RESUMEN
This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Regeneración Ósea/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Factor 5 de Diferenciación de Crecimiento/farmacología , Osteogénesis/efectos de los fármacos , Animales , Proteína Morfogenética Ósea 2/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Cartílago/crecimiento & desarrollo , Fémur/lesiones , Factor 5 de Diferenciación de Crecimiento/metabolismo , Modelos Animales , RatasRESUMEN
Transforming growth factor-beta 1 (TGF-beta(1)) is a key regulator of immune tolerance. TGF-beta(1) controls T lymphocyte activation and is involved in the immunosuppressive function and generation of regulatory T lymphocytes. Connective tissue growth factor (CTGF) has an essential role in the formation of connective tissue and blood vessels. CTGF expression is induced by TGF-beta(1) in several cell types and CTGF mediates several of the downstream actions of TGF-beta(1). Since little is known about the potential synergy between CTGF and TGF-beta(1) in T lymphocyte biology, the purpose of the present study was to determine whether CTGF can modulate TGF-beta(1)-mediated effects on human CD4+ T lymphocytes. Human recombinant CTGF was expressed in HEK293 cells. rCTGF was biologically active demonstrated by induction of proliferation in the endothelial cell line EA hy 926. rCTGF alone did not potentiate or diminish anti-CD3-induced CD4+ T lymphocyte proliferation and did not activate the Smad signaling pathway in CD4+ T lymphocytes. Furthermore, rCTGF did not attenuate TGF-beta(1)-mediated inhibition of CD4+ T lymphocyte proliferation and TGF-beta(1)-induced Smad signaling in CD4+ T lymphocytes. These results indicate that rCTGF had no detectable effects of its own on human CD4+ T lymphocytes and did not potentiate the effects of low amounts of TGF-beta(1) on human CD4+ T lymphocytes. Overall, these data support the hypothesis that CTGF does not act on CD4+ T lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Proteínas Inmediatas-Precoces/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo , Humanos , Fosforilación , Proteínas Recombinantes/farmacologíaRESUMEN
Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor beta superfamily, especially BMP-2, induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to delivery BMPs for practical use need to be developed with the objective to heal cartilage and bone-related diseases in medical, dental and veterinary practice. Thus, the aim of this article was to present an overview of the principals carriers used to delivery BMPs and alternative delivery systems for these proteins.
Asunto(s)
Proteínas Morfogenéticas Óseas/administración & dosificación , Regeneración Ósea/efectos de los fármacos , Preparaciones de Acción Retardada , Sistemas de Liberación de Medicamentos/veterinaria , Fracturas Óseas/tratamiento farmacológico , Animales , Regeneración Ósea/fisiología , Portadores de Fármacos , Sistemas de Liberación de Medicamentos/métodos , Factor de Crecimiento Transformador beta/administración & dosificación , Cicatrización de HeridasRESUMEN
Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity.
Asunto(s)
Proteína Ligando Fas/química , Proteína Ligando Fas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/química , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Reactivos de Enlaces Cruzados/farmacología , Humanos , Células Jurkat , Ratones , Proteínas Mutantes/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad/efectos de los fármacos , Relación Estructura-Actividad , Tenascina/metabolismoRESUMEN
BACKGROUND: Modification of the heparin binding site by alteration of the amino acid sequence of bone morphogenetic protein-2 (BMP-2) results in a change in the local retention time. The purpose of this study was to compare the osteogenic activity of T3 and T4, two mutants with increased binding capacity to heparin, and B2GDF-5 a mutant resulting from the fusion of the n-terminal amino acid sequence of BMP-2 and the c-terminal sequence of GDF-5 with wild-type BMP-2 in vivo. MATERIAL AND METHODS: The proteins were coupled to an equine-derived collagen carrier and implanted in standardized critical size calvarial defects in adult rats. After 28 days, bone formation was evaluated radiographically and the new bone was characterized histologically. RESULTS: Proteins T3 and T4 showed a higher osteogenic activity than BMP-2. Less new bone formation was observed with GDF-5 and B2GDF-5 than with-type BMP-2. No difference in bone formation was observed between GDF-5 and B2GDF-5. CONCLUSION: Increased heparin binding capacity enhances osteogenic activity of BMP-2 in vivo. This might be due to a longer retention period in the tissue and thus better bioavailability. Replacement of the N-terminal amino acid sequence of GDF-5 by the corresponding sequence of BMP-2 did not result in an increased osteogenic activity as heparin binding capacity is not the main reason for the bioavailability of GDF-5.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Mutación/genética , Fragmentos de Péptidos/genética , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos/genética , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/farmacocinética , Proteínas Morfogenéticas Óseas/farmacología , Cartílago/efectos de los fármacos , Matriz Extracelular/metabolismo , Factor 5 de Diferenciación de Crecimiento , Caballos , Técnicas In Vitro , Masculino , Osteogénesis/efectos de los fármacos , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/farmacología , Ratas , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Relación Estructura-Actividad , Factor de Crecimiento Transformador beta/farmacocinética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: Direct mandibular reconstruction with an autologous bone transplant was compared with an osteoinductive implant following an extensive continuity resection of the lower jaw in Göttinger mini-pigs. METHOD: In nine full-grown mini-pigs a one-sided continuity defect (5 cm) was created in the lower jaw. In four animals it was filled with a 50 x 25 x 15 mm(3) collagenous carrier enhanced by rhBMP-2 (400 micro g/cm(3) rhBMP-2). In two animals only the carrier was implanted as a control. Three animals received the resected autologous bone as a free transplant. Bone regeneration and consolidation of the defects was analyzed radiographically and histologically. RESULTS: Following implantation of the osteoinductive implant, complete osseous consolidation of the continuity defect in the lower jaw was observed in all animals. The defects were completely filled with a biomechanically stable bone which showed signs of functional adaptation. The replantation of the orthotopic autologous bone did not lead to functional stability quickly enough. In the periphery only an incomplete bony bridge was formed which was interrupted by large pseudarthrosis. No consolidation of the defects was found in the control group (carrier alone). CONCLUSION: Direct reconstruction of an extensive, biomechanically loaded defect with an osteoinductive implant proved to be the superior method. The osseous regeneration observed shows an immediate functional orientation. The necessity for extensive adaptive remodeling is thus minimized.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Trasplante Óseo/métodos , Materiales Biocompatibles Revestidos , Colágeno , Mandíbula/cirugía , Implantación de Prótesis Mandibular/métodos , Prótesis Mandibular , Oseointegración/efectos de los fármacos , Factor de Crecimiento Transformador beta , Animales , Densidad Ósea/efectos de los fármacos , Proteína Morfogenética Ósea 2 , Regeneración Ósea/efectos de los fármacos , Regeneración Ósea/fisiología , Mandíbula/patología , Seudoartrosis/patología , Porcinos , Porcinos EnanosRESUMEN
BACKGROUND: The alteration of the N-terminal amino acid sequence of BMP-2 allows modification of heparin binding of the new protein. This leads to a change in the local retention time at the site of implantation. Mutants with increased (T3, T4) and with no binding (EHBMP-2) to heparin were assessed for their osteoinductivity in vivo and compared with the wild type BMP-2. METHODS: Cylindrical collagenous carriers (diameter = 5 mm, height = 10) were loaded with different concentrations (0.25-4 micro g) of the proteins. Following intramuscular implantation into the hind legs, the bone formation was measured in radiographic follow-ups. After 28 days the newly formed bone was characterized histologically. RESULTS: Elimination of the heparin binding leads to massive reduction in osteoinductivity. On the other hand, an increase in the heparin binding leads to enhancement in the osteoinductive properties, resulting in faster bone formation with a higher yield. CONCLUSION: It could be shown for the first time that modifications of BMP-2 by gene technology can lead to proteins with enhanced binding to components of the extracellular matrix. The resulting prolonged retention time at the implantation site results in an increased osteoinductivity compared with the wild type.
Asunto(s)
Secuencia de Aminoácidos/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/farmacología , Regeneración Ósea/efectos de los fármacos , Sustitutos de Huesos , Proteínas Portadoras/genética , Materiales Biocompatibles Revestidos , Colágeno , Técnicas Genéticas , Variación Genética/genética , Mutación/genética , Oseointegración/efectos de los fármacos , Prótesis e Implantes , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Microrradiografía , Microscopía Fluorescente , Terminación de la Cadena Péptídica Traduccional/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Interleukin 4 (IL-4) is essential for the switching of B cells to IgE antibody production and for the maturation of T helper (Th) cells toward the Th2 phenotype. These mechanisms are thought to play a crucial role in the pathogenesis of the allergic airway inflammation observed in asthma. In the present study, we examined the anti-inflammatory effects of DNA administration of murine IL-4 mutant Q116D/Y119D (IL-4 double mutant, IL-4DM), which binds to the IL-4 receptor alpha and is an antagonist for IL-4. Immunization of BALB/c mice with alum-adsorbed ovalbumin (OVA) followed by aspiration with aerosolized OVA resulted in the development of allergic airway inflammation. A single administration of IL-4DM DNA before the aerosolized OVA challenge protected the mice from the subsequent induction of allergic airway inflammation. Serum IgE level and extent of eosinophil infiltration in bronchoalveolar lavage (BAL) from IL-4DM DNA-administered mice were significantly lower than those in BAL from control plasmid-immunized mice. In our study, IL-4 or IL-4 mutants were not detected in sera from mice that had received a single administration of IL-4DM DNA. The results of this study provide evidence for the potential utility of IL-4 mutant antagonist DNA inoculation as an approach to gene therapy for asthma.
Asunto(s)
Asma/terapia , ADN/administración & dosificación , Interleucina-4/antagonistas & inhibidores , Animales , Pruebas de Provocación Bronquial , Bronquitis/prevención & control , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/metabolismo , Eosinófilos/metabolismo , Terapia Genética/métodos , Inmunoglobulina E/biosíntesis , Interleucina-4/administración & dosificación , Interleucina-4/genética , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Ovalbúmina , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Th2/metabolismo , Vacunas de ADNRESUMEN
Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Mesodermo/efectos de la radiación , Transducción de Señal/efectos de la radiación , Factor de Crecimiento Transformador beta , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Proteína Morfogenética Ósea 2 , División Celular , Línea Celular , Cobalto , Reactivos de Enlaces Cruzados , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Ratones , Modelos Biológicos , Osificación Heterotópica , Osteoblastos/metabolismo , Pruebas de Precipitina , Proteínas Smad , Proteína Smad1 , Factores de Tiempo , Transactivadores/metabolismoRESUMEN
Bone morphogenetic proteins (BMP) are dimeric factors initiating several distinct signaling cascades by binding to two types of transmembrane serine/threonine kinase receptors (BRI and BRII), and are thus regulating several steps in embryonal development and adult tissue homeostasis. BMP-2 contains two symmetrical pairs of juxtaposed epitopes: the wrist epitope with high affinity to BRI consists of residues from both BMP-2 monomers, while the knuckle epitope resembles the low affinity site for BRII and comprises residues from only one monomer. Here we generated heterodimeric BMP-2 muteins with one monomer mutant in either epitope I for BRI (eI-) or epitope II for BRII (eII-) and the second monomer wild type for receptor interactions (m-). These muteins (B2eI-/B2m- and B2eII-/B2m-) were analyzed by biosensor analysis as well as by measuring their biological activity and compared to their homodimeric forms (either wild type or mutant). Depletion of only one epitope II results in the loss of biological activity as measured byalkaline phosphatase (ALP) activity and Smad induced reportergene assays. However, depletion of only one epitope I shows a reduction of ALP activity to about 25%, while the activation of the Smad pathway remained normal. Homomeric muteins are non-functional for both Smad and ALP activation. This suggests that two functional epitopes II have to be present on one BMP-2 molecule for receptor activation. Futhermore, both pathways (Smad and ALP) are triggered differently by distinct BMP-receptor complexes. Heteromeric BMP-2 mutants therefore allow a distinguishable manipulation of either pathway and thus represent important tools for the generation of specific BMP-2 antagonists or agonists.
Asunto(s)
Proteínas Morfogenéticas Óseas/inmunología , Proteínas Morfogenéticas Óseas/metabolismo , Epítopos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Sitios de Unión , Técnicas Biosensibles , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Dimerización , Matriz Extracelular/metabolismo , Ratones , Músculo Esquelético/citología , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Smad , Transactivadores/metabolismoRESUMEN
IL-12 and IFN-gamma positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-gamma (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4(+) T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-gamma-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-gamma and IL-4. In conclusion, our results suggest that the IL-12/IFN-gamma axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-gamma production from these cells limits disease progression.
Asunto(s)
Enfermedades Autoinmunes/etiología , Interferón gamma/fisiología , Interleucina-12/fisiología , Miocarditis/etiología , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/prevención & control , Linfocitos T CD4-Positivos/inmunología , Interleucina-4/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Miocarditis/prevención & control , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
BACKGROUND: IL-4 and IL-13 are key regulators in atopic disorders and both signal through the receptor chain IL-4Ralpha. IL-4 and IL-13 are also the only cytokines known to induce class switching to IgE. We sought to compare allergen-specific IgE responses and allergic reactivity of wild-type (wt) mice with IL-4-/- and IL-4Ralpha-/- mice, which lack both IL-4 and IL-13 functions. METHODS: BALB/c wt, IL-4-/- and IL-4Ralpha-/- mice were immunized with ovalbumin intranasally or intraperitoneally and specific antibody titers were measured by ELISA. Bronchoalveolar lavage fluids and lung tissue were analyzed cytologically and histologically. Allergic reactivity was determined by active cutaneous anaphylaxis and anaphylactic shock. RESULTS: wt mice immunized intranasally or intraperitoneally showed high titers of specific IgE 3 and 6 weeks after primary sensitization, resulting in cutaneous anaphylaxis and anaphylactic shock upon challenge. Intranasal sensitization resulted in airway eosinophilia and goblet cell metaplasia. In contrast, IL-4-/- and IL-4Ralpha-/- mice showed no specific IgE after 3 weeks, but produced high titers after 6 weeks. At this time cutaneous anaphylaxis and anaphylactic shock could be induced as in wt mice, but lung pathology was absent. CONCLUSIONS: We conclude that upon long-term allergen exposure, alternative switch mechanisms independent of IL-4 and IL-4Ralpha may induce IgE but not asthma-like lung pathology. This may be relevant for the development of allergic disease, since long-term allergen exposure is a frequent condition during allergic sensitization.
Asunto(s)
Alérgenos/inmunología , Anafilaxia/etiología , Inmunoglobulina E/biosíntesis , Interleucina-4/fisiología , Receptores de Interleucina-4/fisiología , Animales , Eosinofilia/etiología , Femenino , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/clasificación , Interleucina-13/fisiología , Metaplasia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ovalbúmina/inmunologíaRESUMEN
PURPOSE: Heterotopic ossification (HO) is a common complication following total hip replacement. Clinical studies showed the effectiveness of irradiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to find a suitable cell system, which can reproduce in-vitro data resulting from clinical in-vivo studies. The establishment of such a cell model allows detailed analyses of the mechanism of radiotherapy. METHOD: The chicken limb bud test was used as an in-vitro model. The cells acquired by the limb bud test were irradiated with different doses (0 Gy, 3 Gy, 7 Gy, 10 Gy, 20 Gy). Irradiation was set either 1 hour before, or 1 or 3 days after BMP-2 incubation. The synthesis of proteoglycans (PGS) upon treatment with bone morphogenetic protein (BMP)-2 was measured in cells incubated with BMP-2 for 4 days followed by 35SO4(2-) labeling for 6 hours. Labeled proteoglycans were precipitated using Alcian blue and measured in a raytest radio-TLC analyzer. The incubation with BMP-2 was defined to correlate the in-vivo stimulus meaning the operation. RESULTS: The proteoglycan synthesis was significantly reduced by irradiation 1 hour before or 1 day after BMP-2 incubation, if the dosage was at least 7 Gy. Higher doses than 7 Gy did not lead to lower proteoglycan levels. There was only a trend for a reduction of proteoglycan synthesis by 3 Gy irradiation, but no significant difference compared to the non-irradiated control. An irradiation 3 days after BMP-2 incubation had no effect on proteoglycan. CONCLUSION: A dose and time dependent effect of radiation on BMP-2-induced proteoglycan synthesis was observed. Therefore the results of clinical in-vivo studies were reproduced exactly by the limb bud test. We established an in-vitro cell model to analyze the mechanism of the prevention of heterotopic ossification by radiotherapy on cellular or sub-cellular level.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de la radiación , Mesodermo/efectos de la radiación , Osificación Heterotópica/radioterapia , Proteoglicanos/metabolismo , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Recuento de Células , Embrión de Pollo , Relación Dosis-Respuesta en la Radiación , Humanos , Esbozos de los MiembrosRESUMEN
BACKGROUND: Knowledge of the factors which control IgE production is essential in order to understand the pathogenesis of immediate hypersensitivity reactions. We have studied the extent to which the route and frequency of antigen application as well as different antigen amounts may influence IgE synthesis. METHODS: We established sensitisation protocols in BALB/c mice, in which various doses of ovalbumin (Ova) were applied via intranasal, epicutaneous or intraperitoneal routes. Ova-specific antibodies were measured by ELISA. After 6 weeks of sensitisation, anaphylactic shock was measured following intravenous challenge with Ova. In addition, bronchoalveolar lavages were performed in intranasally sensitised mice. RESULTS: We were able to show that the most efficient IgE production was achieved by long-term antigen application via the airways, leading to local allergic airway pathology. The epicutaneous route of antigen application also induced very high IgE titres, while intraperitoneal sensitisation led to significantly lower IgE levels. After intraperitoneal sensitisation, IgE synthesis was best induced by increasing the frequency of antigen application, but not by increasing the amount of antigen. In all groups of mice, Ova-specific IgE antibodies were high enough to induce systemic allergic symptoms leading to anaphylactic shock. The severity of shock correlated with the amount of specific IgE. CONCLUSIONS: Taken together, our results demonstrate that antigen application via the airways or skin induces IgE synthesis more efficiently than via the intraperitoneal route. Few exposures with high-dose antigen are less efficient than multiple exposures with low doses. Our finding that both the route and the frequency of antigen application strongly influence IgE synthesis may help to understand how environmental antigens lead to allergic sensitisation.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/biosíntesis , Administración Cutánea , Administración Intranasal , Alérgenos/administración & dosificación , Anafilaxia/etiología , Animales , Antígenos/administración & dosificación , Antígenos/inmunología , Femenino , Hipersensibilidad Inmediata/patología , Inmunoglobulina G/biosíntesis , Inyecciones , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Peritoneo , Eosinofilia Pulmonar/inmunología , Eosinofilia Pulmonar/patologíaRESUMEN
BACKGROUND: Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) belong to the large transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. Signaling of the BMPs requires the binding of the BMP to the BMP cell surface receptors BMPR-IA, BMPR-IB, and BMPR-II. Similar to other cytokines, members of the TGF-beta superfamily exhibit stringent specificity in their ligand-receptor interactions, which may be a reason for the qualitative and quantitative differences in cellular responses. To understand how BMPs and GDFs activate their receptors, it is important to determine structure and binding mechanisms of ligand-receptor complexes. We have used BMP-2 as a key representative of the BMPs to identify the epitopes for type I and type II receptor binding by mutational interaction analyses and have solved the crystal structure of a BMP2:BMPR-IA receptor ectodomain complex. METHODS: To identify amino acid side chains involved in receptor binding, a collection of in vitro mutagenized human BMP-2 variants was prepared and subjected to interaction analyses with use of the receptor ectodomains of BMPR-IA, BMPR-II, and ActR-II immobilized on a biosensor system. The biological activity of the BMP-2 variants was measured by BMP-2 dependent expression of alkaline phosphatase (ALP) in C2C12 cells. For crystallization, a complex of BMP-2 and the ectodomain of BMPR-IA was formed in solution, purified, and crystallized as described(12). RESULTS: The ligand-receptor interaction analysis of the BMP-2 variants identified distinct epitopes for type I and type II receptor binding. Because the structure of TGF-beta-like proteins has been compared with that of an open hand, the binding epitope for the type I receptor was-on the basis of its location-termed "wrist" epitope. The crystal structure of the BMP-2:BMPR-IA ectodomain complex revealed a key feature of the ligand-receptor interaction: a large hydrophobic residue (Phe85) within a hydrophobic patch of BMPR-IA fit into a hydrophobic pocket composed of residues of both BMP-2 monomers. A second epitope identified by alanine mutagenesis scanning was termed the "knuckle" epitope on the basis of its location on the outer side of the "finger" segments of BMP-2. Mutations in either the wrist epitope or the knuckle epitope produced variants with altered biological activities. Variants with antagonistic properties were exclusively generated by mutations in the knuckle epitope of BMP-2. CONCLUSIONS AND CLINICAL RELEVANCE: The identification and characterization of the two receptor binding epitopes in BMP-2 provide new insight into the primary steps of BMP-receptor activation. Because of the structural similarities between members of the TGF-beta superfamily, it can be assumed that the data presented in this work are transferable to other TGF-beta receptor systems. Because of the association with various diseases, the generation of antagonists of other TGF-beta superfamily members might generate potent tools for basic research and therapeutic approaches.
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/química , Proteínas Serina-Treonina Quinasas/química , Receptores de Factores de Crecimiento/química , Secuencia de Aminoácidos , Proteína Morfogenética Ósea 2 , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Epítopos , Humanos , Mutación , Conformación Proteica , Transducción de Señal/fisiología , Factor de Crecimiento Transformador betaRESUMEN
Transforming growth factor-beta (TGF-beta) signals through membrane-bound serine/threonine kinase receptors, which upon stimulation phosphorylate Smad proteins and thereby trigger their nuclear translocation and transcriptional activity. Although the three mammalian isoforms of TGF-beta are highly homologous at the level of sequence, analysis of their in vivo function by gene knockouts revealed striking differences, suggesting no significant functional redundancy between TGF-beta1, -2 and -3. While signal transduction by TGF-beta1 has been well characterized, receptor binding and activation by the TGF-beta2 isoform is less well understood. Here, we show that TbetaRII-B, an alternatively spliced variant of the TGF-beta type II receptor, is a TGF-beta2 binding receptor, which mediates signalling via the Smad pathway in the absence of any TGF-beta type III receptor (TbetaRIII). L6 cells lacking endogenous TbetaRIII as well as TbetaRII-B do not respond to TGF-beta2. Transfection of these cells with TbetaRII-B restores TGF-beta2 sensitivity. The expression of TbetaRII-B is restricted to cells originating from tissues such as bone where the isoform TGF-beta2 has a predominant role. This reflects the importance of this receptor in TGF-beta isoform-specific signalling.
Asunto(s)
Receptores de Activinas Tipo I , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Línea Celular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Disulfuros/química , Expresión Génica , Glicosilación , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoglicanos/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/química , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteína Smad2 , Transactivadores/metabolismo , Transfección , Factor de Crecimiento Transformador beta2RESUMEN
INTRODUCTION: Osteotropic growth factors enhance bone repair, but their efficacy in an area of necrotic bone is not known. The purpose of this study was to investigate the effects and potential side effects of an intraosseous application of absorbable bone morphogenetic protein-2 (BMP-2) and growth and differentiation factor-5 (GDF-5) composites in a sheep model for partial necrosis of the femoral head. MATERIALS AND METHODS: The direct injection of ethanol under fluoroscopy into the superior centre of the right femoral head produced histologically documentable necrosis of the central region of the head in a previous study of ten sheep. Another 27 sheep constituted the sample to study the effects of BMP-2 and GDF-5. Necrosis was produced in the same fashion in these animals. Four weeks later nine sheep received 300 microg recombinant BMP-2 and nine sheep 300 microg recombinant GDF-5 on an absorbable carrier by surgical implantation. Nine sheep received the carrier alone (control group). The animals were sacrificed at 3, 6, and 12 weeks after implantation and both femora were harvested. RESULTS: Bone density analysis and microscopic examination indicated that bone formation was noticeably induced as early as 3 weeks postoperatively in the growth factor treated animals. Bone regeneration was enhanced by growth factor composites. This was documented by histological scoring and histomorphometric analysis. No severe local side effects secondary to the growth factors, such as heterotopic ossification or inflammation, were observed in either group. DISCUSSION: The application of an absorbable growth factor composite in combination with established surgical techniques is a promising approach, that may enhance the healing of devitalised bone defects. Based on these results, further studies regarding biodegradation, dosage of the protein and surgical technique are required.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Huesos/patología , Sustancias de Crecimiento/fisiología , Necrosis , Factor de Crecimiento Transformador beta , Cicatrización de Heridas , Animales , Densidad Ósea , Proteína Morfogenética Ósea 2 , Escherichia coli/metabolismo , Etanol/farmacología , Factor 5 de Diferenciación de Crecimiento , Humanos , Proteínas Recombinantes , Ovinos , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
The extent of BMP-induced new bone formation is mainly determined by the number of mesenchymal target cells in the recipient bed as well as by the biological half-life of the BMP molecules within the tissue. While the number of inducible cells is determined by the age and vascularization of the tissue, the retention time of the BMP molecules can be influenced. One possibility is the coupling of BMPs to suitable carriers, which significantly increases the osteoinductive effect. The reason for this is the physical binding of BMPs to the carrier material, which delays the resorption of the proteins. Other factors are the composition of the carrier materials, their structural stability, and possible dislocations of carrier particles. The local tissue concentration of BMPs can also be increased by an enhanced binding of the proteins to the extracellular matrix. A BMP-2 mutant (BMP-2xa) was produced by the specific modification of the amino acid sequence using recombinant technologies. BMP-2xa induces heterotopic bone formation at significantly lower concentrations than natural BMP-2. Furthermore, BMP-2xa-induced bone tissue possesses a higher bone density.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Regeneración Ósea/efectos de los fármacos , Maxilares/efectos de los fármacos , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Humanos , Maxilares/patología , Ratones , Ratas , Proteínas Recombinantes/farmacologíaRESUMEN
Organic synthesis of hormone derivatives is an established route to yield pharmacologically active agents. Until recently this has only been feasible for small organic compounds, but nowadays it is also possible to produce antagonists for larger protein hormones. In particular, the interleukin-4-receptor was a well-suited target for this approach since it plays a pivotal role in the release and progression of allergic diseases. Accordingly, a strong interest and a high medical need is associated with the development of inhibitors. The structural elucidation of the ligand/receptor complex and an improved understanding of the mechanisms concerning receptor binding and activation allow for the rational design of variants that inhibit interleukin-4. Since it is possible to specifically inhibit the interleukin-4-receptor system in this way, a completely new approach to the development of new drugs against allergy and asthma has been established.
RESUMEN
Bone morphogenetic protein-2 (BMP-2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP-2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor-beta (TGF-beta) superfamily, signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane. The binding epitopes of BMP-2 for BMPR-IA (type I) and BMPR-II or ActR-II (type II) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer. A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer. Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles. Mutations in both epitopes yielded variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behaved as antagonists partially or completely inhibiting BMP-2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF-beta superfamily.