains. The wild-type myosin was well purified with similar enzymatic and motor activities to those of the naturally isolated V3 cardiac myosin. Arg249-->Gln and Arg453-->Cys mutations resulted in decreased actin translocating activity (61 and 23% of the wild-type, respectively) with decreased intrinsic ATPase activity. Arg403-->Gln mutation greatly decreased actin translocating activity (27% of wild type) with a 3.3-fold increased dissociation constant for actin, while intrinsic ATPase activity was unchanged. Val606-->Met mutation only mildly affected the actin translocating activity as well as ATPase activity of myosin. The degree of deterioration by each mutation was closely correlated with the prognosis of the affected kindreds, indicating that myosin dysfunction caused by the point mutations is responsible for the pathogenesis of the disease. Structure/function relationship of myosin is discussed.
M Sata, M Ikebe
Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.
J R Balser, H B Nuss, D W Orias, D C Johns, E Marban, G F Tomaselli, J H Lawrence
Insulin receptors (IR) and IGF-I receptors (IGF-IR) have been shown to form hybrid receptors in tissues coexpressing both molecules. To date there is no information about the distribution of hybrids in tissues of normal or diabetic subjects. We developed a microwell-based immunoassay to quantitate hybrids in small human tissues samples. Microwells were coated with MA-20 anti-IR antibody or alpha-IGF-IR-PA antibody directed against the IGF-IR alpha-subunit, and incubated with skeletal muscle extracts of patients with noninsulin-dependent diabetes mellitus (NIDDM) and normal controls. Immobilized receptors were incubated with 125I-insulin or 125I-IGF-I in the presence or absence of the two unlabeled ligands. Hybrids were quantified as the fraction of 125I-IGF-I binding immunoadsorbed with MA-20 and expressed as percentage of total IGF-IR (type I+hybrids) immobilized with alpha-IGF-IR-PA. The immunoassay was validated using Western blotting analysis. Relative abundance of hybrids detected in NIDDM patients was higher than in controls. The percentage of hybrids was negatively correlated with IR number and in vivo insulin sensitivity measured by an insulin tolerance test, whereas the percentage was positively correlated with insulinemia. Insulin binding affinity was lower in NIDDM patients than in controls, and was correlated with the percentage of hybrids. Maximal IGF-I binding was significantly higher in muscle from NIDDM patients compared to controls and was positively correlated with the percentage of hybrid receptors whereas IGF-I binding affinity did not differ between the two groups. These results raise the possibility that alterations in expression of hybrid receptors may contribute to decreased insulin sensitivity, and to increased sensitivity to IGF-I. Because IGF-I has been proposed as a hypoglycemic agent in NIDDM, these results are relevant to the development of new approaches to the treatment of insulin resistance of NIDDM.
M Federici, L Zucaro, O Porzio, R Massoud, P Borboni, D Lauro, G Sesti
Based on preliminary but variable results with direct DNA transfer into wounds, we evaluated in vivo gene transfer by particle-mediated DNA delivery to rat skin to determine whether overexpression of TGF-beta1 at the site of skin incisions would result in a significant improvement in repair. Optimization of the method with viral promoter-luciferase reporter constructs indicated that expression of luciferase activity persisted up to 5 d and was promoter, pressure, and site dependent (ventral > dorsal). Using cytomegalovirus (CMV)-driven human alpha1-antitrypsin, transgene expression was immunolocalized within keratinocytes of the stratum granulosum at 24 h. We measured tensile strength of skin incisions at 11-21 d in both normal and diabetic rats transfected with TGF-beta1 expression vectors at surgery. Native murine TGF-beta1 under an SV40 promoter produced positive effects, while wound strengthening was more pronounced in diabetic animals using a CMV-driven construct. Transfection of rat skin with constitutively active, mutant porcine TGF-beta1 under the control of the CMV and Moloney murine leukemia virus promoters significantly increased tensile strength up to 80% for 14-21 d after surgery. Transfection 24 h before surgery was more effective. Particle-mediated gene delivery can be used to deliver viral promoter-cytokine expression constructs into rat skin in a safe, efficient, and reproducible fashion. The extent of wound repair, as evidenced by enhanced tensile strength, can be markedly improved in tissues transfected with TGF-beta1 expression constructs.
S I Benn, J S Whitsitt, K N Broadley, L B Nanney, D Perkins, L He, M Patel, J R Morgan, W F Swain, J M Davidson