RESUMEN
OBJECTIVE: To determine whether oral tolerance to type I collagen (CI) could be induced in patients with systemic sclerosis (SSc). METHODS: Twenty adult patients with limited or diffuse SSc were enrolled in a study to receive 0.1 mg of solubilized native bovine CI daily for 1 month, followed by 0.5 mg daily for 11 months. Peripheral blood mononuclear cells (PBMC) were obtained from the patients and cultured with human alpha1(I) and alpha2(I) chains, before and after CI treatment. Culture supernatants were analyzed for levels of interferon-gamma (IFNgamma) and interleukin-10 (IL-10). Sera obtained before and after treatment were analyzed for levels of soluble IL-2 receptor (sIL-2R). Although this study was not intended to assess the clinical efficacy of oral CI administration in SSc, selected measures of disease severity and organ involvement were evaluated. RESULTS: Oral administration of CI to SSc patients induced significant reductions in levels of IFNgamma and IL-10 in alpha1(I)- and alpha2(I)-stimulated PBMC culture supernatants, indicating that T cell immunity to CI was decreased by this treatment. Serum levels of sIL-2R also decreased significantly after oral CI treatment, suggesting a reduction in T cell activation. Significant improvements occurred in the modified Rodnan skin thickness score and the modified Health Assessment Questionnaire after 12 months of oral CI in this open trial. The lung carbon monoxide diffusing capacity improved statistically and showed a trend toward clinically significant improvement. CONCLUSION: Oral administration of bovine CI to patients with diffuse or limited SSc induces a reduction in T cell reactivity to human CI, appears to be well tolerated, and does not worsen the disease. Further evaluation of oral tolerance to CI in patients with SSc is justified to determine whether it has therapeutic efficacy.
Asunto(s)
Esclerodermia Sistémica/inmunología , Administración Oral , Animales , Bovinos , Colágeno/administración & dosificación , Colágeno/inmunología , Regulación hacia Abajo , Femenino , Humanos , Tolerancia Inmunológica/fisiología , Interferón gamma/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Cooperación del PacienteRESUMEN
OBJECTIVE: To investigate the efficacy of oral type II collagen (CII) in the treatment of rheumatoid arthritis (RA), when added to existing therapy. METHODS: Patients with active RA (n = 190) were randomized into a 6-month, double-blind, placebo-controlled trial. Patients continued to take their current arthritis medications. Patients received either placebo or bovine CII, 0.1 mg/day for 1 month, then 0.5 mg/day for 5 months. RESULTS: There were no significant differences between the baseline characteristics of either group. The primary response parameter was the American College of Rheumatology (ACR) preliminary definition of improvement in RA (ACR 20). There was no statistically significant difference in the ACR 20 after 6 months (20.0% of placebo patients; 16.84% of bovine CII patients). There were significant differences in several clinical variables after treatment, all favoring the placebo group. CONCLUSION: Oral solubilized bovine CII, added to existing therapy, did not improve disease activity in patients with RA.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Colágeno/uso terapéutico , Administración Oral , Adolescente , Adulto , Anciano , Animales , Artritis Reumatoide/patología , Bovinos , Colágeno/administración & dosificación , Método Doble Ciego , Quimioterapia Combinada , Femenino , Humanos , Articulaciones/efectos de los fármacos , Articulaciones/patología , Masculino , Persona de Mediana Edad , Dimensión del Dolor , Índice de Severidad de la Enfermedad , Encuestas y Cuestionarios , Resultado del TratamientoRESUMEN
Data from a number of laboratories support a potential role for tumor necrosis factor-alpha (TNF-alpha) in the loss of insulin sensitivity and the pathogenesis of insulin resistance (IR) in diabetic animal models and human patients. We designed experiments to establish a dose-response relationship for TNF-alpha and IR in H-411E cells in culture. IR was measured by inhibition of the ability of graded amounts of insulin to stimulate expression of calmodulin (CaM) mRNA in these cells. This was assessed by autoradiographs of Northern blot(s) of CaM mRNA probed with labeled oligonucleotide cDNA for rat CaM. We found that TNF-alpha at 0.1, 1.0, and 10.0 ng/ml opposed 10,000 microU/ml insulin (i.e., %IR = 20%, 67%, and 88%, respectively). At 1.0 ng/ml TNF-alpha, insulin at the concentration of 1000 microU/ml (0.006 micromol/L) stimulated CaM mRNA at a 41% level and at 10,000 microU/ml (0.06 micromol/L) at a 63% level. Furthermore, oligopeptide TNF-alpha homologs (at 1000 x the molar concentration of TNF-alpha) TNF-alpha 69-100 and TNF-alpha 133-157 conferred 66% and 101% IR, respectively, while all other peptide fragments of TNF-alpha were essentially without effect. Studies done with both monoclonal and polyclonal antibodies to the TNF-alpha receptor demonstrated blocking activity by polyclonal but not by monoclonal anti TNF-alpha receptor antibody. This supports the concept that the activity of the peptide fragments occurs through the TNF-alpha receptor and not through nonspecific translocation across the plasma membrane. These data suggest that the epitopes on TNF-alpha that mediate IR reside in two regions of the molecule spanning amino acid residues 69-100 and 133-157.
Asunto(s)
Antagonistas de Insulina/farmacología , Resistencia a la Insulina/fisiología , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Northern Blotting , Calmodulina/biosíntesis , Calmodulina/genética , Carcinoma Hepatocelular , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Humanos , Insulina/farmacología , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
DBA/1 mice were administered type II collagen (CII) or collagen peptides intranasally before systemic immunization to determine whether tolerance could be induced and autoimmune arthritis suppressed. Although prior experiments have demonstrated that collagen given intravenously or orally is effective, the respiratory mucosal route offers several theoretical advantages for dosing peptides, in addition to ease of use. Intact CII, CB11 and a synthetic peptide containing the immunodominant T-cell epitope recognized by H-2q mice were all effective in reducing the incidence and severity of arthritis and the immune response to CII. Since previous studies have demonstrated the importance of IgG2 antibody subclasses to the induction of collagen-induced arthritis, total immunoglobulin G (IgG), IgG1, and IgG2a and IgG2b were measured. IgG2 antibody subclasses were significantly downregulated by the treatment regimen, whereas a slight decrease in IgG1 antibodies was noted that was not significant. In an effort to determine the mechanism by which arthritis was attenuated, cervical lymph node and spleen cells from treated mice were cultured separately with CII and supernatants tested for the presence of T-cell lymphokines. The cells provided a T-helper 2 (Th2)-like response to CII, with T cells from lymph nodes secreting interleukin-4 (IL-4) and splenocytes secreting both IL-4 and IL-10, whereas a Th1-like response was detected in immunized mice not tolerized with CII. These findings indicate that the downregulation of arthritis that occurs with intranasal administration of CII is associated with Th2-type lymphokine profile and a decrease in complement-fixing antibody subclass.
Asunto(s)
Artritis/prevención & control , Enfermedades Autoinmunes/prevención & control , Colágeno/uso terapéutico , Terapia de Inmunosupresión/métodos , Administración Intranasal , Animales , Artritis/etiología , Artritis/inmunología , Autoanticuerpos/sangre , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/inmunología , Colágeno/inmunología , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos DBA , Células Th2/inmunologíaRESUMEN
Interleukin-1 (IL-1) is hypothesized to be involved in physiological sleep regulation and in sleep responses occurring during infectious disease. If this hypothesis is correct, then inhibition of endogenous IL-1 should reduce both normal sleep and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-induced sleep. MDP is a somnogenic substance derived from bacterial cell walls. We report here the effects of a synthetic IL-1 receptor fragment corresponding to amino acid residues 86-95 of the human type I IL-1 receptor (IL-1RF) on spontaneous sleep and IL-1 beta- and MDP-induced sleep and fever in rabbits. Two doses of the IL-1RF (25 and 50 micrograms) were injected into normal rabbits intracerebroventricularly (icv). Both doses significantly decreased spontaneous non-rapid eye movement sleep (NREMS) across a 22-h recording period. Pretreatment of rabbits with 25 micrograms of IL-1RF blocked the somnogenic actions of 10 ng icv IL-1. Similarly, central pretreatment of animals with 25 micrograms IL-1RF significantly attenuated the NREMS-promoting and REMS-suppressive actions of 150 pmol MDP injected centrally. The increase in NREMS and decrease in REMS induced by systemic injection of 12.5 micrograms/kg MDP were also significantly suppressed by central administration of 50 micrograms IL-1RF. In contrast, the febrile response induced by either intracerebroventricularly or intravenously injected MDP were not significantly affected by IL-1RF. These results support the hypothesis that endogenous, brain-derived IL-1 contributes to the maintenance of normal sleep and may mediate sleep responses to systemic as well as central bacterial infections.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Interleucina-1/química , Sueño/efectos de los fármacos , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Animales , Fiebre/inducido químicamente , Fiebre/fisiopatología , Humanos , Inyecciones , Interleucina-1/farmacología , Masculino , Conejos , Fases del Sueño/efectos de los fármacosRESUMEN
Inflammatory cytokines may affect cerebral circulation under pathological conditions. Responses of cerebral pial arterioles to one such cytokine, interleukin (IL)-1 beta and its inhibitor [soluble IL-1 receptor (sIL-1R)] were examined in anesthetized newborn pigs using closed cranial windows. Levels of prostanoids and cyclic nucleotides in periarachnoid cerebral spinal fluid (CSF) were measured. To examine the structure-activity relationship of the parent IL-1 beta molecule, two IL-1 beta fragments with amino acid sequences of 187-204 [IL-1 beta-(187-204)] and 208-240 [IL-1 beta-(208-240)] were tested for their effects on pial arterioles. Diameter changes of pial arterioles were sequentially recorded every 5 min for 30 min after topical application of IL-1 peptides. CSF was sampled at the end of the 30 min. IL-1 beta dose dependently induced arteriolar dilation and increased prostaglandin E2 (PGE2), 6-keto-PGF1 alpha adenosine 3',5'-cyclic monophosphate (cAMP), and guanosine 3',5'-cyclic monophosphate (cGMP). Intravenous indomethacin blocked the IL-1 beta-induced vasodilation, the increased prostanoids, and the increased cAMP, but not the increased cGMP. Neither heat-inactivated IL-1 beta nor IL-1 beta vehicle affected arteriolar diameter or CSF levels of prostanoids. The sIL-1R blocked the IL-1 beta-induced vasodilation and the increased CSF prostanoids. IL-1 beta-(208-240) also induced pial arteriolar dilation; however, its vasodilatory potency was 1,000 times less than that of the whole IL-1 beta molecule. IL-1 beta-(187-204) did not induce pial arteriolar dilation even when its dose was increased to the level of IL-1 beta-(208-240). These results suggest that IL-1 beta, through the activation of membrane-bound IL-1 beta receptors, induces pial arteriolar dilation via mechanisms that involve prostanoids and cyclic nucleotides. The results also indicate that the 208-240 amino acid sequence of IL-1 beta has a sequence-specific physiological function.
Asunto(s)
Circulación Cerebrovascular/efectos de los fármacos , Interleucina-1/farmacología , Fragmentos de Péptidos/farmacología , Piamadre/irrigación sanguínea , Vasodilatación , 6-Cetoprostaglandina F1 alfa/antagonistas & inhibidores , 6-Cetoprostaglandina F1 alfa/líquido cefalorraquídeo , Animales , Animales Recién Nacidos , Arteriolas/efectos de los fármacos , Dinoprostona/antagonistas & inhibidores , Dinoprostona/líquido cefalorraquídeo , Indometacina/farmacología , Receptores de Interleucina-1/fisiología , Solubilidad , PorcinosRESUMEN
The involvement of tumor necrosis factor (TNF) in sleep regulation was investigated by blocking TNF using a synthetic TNF receptor fragment (TNFRF) in rabbits. Intracerebroventricular injection of 50 micrograms TNFRF decreased spontaneous non-rapid-eye-movement (NREM) sleep across the 21 h recording period. After 6 h of sleep deprivation (SD), both duration of NREM sleep and electroencephalographic (EEG) slow wave activity during NREM sleep were enhanced. TNFRF suppressed SD-enhanced NREM sleep and EEG slow wave activity. SD per se did not affect rapid-eye-movement (REM) sleep, but TNFRF treatment increased REM sleep after SD. These results are consistent with the hypothesis that TNF is involved in the regulation of physiological NREM sleep.
Asunto(s)
Sueño/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos , Polisomnografía , Conejos , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Privación de Sueño/fisiologíaRESUMEN
Tumor necrosis factor (TNF) is a cytokine that possesses many biological activities, including enhancement of non-rapid-eye-movement sleep (NREMS). The role of endogenous TNF in the regulation of spontaneous sleep is unknown. If TNF is involved in sleep regulation, then reduction of endogenous TNF should suppress spontaneous sleep. A soluble TNF-binding protein I (TNF-BP I) and a synthetic fragment of TNF-BP I, TNF-R-(159-178), that contains the biologically active region of TNF-BP I, were used. These substances bind TNF and possess TNF-inhibitory activity; their effects on rabbit sleep after intracerebroventricular injection were determined across a 6-h recording period. Two doses of TNF-BP I (0.05 micrograms and 0.5 micrograms) were administered; the higher dose of TNF-BP I significantly decreased NREMS. Four doses of TNF-R-(159-178) (0.25 micrograms, 2.5 micrograms, 25 micrograms and 50 micrograms) were used. The 25 micrograms and 50 micrograms doses significantly suppressed NREMS. The highest dose (50 micrograms) also decreased REM sleep. These results are consistent with the hypothesis that endogenous brain TNF is involved in the regulation of normal sleep.
Asunto(s)
Química Encefálica/fisiología , Sueño/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Química Encefálica/efectos de los fármacos , Electroencefalografía/efectos de los fármacos , Inyecciones Intraventriculares , Masculino , Conejos , Receptores del Factor de Necrosis Tumoral/química , Proteínas Recombinantes/antagonistas & inhibidores , Sueño/efectos de los fármacos , Sueño REM/efectos de los fármacos , Sueño REM/fisiología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Colágeno/inmunología , Epítopos/inmunología , Ganglios Linfáticos/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Western Blotting , Colágeno/química , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo/métodos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ratones , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunología , Ratas , Ratas Endogámicas WKY , Vacunas SintéticasRESUMEN
Tolerization of B10.RIII mice (H-2r) with intravenously injected type II collagen (CII) renders the animals resistant to induction of collagen-induced arthritis (CIA). In order to clarify H-2r-restricted T-cell responses that modulate CIA, we have analysed the T-cell proliferative response of B10.RIII mice against cyanogen bromide (CB) peptides of CII, and detected the strongest response to alpha 1(II)-CB10 (CII 552-897). A panel of chemically synthesized overlapping peptide homologues was used to deduce the minimum structure of this determinant which was found to be CII 610-618. A 15-residue synthetic peptide flanking this region, CII 607-621, was found to effectively suppress arthritis when administered as a tolerogen. Collectively, these data identify the structural component within alpha 1(II)-CB10 which is capable of inducing tolerance in B10.RIII mice. A similar approach to the treatment of autoimmune arthritis, involving the institution of self-tolerance, has potential applicability to human rheumatoid arthritis.
Asunto(s)
Artritis Experimental/inmunología , Colágeno/inmunología , Epítopos de Linfocito T/aislamiento & purificación , Tolerancia Inmunológica , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/sangre , Artritis Experimental/terapia , Colágeno/administración & dosificación , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos , Datos de Secuencia MolecularRESUMEN
TGF-beta 1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-beta 1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-beta 1 that contained residues 368-374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-beta 1 monomer were able to compete with [125I]hrTGF-beta 1 for binding to TGF-beta cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed.
Asunto(s)
Factores Quimiotácticos/química , Quimiotaxis de Leucocito , Monocitos/fisiología , Neutrófilos/fisiología , Factor de Crecimiento Transformador beta/química , Secuencia de Aminoácidos , Mapeo Epitopo , Fibroblastos/fisiología , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.
Asunto(s)
Membrana Basal/fisiología , Colágeno/genética , Adipocitos/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Humanos , Riñón/citología , Riñón/fisiología , Hígado/química , Datos de Secuencia Molecular , Músculos/química , Nefritis Hereditaria/patología , Péptidos/inmunología , Piel/químicaRESUMEN
The murine model of collagen-induced arthritis is characterized by the development of an immune response against joint cartilage. Arthritis can be significantly suppressed by the administration of type II collagen (CII) or one of the CNBr peptides, CB11 (CII 124-402) as a tolerogen prior to immunization. We have previously shown that two synthetic peptides, representing sequences CII 260-270 and CII 181-209, are effective tolerogens. In this paper, we now characterize the T cell determinant with CII 181-209. A series of synthetic peptides overlapping CII 181-209 and analogs of chick CII 181-209 containing site-directed amino acid substitutions was developed and cultured with T cells from DBA/1 mice immunized with CII. Supernatants were collected and analyzed for the presence of the T cell lymphokine IFN-gamma. These data indicate the critical T cell determinant to be located within CII 190-200. This conclusion is further supported by the observation that an unodecapeptide representing CII 190-200 was just as effective as CII 181-209 in suppressing arthritis and anti-CII antibody response when tested as a tolerogen. Analogs containing single amino acid substitutions at residues 191, 194, 197, 198, or 200 were significantly less effective in inducing T cell responses. Each of these peptide analogs was then given as neonatal tolerogens to DBA/1 mice. Mice were subsequently immunized and observed for the development of arthritis. These studies identified residues 194, 197, 198, and 200, and probably residue 191, as critical for tolerance and the suppression of arthritis. Elucidation of the fine structures of T cell determinants which are critical for suppression of arthritis should allow these techniques to be used for developing specific immunotherapeutic approaches to autoimmune arthritis.
Asunto(s)
Colágeno/inmunología , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Artritis Reumatoide/prevención & control , Pollos , Epítopos/química , Tolerancia Inmunológica , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Péptidos/análisisRESUMEN
Susceptibility to collagen-induced arthritis (CIA), a murine model of autoimmune arthritis, is strongly linked to only two major histocompatibility complex (MHC) haplotypes, H-2q and H-2r. In order to identify the determinants of type II collagen (CII) required to induce arthritis in H-2r-bearing mice, B10.RIII mice were immunized with bovine, chick or human CII. Only bovine CII induced significant arthritis and autoantibodies. When the major CNBr peptides of bovine collagen were isolated and used for immunization, only mice immunized with CB8, representing CII 403-551, developed arthritis. To identify immunogenic epitope(s) within CB8, a panel of synthetic peptides representing overlapping sequences of the bovine peptide was generated. When each peptide was cultured with T cells from B10.RIII mice immunized with CII, one peptide, representing CII 430-466, contained a major T-cell epitope. By using an in vitro lymphokine production assay, the T-cell epitope was further narrowed to CII 442-456. These findings suggest that a T-cell determinant important for the initiation of arthritis in B10.RIII (H-2r) mice is located within a 15 amino acid sequence, residues 442-456 of bovine CII.
Asunto(s)
Artritis/inmunología , Enfermedades Autoinmunes/inmunología , Colágeno/inmunología , Epítopos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Bovinos , Pollos , Colágeno/química , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunologíaRESUMEN
Phosphorylation status of the Sendai virus P protein was examined during virus infection and compared with cell-free phosphorylation. P protein from Sendai virus-infected (VI) and P/C gene-transfected (PT) mammalian cells and from purified virions (PV) was phosphorylated at only serine residues. In contrast, cell-free phosphorylation of the P protein with virion-associated protein kinase (VAPK) occurred at both threonine and serine. Tryptic phosphopeptide maps of the P protein from VI, PT, and PV showed that the phosphorylation was primarily localized on one peptide (TP1), while VAPK phosphorylated the P protein on several peptides. There was no change in the steady-state phosphopeptide map of the P protein during virus replication, indicating that the TP1 is constitutively phosphorylated. Inhibition of cellular phosphatases (PP1 and PP2A) by okadaic acid (OA) in virus-infected cells caused a sixfold increase in the P protein phosphorylation, solely at serine residues. The phosphopeptide map of the OA-P protein revealed that phosphorylation occurred on several peptides, but the OA-P map was significantly different from the VAPK-P map. However, additional phosphorylation of the P protein did not block its association with nucleocapsids. These results suggest that the Sendai virus P protein is constitutively phosphorylated primarily at one locus but has the potential for phosphorylation at additional sites. Further, our results do not show any correlations between the intracellular and cell-free phosphorylation of the P protein and, therefore, question the validity of cell-free phosphorylations.
Asunto(s)
Fosfoproteínas/fisiología , Virus Sendai/fisiología , Proteínas Virales/fisiología , Animales , Células COS , Sistema Libre de Células , Fosforilación , Replicación ViralRESUMEN
Epidermal growth factor (EGF) stimulates adenylyl cyclase in the heart via activation of the stimulatory GTP-binding protein Gs. Therefore, employing peptides corresponding to regions in the cytosolic domain of the EGF receptor, we have investigated the ability of sequences within the EGF receptor to activate Gs. A 13-aa peptide (EGFR-13) corresponding to the juxtamembrane region in the cytosolic domain of the EGF receptor stimulated GTP binding and GTPase activity of Gs. This peptide did not stimulate GTP binding to Gi but increased the GTPase activity of this protein. Additionally, phosphorylation of the protein kinase C site (threonine residue) within EGFR-13 decreased the ability of the peptide to stimulate Gs and increase GTPase activity of Gi. Further, in functional assays of Gs employing S49 cyc- cell membranes, EGFR-13 increased the ability of Gs to stimulate adenylyl cyclase; phospho-EGFR-13 and a 14-aa peptide corresponding to a sequence in the cytosolic domain of the EGF receptor did not alter the functional activity of Gs. Hence, the juxtamembrane region of the EGF receptor can activate Gs and, by stimulating GTPase activity of Gi, inactivates this latter G protein. Phosphorylation of the threonine residue within this region attenuates the activity of the peptide as a modulator of G-protein function.
Asunto(s)
Adenilil Ciclasas/metabolismo , Encéfalo/metabolismo , Receptores ErbB/metabolismo , Proteínas de Unión al GTP/metabolismo , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Clonación Molecular , Citosol/metabolismo , Receptores ErbB/química , Escherichia coli , GTP Fosfohidrolasas/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Datos de Secuencia Molecular , Fosforilación , Proteínas Recombinantes/metabolismoRESUMEN
DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis. We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11. MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E. coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice. LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice. A single i.p. injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones. These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11. Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.
Asunto(s)
Anticuerpos Monoclonales/toxicidad , Artritis Experimental/inducido químicamente , Artritis Experimental/etiología , Colágeno/toxicidad , Epítopos/inmunología , Lipopolisacáridos/toxicidad , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Colágeno/inmunología , Escherichia coli/inmunología , Masculino , Metaloendopeptidasas/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBARESUMEN
Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Pro alpha 1(I) gene in FIC. LTIC expressed abundant transcripts of Pro alpha 1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Pro alpha 1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor beta (TGF beta)-treated LTIC had a preferential increase in the rate of Pro alpha 1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549-2556] was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGF beta treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGF beta. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGF beta-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGF beta-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.
Asunto(s)
Colágeno/genética , Regulación de la Expresión Génica , Hígado/fisiología , Factor de Transcripción AP-1/fisiología , Animales , Secuencia de Bases , Células Cultivadas , ADN/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Genes jun , Hígado/citología , Datos de Secuencia Molecular , Fenotipo , Regiones Promotoras Genéticas/fisiología , Unión Proteica , Ratas , Ratas Wistar , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Autoimmune arthritis can be induced in rats by immunization with cartilage-specific types II and XI collagen. Type XI collagen is composed of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains, in which alpha 3(XI) is essentially identical to alpha 1(II) of type II, and alpha 1(XI) and alpha 2(XI) are similar to alpha 1(V) and alpha 2(V) of type V collagen. To characterize the immune response to type XI collagen, Lewis rats were injected with bovine type XI collagen, and the cellular and humoral responses were compared with those of rats injected with types V and II collagen. Arthritis, IgG deposits in cartilage, and joint destruction were seen in rats immunized with types XI and II collagen. Type XI elicited strong cellular responses to rat types XI, V, and II; conversely, types II and V collagen elicited strong responses to rat type XI. Antitype XI Abs reacted with rat type XI, moderately with rat type V, but poorly with rat type II. Direct and inhibition ELISA showed that cross-reactions between types XI and V collagen resulted from recognition of determinants shared by their respective alpha 2(XI) and alpha 1(V) chains. Abs eluted from joints of rats immunized with type XI collagen, however, reacted only with native rat type XI collagen. These data demonstrate that type XI collagen induces diverse populations of Abs differing in collagen-type specificity, and suggest that only those Abs to native rat type XI collagen are central to the pathogenesis of type XI collagen-induced arthritis.
Asunto(s)
Artritis/inmunología , Colágeno/inmunología , Animales , Formación de Anticuerpos , Femenino , Hipersensibilidad Tardía , Inmunización , Inmunohistoquímica , Ratas , Ratas Endogámicas LewRESUMEN
An epitope of the alpha-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the alpha-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab' fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four alpha-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-alpha mAb is contained within a protease-resistant domain. Partial sequencing of a 24.1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the alpha-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known alpha-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the alpha-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.