RESUMEN
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrotic changes in skin and other organs involving excessive collagen deposition. Here we investigated the effect of intravenous immunoglobulin (IVIG) on fibrosis in a murine model of bleomycin (BLM)-induced scleroderma. Scleroderma was induced in C3H/He J mice by subcutaneous BLM injections daily for 35 days. The collagen content in skin samples from the BLM-injected group (6·30 ± 0·11 mg/g tissue) was significantly higher than the PBS group (5·80 ± 0·10 mg/g tissue), and corresponded with dermal thickening at the injection site. In contrast, mice treated with IVIG for 5 consecutive days after initiating BLM injection showed lesser collagen content significantly (IVIG group, 5·61 ± 0·09 mg/g tissue; BLM vs. IVIG). In order to investigate the cellular and protein characteristics in the early stage of the model, the skin samples were obtained 7 days after the onset of experiment. Macrophage infiltration to the dermis, monocyte chemoattractant protein (MCP-1)-positive cells, and increased TGF-ß1 mRNA expression were also observed in the BLM group. IVIG inhibited these early fibrogenic changes; MCP-1 expression was significantly lesser for the IVIG group (1·52 ± 0·19 pg/mg tissue) than for the BLM group (2·49 ± 0·26 pg/mg tissue). In contrast, TGF-ß1 mRNA expression was significantly inhibited by IVIG. These results suggest that IVIG treatment may inhibit macrophage recruitment to fibrotic sites by down regulating MCP-1 and TGF-ß production, and thus could be a potential drug for managing fibrotic disorders such as SSc.
Asunto(s)
Colágeno/metabolismo , Inmunoglobulinas Intravenosas/uso terapéutico , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Animales , Bleomicina/toxicidad , Quimiocina CCL2/análisis , Colágeno/análisis , Regulación hacia Abajo , Femenino , Humanos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Esclerodermia Sistémica/inducido químicamente , Piel/patología , Factor de Crecimiento Transformador beta1/análisisRESUMEN
A major problem with allergen-specific immunotherapy involving repeated injection of allergens is the risk of an anaphylactic reaction. We engineered the major house dust mite allergen, Der f 2, to reduce its capacity to induce skin test reactivity and histamine release from peripheral blood basophils in allergic patients. The engineered allergen, in which the disulfide bond that linked the N- and C-terminal sequences of Der f 2 was disrupted, retained T-cell epitopes essential for immunotherapy and ability to stimulate T-cell proliferation. Such engineered allergens are potentially useful for safer and more effective immunotherapy for allergies.
Asunto(s)
Alérgenos/genética , Antígenos/genética , Desensibilización Inmunológica , Ingeniería Genética , Glicoproteínas/genética , Ácaros/inmunología , Alérgenos/inmunología , Alérgenos/metabolismo , Animales , Antígenos/inmunología , Antígenos/metabolismo , Antígenos Dermatofagoides , Basófilos/efectos de los fármacos , Basófilos/inmunología , División Celular/efectos de los fármacos , Cisteína/genética , Cisteína/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Epítopos de Linfocito T/inmunología , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Liberación de Histamina/efectos de los fármacos , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Inmunoglobulina E/metabolismo , Mutagénesis Sitio-Dirigida , Pruebas CutáneasRESUMEN
We investigated expression of ephrin-B2 and Eph-B4 in the retinal tissues of six primate eyes with neovascularization and iris rubeosis secondary to laser-induced central retinal vein occlusion and in tissue from 10 human eyes with proliferative diabetic retinopathy. Two primate eyes with rubeosis and retinal neovascularization were enucleated 1, 2 and 4 weeks after the creation of central retinal vein occlusion. Antibodies were localized using the avidin-biotin reaction. In the primate eyes, ephrin-B2 was negative at I week and positive at 2 and 4 weeks in the rubeotic tissue, but was positive only at 2 weeks in the retinal neovascular membrane. Eph-B4 was negative in all the primate eye specimens. In the human tissue, ephrin-B2 was detected in two of the five eyes with rubeosis and three of the five eyes with retinal neovascularization. These data suggest that ephrin-B2 is a key regulator of neovascularization.
Asunto(s)
Efrina-B2/análisis , Efrina-B2/fisiología , Enfermedades del Iris , Neovascularización Retiniana/patología , Proteínas Angiogénicas/análisis , Animales , Retinopatía Diabética , Modelos Animales de Enfermedad , Efrina-B2/genética , Efrinas/análisis , Efrinas/genética , Efrinas/fisiología , Regulación de la Expresión Génica , Inmunohistoquímica , Enfermedades del Iris/patología , Macaca , Neovascularización Patológica/patología , Receptor EphB4/análisis , Oclusión de la Arteria Retiniana , Neovascularización Retiniana/etiología , Factores de TiempoRESUMEN
B cell epitopes of the major house dust mite allergen Der f 2 from Dermatophagoides farinae were analysed using deletion mutants of Der f 2 expressed as fusion proteins in Escherichia coli. The reactivities of these partial Der f 2 molecules to human anti-mite IgE antibodies in atopic patients and to murine anti-Der f2 monoclonal antibodies (mAbs) were examined by immunoblotting. A C-terminal deletion mutant of Der f 2, 1-123, had almost the same reactivity to human IgE as the whole Der f 2 (1-129) and an N-terminal deletion mutant of Der f 2 (25-129) still had weak reactivity. On the other hand, in two deleted Der f 2 molecules, 1-120 and 30-129, reactivity was lost in spite of long overlapping sequences. These results suggest that the human IgE antibodies to Der f 2 in atopic patient sera recognize the conformational structures dependent on the tertiary structure of Der f 2, including disulfide bond formations, rather than the contiguous sequences of amino acids. The sequences 1-24, 25-29 and 121-123 were revealed as the minimum N- and C- terminal amino acid sequences required for IgE binding. Contrastingly, all three murine mAbs bound to the smaller deletion mutants, 1-90 and 67-129, suggesting that the cores of the epitopes for these mAbs exist in the 24 amino acid sequence of Der f 2, 67-90 overlapping the sequential human IgE epitope on Der p 2, the equivalent allergen from Dermatophagoides pteronyssinus. These findings are important for the understanding of the antigenic structure of Der f 2 and for the manipulation of the allergen for immunotherapy.
Asunto(s)
Alérgenos/inmunología , Anticuerpos Monoclonales/inmunología , Glicoproteínas/inmunología , Inmunoglobulina E/química , Alérgenos/química , Secuencia de Aminoácidos , Animales , Antígenos Dermatofagoides , Mapeo Epitopo , Glicoproteínas/química , Humanos , Inmunoglobulina E/metabolismo , Ratones , Ácaros/inmunología , Unión Proteica , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
Allergen-specific immunotherapy, in which repeated injections of allergens over prolonged periods are used to induce tolerance, has proven an effective treatment of allergy. A major side effect of allergen-specific immunotherapy is anaphylactic reaction. House dust mite allergens are major causative factors associated with various allergic diseases. Der f 2 is the major house dust mite allergen composed of 129 amino acid residues. Analysis using deletion mutants of Der f 2 suggested that T-cell epitopes of Der f 2 were multiple in mite-allergic patients. We found that some IgE epitopes were renatured by dialysis of a mixture of two denatured C- and N-terminal deletion mutants, 1-112 and 85-129 in 13 patients out of 14. On the other hand, IgE binding activity was negative in the separately dialyzed fragments and their mixture in each patient tested. Furthermore, we demonstrated that neither of the two separately prepared polypeptides induced in vivo skin prick test reactivity. These findings are important for improvement of T-cell targeting allergen-specific immunotherapy and development of monovalent IgE haptens. The use of combinations of overlapping non-anaphylactic fragments of allergen covering all of the T-cell epitopes achieves the removal of IgE reactivity, the cause of harmful anaphylactic reactions, without affecting the T-cell reactivity essential for immunotherapy, offering potentially safer and more effective treatment for allergic disease.
Asunto(s)
Alérgenos/genética , Alérgenos/inmunología , Desensibilización Inmunológica/métodos , Glicoproteínas/genética , Glicoproteínas/inmunología , Ácaros/inmunología , Alérgenos/administración & dosificación , Anafilaxia/prevención & control , Animales , Antígenos Dermatofagoides , Polvo , Epítopos/química , Epítopos/genética , Glicoproteínas/administración & dosificación , Humanos , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad Inmediata/terapia , Inmunoglobulina E/sangre , Activación de Linfocitos , Ácaros/genética , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Eliminación de Secuencia , Pruebas Cutáneas , Linfocitos T/inmunologíaRESUMEN
Der f 2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f 2 recognized by human IgE antibodies, artificial mutations were introduced to Der f 2 protein. The IgE-binding activity of Der f 2 was significantly decreased by deletion of 10 amino acids at the N-terminus or nine amino acids at the C-terminus. Site-directed mutagenesis with a single amino acid replacement by Ala or Leu in both N- and C-terminal regions as well as a central portion was performed to generate 42 single-site mutations. Amino acid replacement around a disulfide bond of Cys8-Cys119 caused a marked decrease in IgE-binding activity. Furthermore, a distinct decrease in IgE-binding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these results, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitope structures recognized by human IgE antibodies.
Asunto(s)
Alérgenos/análisis , Aminoácidos/análisis , Glicoproteínas/análisis , Epítopos Inmunodominantes/análisis , Inmunoglobulina E/análisis , Ácaros/inmunología , Mutagénesis Sitio-Dirigida/inmunología , Alérgenos/genética , Alérgenos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antígenos Dermatofagoides , Secuencia de Bases , Sitios de Unión de Anticuerpos , Unión Competitiva , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/aislamiento & purificación , Inmunoglobulina E/química , Ácaros/genética , Datos de Secuencia MolecularRESUMEN
Der f 2 is one of the major mite allergens recognized by human IgE antibodies of allergic patients. Using five anti-Der f 2 mouse monoclonal antibodies, human IgE epitopes of Der f 2 were analyzed. Among them, two monoclonal antibodies 15E11 and 13A4 inhibited the binding between Der f 2 and human IgE antibodies. To determine major IgE epitopes of Der f 2, epitopes for the monoclonal IgG antibodies were analyzed using 43 single site Der f 2 mutants constructed by site-directed mutagenesis. Binding ability of 13A4 and 15E11 was decreased by the amino acid replacement around the C-terminus, and around 73rd, respectively. These results suggest that the C-terminal portion and the central portion around 73rd of Der f 2 were recognized by human IgE antibodies as major epitopes. The location of the putative IgE epitopes on 3-D structure of Der f 2 is also discussed.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Glicoproteínas/inmunología , Epítopos Inmunodominantes/inmunología , Inmunoglobulina E/inmunología , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos/inmunología , Antígenos Dermatofagoides , Mapeo Epitopo , Glicoproteínas/genética , Humanos , Ratones , Ácaros , Datos de Secuencia Molecular , MutaciónRESUMEN
Spontaneous mast cell tumors (MCT) are the most common malignant neoplasm in the dog, representing between 7% and 21% of all canine tumors, an incidence much higher than that found in humans. These tumors often behave in an aggressive manner, metastasizing to local lymph nodes, liver, spleen, and bone marrow. The proto-oncogene c-kit is known to play a critical role in the development and function of mast cells. Point mutations in the kinase domain of c-kit leading to tyrosine phosphorylation in the absence of ligand binding have been identified in three mastocytoma lines, (P815, RBL, and HMC-1), and some human patients with various forms of mastocytosis. We now demonstrate that although c-kit derived from canine MCT did not contain the previously described activating point mutations, 5 of the 11 tumors analyzed possessed novel mutations consisting of tandem duplications involving exons 11 and 12. We also show that one such duplication, detected in a canine mastocytoma cell line, was associated with constitutive phosphorylation of c-kit protein (KIT), suggesting that these mutations may contribute to the development or progression of canine MCT.
Asunto(s)
Enfermedades de los Perros/genética , Sarcoma de Mastocitos/veterinaria , Proteínas Proto-Oncogénicas c-kit/genética , Secuencias Repetidas en Tándem , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , ADN/genética , Perros , Exones/genética , Sarcoma de Mastocitos/genética , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factor de Células Madre/metabolismo , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Der f 3 is one of the allergens produced by house dust mite Dermatophagoides farinae showing serine protease activity. Based on its amino acid sequence, a cDNA clone encoding Der f 3 was isolated from a cDNA library of D. farinae. Sequencing analysis of the clone revealed the presence of an open reading frame of 780 bp, which encodes a mature protein of 232 amino acids with 27 amino acids of pre-pro sequence at the N-terminus. When proDer f 3 was produced in Escherichia coli as a fused protein with glutathione-S-transferase, the fused protein was accumulated as inclusion bodies. The protein purified with 8 M urea and glutathione-affinity column chromatography, however, did not show protease activity. When an arginine residue was introduced at the C-terminus of the pro-region in place of threonine, removal of the pro-region to produce an active mature protease was observed. The specificity and the activity of this recombinant protease were almost the same as those of native Der f 3.
Asunto(s)
Clonación Molecular , ADN Complementario/genética , Escherichia coli/genética , Expresión Génica , Glicoproteínas/genética , Serina Endopeptidasas , Alérgenos , Secuencia de Aminoácidos , Antígenos Dermatofagoides , Secuencia de Bases , Cromatografía de Afinidad , ADN Complementario/química , Glutatión Transferasa/genética , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina E/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión , Serina Endopeptidasas/metabolismoRESUMEN
The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.
Asunto(s)
Bacillus/genética , ADN Bacteriano/genética , Genes Bacterianos , alfa-Amilasas/genética , Secuencia de Aminoácidos , Bacillus/enzimología , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Concentración de Iones de Hidrógeno , Plásmidos , ARN Bacteriano/genética , TemperaturaRESUMEN
Heparin cofactor II is postulated to be an extravascular thrombin inhibitor that is physiologically stimulated by dermatan sulfate. However, the role of heparin cofactor II has not yet been clearly demonstrated in vivo. In this study, we estimated the antithrombotic effect of heparin cofactor II administered exogenously in a rat model of thrombosis. Thrombus was induced in the rat femoral artery by endothelial damage due to the photochemical reaction between systemically injected rose bengal and transillumination with green light. Pretreatment with heparin cofactor II significantly prolonged the time required to occlude the femoral artery (occlusion time) in a dose-dependent manner. At an effective dose in this thrombosis model, heparin cofactor II did not prolong the activated partial thromboplastin time and the prothrombin time in normal rats. Argatroban, a selective synthetic thrombin inhibitor, significantly prolonged the occlusion time. However, argatroban also prolonged the activated partial thromboplastin time and prothrombin time at an effective dose. These results suggest that the administration of heparin cofactor II in vivo effectively inhibited thrombus formation on the vessel walls whose endothelium is damaged without a prolongation of the coagulation time while heparin cofactor II may also inhibit the thrombin activity in the subendothelial tissue in vivo.
Asunto(s)
Fibrinolíticos/farmacología , Cofactor II de Heparina/farmacología , Inhibidores de Serina Proteinasa/farmacología , Trombosis/tratamiento farmacológico , Animales , Relación Dosis-Respuesta a Droga , Fibrinolíticos/uso terapéutico , Cofactor II de Heparina/uso terapéutico , Masculino , Ratas , Ratas Wistar , Inhibidores de Serina Proteinasa/uso terapéutico , Tiempo de Coagulación de la Sangre TotalRESUMEN
To determine whether inflammatory cytokines are increased in proliferative diabetic retinopathy. We measured concentrations of interleukin-6, 8 (IL-6, 8) and tumor necrosis factor (TNF)-alpha by enzyme-linked immunosorbent assay (ELISA) in vitreous and serum from 47 patients with proliferative diabetic retinopathy and 21 patients with vitreous noninflammatory retinopathies. Vitreous concentration of IL-6 were 64.7+/-12.8 pg/ml in proliferative diabetic retinopathy, much greater (P<.005) than in noninflammatory retinopathy (2.8+/-4.5 pg/ml). Amounts of IL-8 in vitreous fluid also were greater in proliferative retinopathy than in noninflammatory retinopathy (34.0+/-11.5 vs. 6.1+/-2.0 pg/ml, P<.005). Concentrations of TNF-alpha in vitreous fluid were not statistically different in proliferative retinopathy from those in noninflammatory retinopathy. In sera, concentrations of IL-6 and IL-8 were not different between proliferative and noninflammatory retinopathy. However, serum TNF-alpha was much greater in proliferative retinopathy than in noninflammatory retinopathy (0.81+/-0.72 vs. 0.09+/-0.00 pg/ml, P<.001). Elevated TNF-alpha in serum then may be diagnostically useful in proliferative diabetic retinopathy. And inflammatory cytokines in vitreous may be pathogenically important in this concentration.
Asunto(s)
Sangre/metabolismo , Citocinas/metabolismo , Retinopatía Diabética/metabolismo , Mediadores de Inflamación/metabolismo , Vitreorretinopatía Proliferativa/metabolismo , Cuerpo Vítreo/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
To investigate the effect of SG-210, a potent inhibitor selective to aldose reductase (ARI), on the impaired polyol pathway, we examined biochemically and histologically the potencies of this compound in streptozotocin-induced diabetic or galactosemic rats. The study with diabetic rats showed that SG-210 (1-10 mg x kg(-1)) dose-dependently inhibited sorbitol accumulations in erythrocytes, sciatic nerves, lens, and retina with ED50 values of 1.4, 1.3, 3.5, and 4.6 mg x kg(-1), respectively. Zenarestat, currently under clinical trials both in Japan and the United States, was about two or over five times less potent than SG-210 in suppressing sorbitol contents of erythrocytes or other tissues, respectively. Epalrestat, commercially available, was much less potent in reducing the contents with ED50 values of more than 30 mg x kg(-1) in all of the cells and the tissues examined. An extensive study using galactosemic rats indicated that SG-210 (3-30 mg x kg(-1)) inhibited galactitol accumulations in lens and retina as well as in erythrocytes, preventing the progression of histological abnormalities in lens accompanied by the reduction in galactitol contents. Epalrestat (3-30 mg x kg(-1)) failed to show any significant effects. Pharmacokinetic studies suggested that SG-210 has a high bioavailability and possesses a long half-life in rats (ca. 10 h). Taken together with its excellent pharmacokinetic profiles, the potent suppressive effects of SG-210 observed in this study may be available as a new treatment of diabetic complications.
Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Diabetes Mellitus Experimental/metabolismo , Alcoholes del Azúcar/metabolismo , Tiazinas/farmacología , Tiazoles/farmacología , Animales , Benzotiazoles , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/metabolismo , Inhibidores Enzimáticos/farmacología , Eritrocitos/metabolismo , Galactitol/metabolismo , Galactosemias/metabolismo , Cristalino/metabolismo , Cristalino/patología , Quinazolinas/farmacología , Ratas , Retina/metabolismo , Nervio Ciático/metabolismo , Sorbitol/metabolismoRESUMEN
BACKGROUND/AIMS: Although there are various methods to detect endotoxin mainly after the intravenous injection of purified endotoxin in a host, its uptake and distribution among the various organs is not well understood. In the present study, the time course of the distribution and disappearance of endotoxin in various rat organs following injection via two different routes was evaluated by an immunohistochemical staining method using a newly developed monoclonal antibody against Factor C. MATERIALS AND METHODS: The time course of the distribution of lipopolysaccharide (LPS) into the liver, spleen, lung, and kidney after intravenous (i.v.) or intraperitoneal (i.p.) injection of LPS was studied by immunohistochemical staining using a newly developed monoclonal antibody against Factor C in rats. Moreover, plasma endotoxin levels were measured by a modification of a chromogenic endotoxin-specific assay. RESULTS: At 30 minutes after injection in the i.v. group and at 12 and 24 hours in the i.p. group, endotoxin was present on Kupffer cells by staining and on some sinusoidal endothelial cells in the liver as well as on macrophages in the marginal zone of the spleen. The plasma endotoxin levels in the i.v. group decreased gradually after injection. However, levels in the i.p. group gradually increased, reaching a maximum level at 6 hours after injection, and then gradually decreasing. CONCLUSION: These results suggest that, regardless of the route of injection, endotoxin can be detected by an immunohistochemical staining method using a monoclonal antibody against Factor C.
Asunto(s)
Endotoxinas/farmacocinética , Inmunohistoquímica , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Choque Séptico/sangre , Bazo/metabolismo , Animales , Riñón/patología , Hígado/patología , Pulmón/patología , Masculino , Ratas , Ratas Wistar , Choque Séptico/patología , Bazo/patologíaRESUMEN
A cDNA library corresponding to mite protein was screened employing anti-Der f II antibody. Two possible clones were obtained, which contained plasmids, pFL1 and pFL11, respectively. Both plasmids had insertions of about 500 base pairs. The DNA sequences of the two insertions were determined, from which the amino acid sequences were deduced. The amino acid sequence of the purified native Der f II protein could be determined to 45 residues from the N-terminus. As a result of comparison, we concluded that the cDNAs prepared from live mite Dermatophagoides farinae corresponded to the mite allergen, Der f II.
Asunto(s)
Alérgenos/genética , ADN/genética , Secuencia de Aminoácidos , Animales , Antígenos Dermatofagoides , Secuencia de Bases , Clonación Molecular , Código Genético , Datos de Secuencia MolecularRESUMEN
BACKGROUND: Inhibition of the interaction between IgE and the alpha-chain of Fc epsilon RI (Fc epsilon RI alpha) is a straightforward strategy to develop therapeutic reagents for IgE-mediated allergic diseases. OBJECTIVE: The purpose of this study is the humanization of CRA2 and/or CRA4, mouse anti-human Fc epsilon RI alpha monoclonal antibodies (mAbs) which recognize the IgE-binding membrane proximal immunoglobulin-like domain of Fc epsilon RI alpha. METHODS: The two mAbs were humanized by CDR grafting onto human V region frameworks encoded by human germline V and J genes. The activities of the recombinant antibodies to bind Fc epsilon RI alpha and inhibit IgE binding to Fc epsilon RI alpha were analyzed by flow cytometry and ELISA. Human peripheral blood basophils were pretreated with the Fab fragments of the humanized CRA2 and stimulated with IgE and an anti-IgE polyclonal antibody. The released histamine was measured. RESULTS: The humanized CRA2 had almost the same activities of binding and inhibition of IgE binding to Fc epsilon RI alpha as the original mouse CRA2. Although the Fc epsilon RI-binding activity was maintained following humanization of the CRA4 light chain V region, it was lost by the humanization of the CRA4 heavy chain V region. Pretreatment of human peripheral blood basophils with the Fab fragments of the humanized CRA2 inhibited their subsequent degranulation activated by cross-linking of the Fc epsilon RI. CONCLUSION: In the humanized CRA2, all amino acid residues except CDR are replaced with the residues encoded by human germline genes. The humanization of CRA2 might be an important step in the development of immunotherapy to manipulate the IgE network in which mast cells, basophils, and various types of Fc epsilon RI alpha expressing cells are involved.
Asunto(s)
Basófilos/inmunología , Liberación de Histamina , Inmunoglobulina E/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Receptores de IgE/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We amplified genomic DNA encoding the major house dust mite allergen, Der f2, from Dermatophagoides farinae by means of the polymerase chain reaction and cloned it into Escherichia coli. The nucleotide sequences of the amplified fragments were determined and compared with those of the cDNA previously reported. Four Der f2 genomic clones were obtained, suggesting that the genomic Der f2 gene had sequence polymorphisms like the cDNA clones. Each of the genomic clones had a single small intron. With respect to the exon sequences, two of the four genomic clones were identical with two cDNA clones, respectively. The others were combinations of the two clones. Genomic Southern blotting suggested that the Der f2 gene is located at one locus in the mite genome and that sequence substitutions were due to polymorphisms among individual mite genes.
Asunto(s)
Alérgenos/genética , Genoma , Glicoproteínas/genética , Ácaros/genética , Polimorfismo Genético/genética , Animales , Antígenos Dermatofagoides , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN/análisis , Cartilla de ADN/química , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
A cDNA library corresponding to mite protein was screened using anti-Der f II, a major allergen from the house dust mite Dermatophagoides farinae, antibody. Three possible clones were obtained that contained cDNA fragments coding for Der f II, and the nucleotide sequences of the fragments were determined. There were minor differences observed affecting the deduced amino acid sequence among the three cDNA fragments. The amino acid sequence of the purified native Der f II protein could be analyzed to 45 residues from the N-terminus. As a result of comparison, all the three cDNA fragments code for a mature protein with a derived molecular weight of about 14,000. The amino acid sequence was not homologous to any known protein sequences and it contained six cysteine residues and no N-glycosylation sites.
Asunto(s)
Alérgenos/genética , ADN Recombinante/genética , Escherichia coli/genética , Ácaros/inmunología , Alérgenos/biosíntesis , Secuencia de Aminoácidos , Animales , Antígenos Dermatofagoides , Secuencia de Bases , Western Blotting , Clonación Molecular , Escherichia coli/metabolismo , Expresión Génica , Ácaros/fisiología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed. OBJECTIVE: Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1. METHODS: The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris. Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively. RESULTS: The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium. Their prosequences were removed autocatalytically by dialysis against acidic buffer. Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE. Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities. CONCLUSION: The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy. Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.