RESUMO
Different tissue engineering techniques are used to support rapid vascularisation. A novel technique is the use of platelet-rich fibrin (PRF), an autologous source of growth factors. This study was the first to investigate the influence of PRF matrices, isolated following different centrifugation protocols, on human dermal vascular endothelial cells (ECs) in mono-culture and co-culture with human primary fibroblasts (HFs) as an in vitro model for tissue regeneration. Focus was placed on vascular structure formation and growth factor release. HFs and ECs were cultivated with PRF prepared using a high (710 ×g) or low (44 ×g) relative centrifugation force (RCF) over 14 d. Immunofluorescence staining and immunohistochemistry were used to evaluate the microvascular formation. Cell culture supernatants were collected for evaluation of growth factor release. The results showed a PRF-mediated effect on the induction of angiogenesis in ECs. Microvessel-like structure formation was promoted when ECs were combined with low-RCF PRF as compared to high-RCF PRF or control group. The percentage of vascular lumen area was significantly higher in low-RCF PRF, especially at day 7, which coincided with statistically significantly higher growth factor [vascular endothelial factor (VEGF), transforming growth factor ß1 (TGF-ß1) and platelet derived growth factor (PDGF)] concentration measured in low-RCF PRF as compared to high-RCF PRF or control group. In conclusion, reducing the RCF according to the low-speed centrifugation concept (LSCC) resulted in increased growth factor release and angiogenic structure formation with EC mono-culture, suggesting that PRF may be a highly beneficial therapeutic tool for tissue engineering applications.
Assuntos
Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Neovascularização Fisiológica/efeitos dos fármacos , Fibrina Rica em Plaquetas , Técnicas de Cultura de Células , Células Endoteliais/citologia , Fibroblastos/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologiaRESUMO
BACKGROUND: . Missense variants in the ryanodine receptor 1 gene ( RYR1 ) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. METHODS: . We identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. RESULTS.: We identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. CONCLUSIONS: . Segregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.
Assuntos
Hipertermia Maligna/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Cafeína/farmacologia , Cálcio/metabolismo , Clonagem Molecular , Família , Predisposição Genética para Doença , Variação Genética , Genótipo , Células HEK293 , Humanos , Hipertermia Maligna/epidemiologia , Imagem Molecular , Mutagênese , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
This study represents a new approach to characterising patients at risk of malignant hyperthermia (MH) through the use of a recently published method for identifying high-risk haplotypes in candidate genes. We present analysis based upon the largest standardised and genotyped database of MH patients worldwide. We used unphased RYR1 SNP data directly to (1) assess RYR1 haplotype frequency differences between susceptible cases and control groups and (2) analyse population-based association via clustering of RYR1 haplotypes based on disease risk. Our results show a significant difference in RYR1 haplotype frequency between susceptible cases and UK Caucasian population controls. Furthermore we identify a high-risk cluster of haplotypes that is associated with the commonest UK MH mutation p.G2434R/c.7300G>A. These results demonstrate the applicability of this new and practical method for population based association analysis.
Assuntos
Hipertermia Maligna/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Estudos de Casos e Controles , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Reino Unido , População Branca/genéticaRESUMO
BACKGROUND: Malignant hyperthermia (MH) is associated, in the majority of cases, with mutations in RYR1, the gene encoding the skeletal muscle ryanodine receptor. Our primary aim was to assess whether different RYR1 variants are associated with quantitative differences in MH phenotype. METHODS: The degree of in vitro pharmacological muscle contracture response and the baseline serum creatine kinase (CK) concentration were used to generate a series of quantitative phenotypes for MH. We then undertook the most extensive RYR1 genotype-phenotype correlation in MH to date using 504 individuals from 204 MH families and 23 RYR1 variants. We also determined the association between a clinical phenotype and both the laboratory phenotype and RYR1 genotype. RESULTS: We report a novel correlation between the degree of in vitro pharmacological muscle contracture responses and the onset time of the clinical MH response in index cases (P<0.05). There was also a significant correlation between baseline CK concentration and clinical onset time (P=0.039). The specific RYR1 variant was a significant determinant of the severity of each laboratory phenotype (P<0.0001). CONCLUSIONS: The MH phenotype differs significantly with different RYR1 variants. Variants leading to more severe MH phenotype are distributed throughout the gene and tend to lie at relatively conserved sites in the protein. Differences in phenotype severity between RYR1 variants may explain the variability in clinical penetrance of MH during anaesthesia and why some variants have been associated with exercise-induced rhabdomyolysis and heat stroke. They may also inform a mutation screening strategy in cases of idiopathic hyperCKaemia.
Assuntos
Hipertermia Maligna/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Anestésicos Inalatórios/farmacologia , Cafeína/farmacologia , Creatina Quinase/sangue , Análise Mutacional de DNA/métodos , DNA Complementar/genética , Feminino , Predisposição Genética para Doença , Genótipo , Halotano/farmacologia , Humanos , Masculino , Hipertermia Maligna/enzimologia , Hipertermia Maligna/fisiopatologia , Contração Muscular/efeitos dos fármacos , Fenótipo , Inibidores de Fosfodiesterase/farmacologia , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Tissue-specific monoallelic silencing of the RYR1 gene has been proposed as an explanation for variable penetrance of dominant RYR1 mutations in malignant hyperthermia (MH). We examined the hypothesis that monoallelic silencing could explain the inheritance of an MH discordant phenotype in some instances. METHODS: We analysed parent-offspring transmission data from MH kindreds to assess whether there was any deviation from the expected autosomal dominant Mendelian inheritance pattern. We also evaluated informative single-nucleotide polymorphism (SNP) genotypes in a cohort of unrelated MH patients using genomic DNA (gDNA, prepared from leucocytes) and coding DNA (cDNA, prepared from skeletal muscle). Finally, we examined the segregation of specific mutations at the gDNA and cDNA level within MH families where positive RYR1 gDNA genotype/normal MH phenotype discordance had been observed. RESULTS: In 2113 transmissions from affected parents, there was a consistent parent-of-origin effect (P<0.001) with affected fathers having fewer affected daughters (20%, 95% CI 17-22%) than affected sons (25%, 95% CI 23-26%) or unaffected daughters (27%, 95% CI 25-30%). No discrepancies were observed between the RYR1 SNP genotypes recorded at the gDNA and cDNA levels. In 14 MH negative individuals from 11 discordant families, the familial mutation was detected in skeletal muscle cDNA in all cases. CONCLUSIONS: Epigenetic allele silencing may play a role in the inheritance of MH susceptibility, but this is unlikely to involve silencing of RYR1.
Assuntos
Epigênese Genética , Inativação Gênica , Hipertermia Maligna/genética , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Músculo Esquelético/química , Penetrância , Fenótipo , Polimorfismo de Nucleotídeo Único , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
Purpose The present study evaluated the platelet distribution pattern and growth factor release (VEGF, TGF-ß1 and EGF) within three PRF (platelet-rich-fibrin) matrices (PRF, A-PRF and A-PRF+) that were prepared using different relative centrifugation forces (RCF) and centrifugation times. Materials and methods immunohistochemistry was conducted to assess the platelet distribution pattern within three PRF matrices. The growth factor release was measured over 10 days using ELISA. Results The VEGF protein content showed the highest release on day 7; A-PRF+ showed a significantly higher rate than A-PRF and PRF. The accumulated release on day 10 was significantly higher in A-PRF+ compared with A-PRF and PRF. TGF-ß1 release in A-PRF and A-PRF+ showed significantly higher values on days 7 and 10 compared with PRF. EGF release revealed a maximum at 24 h in all groups. Toward the end of the study, A-PRF+ demonstrated significantly higher EGF release than PRF. The accumulated growth factor releases of TGF-ß1 and EGF on day 10 were significantly higher in A-PRF+ and A-PRF than in PRF. Moreover, platelets were located homogenously throughout the matrix in the A-PRF and A-PRF+ groups, whereas platelets in PRF were primarily observed within the lower portion. âDiscussion the present results show an increase growthfactor release by decreased RCF. However, further studies must be conducted to examine the extent to which enhancing the amount and the rate of released growth factors influence wound healing and biomaterial-based tissue regeneration. âConclusion These outcomes accentuate the fact that with a reduction of RCF according to the previously LSCC (described low speed centrifugation concept), growth factor release can be increased in leukocytes and platelets within the solid PRF matrices.
Assuntos
Plaquetas/metabolismo , Centrifugação/métodos , Fator de Crescimento Epidérmico/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Feminino , Voluntários Saudáveis , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Estudo de Prova de Conceito , CicatrizaçãoRESUMO
Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.
Assuntos
Síndrome de Marfan/genética , Receptores de Ativinas Tipo I/genética , Dissecção Aórtica/genética , Animais , Aneurisma da Aorta Torácica/genética , Proteínas Contráteis/fisiologia , Bases de Dados Genéticas , Proteínas da Matriz Extracelular/fisiologia , Fibrilina-1 , Fibrilinas , Humanos , Proteínas de Ligação a TGF-beta Latente/genética , Síndrome de Marfan/complicações , Camundongos , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/genética , Modelos Animais , Modelos Biológicos , Desnaturação Proteica/genética , Proteínas Serina-Treonina Quinases , Fatores de Processamento de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genéticaRESUMO
The present study analyzes the influence of the addition of monocytes to a biphasic bone substitute with two granule sizes (400-700 µm and 500-1000 µm). The majority of the added monocytes was detectable as mononuclear cells, while also low amounts of (chimeric) multinucleated giant cells (MNGCs) were found. No increase in the total number of MNGCs was established, but a significantly increased percent vascularization. Altogether, the results show that the added monocytes become involved in the tissue response to a biomaterial without marked changes in the overall reaction. Monocyte addition enables an increased implant bed vascularization especially via induction of vessel maturation and, thus intervenes positively in the healing reaction to a biomaterial. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2928-2935, 2016.
Assuntos
Substitutos Ósseos/metabolismo , Hidroxiapatitas/metabolismo , Monócitos/citologia , Neovascularização Fisiológica , Animais , Células Cultivadas , Feminino , Células Gigantes/citologia , Humanos , Teste de Materiais , Camundongos SCID , Próteses e ImplantesRESUMO
The present study investigated the influence of granule size of 2 biphasic bone substitutes (BoneCeramic® 400-700 µm and 500-1000 µm) on the induction of multinucleated giant cells (MNGCs) and implant bed vascularization in a subcutaneous implantation model in rats. Furthermore, degradation mechanisms and particle phagocytosis of both materials were examined by transmission electron microscopy (TEM). Both granule types induced tissue reactions involving primarily mononuclear cells and only small numbers of MNGCs. Higher numbers of MNGCs were detected in the group with small granules starting on day 30, while higher vascularization was observed only at day 10 in this group. TEM analysis revealed that both mono- and multinucleated cells were involved in the phagocytosis of the materials. Additionally, the results allowed recognition of the MNGCs as the foreign body giant cell phenotype. Histomorphometrical analysis of the size of phagocytosed particles showed no differences between the 2 granule types. The results indicate that granule size seems to have impact on early implant bed vascularization and also on the induction of MNGCs in the late phase of the tissue reaction. Furthermore, the results revealed that a synthetic bone substitute material can induce tissue reactions similar to those of some xenogeneic materials, thus pointing to a need to elucidate their "ideal" physical characteristics. The results also show that granule size in the range studied did not alter phagocytosis by mononuclear cells. Finally, the investigation substantiates the differentiation of material-induced MNGCs, which are of the foreign body giant cell type.
Assuntos
Substitutos Ósseos/farmacologia , Osso e Ossos/irrigação sanguínea , Osso e Ossos/imunologia , Células Gigantes/metabolismo , Hidroxiapatitas/farmacologia , Leucócitos Mononucleares/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Neovascularização Fisiológica , Tamanho da Partícula , Fagocitose , RatosRESUMO
Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome, an autosomal dominant disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular system. There is a remarkable degree of clinical variability both within and between families with Marfan syndrome as well as in individuals with related disorders of connective tissue caused by FBN1 mutations and collectively termed type-1 fibrillinopathies. The so-called neonatal region in FBN1 exons 24-32 comprises one of the few generally accepted genotype-phenotype correlations described to date. In this work, we report 12 FBN1 mutations identified by temperature-gradient gel electrophoresis screening of exons 24-40 in 127 individuals with Marfan syndrome or related disorders. The data reported here, together with other published reports, document a significant clustering of mutations in exons 24-32. Although all reported mutations associated with neonatal Marfan syndrome and the majority of point mutations associated with atypically severe presentations have been found in exons 24-32, mutations associated with classic Marfan syndrome occur in this region as well. It is not possible to predict whether a given mutation in exons 24-32 will be associated with classic, atypically severe, or neonatal Marfan syndrome.
Assuntos
Éxons/genética , Síndrome de Marfan/genética , Proteínas dos Microfilamentos/genética , Adolescente , Adulto , Criança , Pré-Escolar , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Fibrilina-1 , Fibrilinas , Genótipo , Humanos , Recém-Nascido , Masculino , Síndrome de Marfan/patologia , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Polimorfismo GenéticoRESUMO
Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome, a dominantly inherited disorder of connective tissue that primarily involves the cardiovascular, ocular, and skeletal systems. There is a remarkable degree of variability both within and between families with Marfan syndrome, and FBN1 mutations have also been found in a range of other related connective tissue disorders collectively termed type-1 fibrillinopathies. FBN1 mutations have been found in almost all of the 65 exons of the FBN1 gene and for the most part have been unique to one affected patient or family. Aside from the "hot spots" for the neonatal Marfan syndrome in exons 24-27 and 31-32, genotype-phenotype correlations have been slow to emerge. Here we present the results of temperature-gradient gel electrophoresis analysis of FBN1 exons 59-65. Six mutations were identified, only one of which had been previously reported. Two of the six mutations were found in patients with mild phenotypes. Taken together with other published reports, our results suggest that a sizable subset (ca. 40%) of mutations in this region is associated with mild phenotypes characterized by the lack of significant aortic pathology, compared with about 7% in the rest of the gene. In two cases, mutations affecting analogous positions within one of the 43 cbEGF modules of FBN1 are associated with mild phenotypes when found in one of the 6 C-terminal modules (encoded by exons 59-63), but are associated with classic or severe phenotypes when found in cbEGF modules elsewhere in the gene.
Assuntos
Síndrome de Marfan/genética , Proteínas dos Microfilamentos/genética , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Éxons , Feminino , Fibrilina-1 , Fibrilinas , Genótipo , Humanos , Masculino , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Estrutura Terciária de ProteínaRESUMO
In the present study, the structure of two allogeneic and three xenogeneic bone blocks, which are used in dental and orthopedic surgery, were histologically analyzed. The ultimate goal was to assess whether the components postulated by the manufacturer can be identified after applying conventional histological and histochemical staining techniques. Three samples of each material, i.e. allogeneic material-1 and -2 as well as xenogeneic material-1, -2 and -3, were obtained commercially. After decalcification and standardized embedding processes, conventional histological staining was performed in order to detect inorganic matrix, cellular or organic matrix components. Allogeneic material-1 showed trabecular bone-like structures, which were free of cellular components as well as of organic matrix. The allogeneic material-2 showed trabecular bone structures, in which connective tissue and cellular remnants were embedded. Additionally, some connective tissue, which resembled fat-like tissue, was found within this material. The xenogeneic material-1 showed trabecular bone-like structures and contained organic components comparable to that demonstrated for the allogeneic material-2. The xenogeneic material-2 showed trabecular bone structures with single cells located in lacunae. The xenogeneic material-3 also showed trabecular structures. Neither cellular nor organic matrix components were found within this material. According to the data of the present study, the allogeneic material-1 and the xenogeneic material-3 were the only investigated materials for which the obtained histological data were in accordance with the manufacturers advertised information. The remaining three materials showed discrepancies-although the manufacturers of all five bone substitute materials stated that their blocks were free of organic/cellular remnants. These data are of great clinical and material science interest. It seems that even patented processing techniques are not always able to deliver reproducible materials. Although the manufacturers of all five bone blocks stated that their blocks were free of organic/cellular remnants, our histological analysis revealed that three out of five bone blocks did contain such remnants. Such specimens might be able to induce an immune response within the recipient.
Assuntos
Aloenxertos/química , Substitutos Ósseos/síntese química , Substitutos Ósseos/normas , Xenoenxertos/química , Teste de Materiais/normas , Guias de Prática Clínica como Assunto , Aloenxertos/normas , Transplante Ósseo/normas , Xenoenxertos/normas , InternacionalidadeRESUMO
The Marfan syndrome (MFS) is an autosomal dominant heritable disorder of connective tissue with highly variable clinical manifestations including aortic dilatation and dissection, ectopia lentis, and a range of skeletal anomalies. Mutations in the gene for fibrillin-1 (FBN1) cause MFS and other related disorders of connective tissue collectively termed type-1 fibrillinopathies. Fibrillin-1 is a main component of the 10- to 12-nm extracellular microfibrils that are important for elastogenesis, elasticity, and homeostasis of elastic fibers. Mutations in fibrillin-1 are hypothesized to exert their effects by dominant negative mechanisms, but recent work has also emphasized the potential role of proteases and disturbances in tissue homeostasis in the pathogenesis of the MFS. This article provides an overview of the clinical aspects of the MFS and current thinking on the pathogenesis of this disorder.
Assuntos
Síndrome de Marfan/genética , Síndrome de Marfan/fisiopatologia , Proteínas dos Microfilamentos/genética , Animais , Modelos Animais de Doenças , Tecido Elástico/fisiologia , Fibrilina-1 , Fibrilinas , Homeostase , Humanos , Síndrome de Marfan/diagnóstico , Microfibrilas/metabolismo , Microfibrilas/ultraestrutura , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In patients with asthma there is only a weak relationship between airway responsiveness to hypertonic saline and methacholine. We tested the hypothesis that airway responsiveness to hypertonic saline in asthma is related to the presence and activity of inflammatory cells in the peripheral blood. Nineteen atopic asthmatic adults (19-28 yr; PC20 0.06-12.4 mg/ml), not receiving steroid treatment, entered a metacholine and hypertonic saline period in random order. Dose-response curves to doubling doses of inhaled methacholine (0.03-256 mg/ml) or hypertonic saline (0.9-14.4% NaCl) were obtained twice in each period, 7 days apart. The response was measured by FEV1. Methacholine responsiveness was measured by PC20 METH of FEV1 and responsiveness to hypertonic saline was expressed as the percentage fall in FEV1 after 14.4% NaCl (HYP14.4%). Peripheral blood was collected before the second challenge test of each period. Apart from leucocyte counts and serum eosinophilic cationic protein (ECP) level, sub-sets of lymphocytes (CD4+/CD3+, CD8+/CD3+, CD25+/CD4+ and VLA-1+/CD4+) were determined using flow cytometry. HYP14.4% was positively correlated to basophil, eosinophil and monocyte counts (r = 0.64, 0.54 and 0.44, respectively; P < 0.05). The basophil count remained positively related to HYP14.4% when PC20METH or FEV1%pred were entered in multiple linear regression analyses (r = 0.66 and 0.75, respectively; P < 0.05). There were no significant relationships between HYP14.4% or PC20METH on one side and ECP level or T-lymphocyte subsets on the other (P > 0.05). We conclude that airway responsiveness to hypertonic saline is positively related to the number of peripheral blood basophils, eosinophils and monocytes. Basophil count is an independent correlate of responsiveness to hypertonic saline, after correction for methacholine responsiveness and baseline lung function. This fits in with active involvement of basophils in airway narrowing to hypertonic saline in vivo.
Assuntos
Asma/sangue , Asma/diagnóstico , Hiper-Reatividade Brônquica/sangue , Testes de Provocação Brônquica , Solução Salina Hipertônica , Adulto , Asma/fisiopatologia , Testes de Provocação Brônquica/métodos , Relação Dose-Resposta Imunológica , Feminino , Humanos , Contagem de Leucócitos , Leucócitos , Masculino , Cloreto de Metacolina , Subpopulações de Linfócitos TRESUMO
Mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant disorder of connective tissue with highly variable clinical manifestations. FBN1 contains 47 epidermal growth factor (EGF)-like modules, 43 of which display a consensus sequence for calcium binding (cbEGF). Calcium binding by cbEGF modules is thought to be essential for the conformation and stability of fibrillin-1. Missense mutations in cbEGF modules are the most common mutations found in MFS and generally affect one of the six highly conserved cysteines or residues of the calcium-binding consensus sequence. We have generated a series of recombinant fibrillin-1 fragments containing six cbEGF modules (cbEGF nos. 15-20) with various mutations at different positions of cbEGF module no. 17, which is known to contain a cryptic cleavage site for trypsin. A mutation affecting a residue of the calcium-binding consensus sequence (K1300E) found in a patient with relatively mild clinical manifestations of classic MFS caused a modest increase in susceptibility to in vitro proteolysis by trypsin, whereas a mutation affecting the sixth cysteine residue of the same cbEGF module (C1320S) reported in a severely affected patient caused a dramatic increase in susceptibility to in vitro proteolysis by trypsin. A mutation at the cryptic cleavage site for trypsin abolished sensitivity of wild-type fragments and fragments containing K1300E to trypsin proteolysis. Whereas the relevance of in vitro proteolysis to the in vivo pathogenesis of MFS remains unclear, our findings demonstrate that individual mutations in cbEGF modules can affect these modules differentially and may suggest an explanation for some genotype-phenotype relationships in MFS.
Assuntos
Ectopia do Cristalino/genética , Síndrome de Marfan/etiologia , Síndrome de Marfan/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Adolescente , Cálcio/farmacologia , Endopeptidases/efeitos dos fármacos , Endopeptidases/metabolismo , Feminino , Fibrilina-1 , Fibrilinas , Humanos , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Patients with asthma who have moderate to severe airway hyperresponsiveness often demonstrate progressive, unlimited airway narrowing in response to inhaled bronchoconstrictor stimuli, which is likely to be due to inflammatory changes within the airway wall. It is unknown whether regular therapy with inhaled steroids can limit this excessive response. OBJECTIVE: We investigated the effect of inhaled budesonide on the development of a plateau on the dose-response curve to methacholine in patients with asthma who did not show such a plateau before the study. METHODS: Thirty-one atopic patients with asthma (age, 19 to 31 years; FEV1 > 70% of predicted value; PC20 < 8 mg/ml) with documented absence of a maximal-response plateau to methacholine on two occasions during the run-in period, participated in a double-blind, placebo-controlled, parallel study. Standardized methacholine challenges were performed at -1, 0, 4, 8, and 12 weeks of treatment with inhaled budesonide, 800 microg two times a day, or corresponding placebo, and after a 2-week washout period. Airway response was measured by FEV1 (percent fall from baseline). A maximal-response plateau was considered if three or more consecutive data points fell within a 5% response range. RESULTS: Thirty patients completed the study. There was a steady increase in the number of budesonide-treated patients exhibiting a maximal-response plateau on the dose-response curve from zero of 15 patients at run-in to nine of 14 patients at week 12, as compared with four of 16 patients in the placebo group (p = 0.03, chi square test). This was accompanied by a significant improvement in PC20 in the budesonide group as compared with the placebo group (p < 0.01 at week 12), whereas the changes in FEV1 were not significantly different between the groups (p = 0.77 at week 12). CONCLUSION: Regular treatment with the inhaled corticosteroid budesonide limits maximal airway narrowing in response to methacholine by introducing a plateau on the dose-response curve in patients with asthma, who were initially characterized by the absence of a plateau. This indicates that inhaled steroids are likely to reduce the hazard of unlimited airway narrowing in asthma.
Assuntos
Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Broncoconstrição/efeitos dos fármacos , Pregnenodionas/uso terapêutico , Adulto , Testes de Provocação Brônquica , Budesonida , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Cloreto de MetacolinaRESUMO
The severity of breathlessness at given degrees of airway obstruction varies between patients with asthma. It has been postulated that the symptoms during bronchoconstriction are determined in part by involvement of airway inflammation. We compared the severity of breathlessness at various degrees of acute airway obstruction between a direct stimulus of airway smooth muscle, methacholine, and an indirectly acting stimulus, hypertonic saline. Twelve atopic asthmatic adults (mean +/- SD; age 25.3 +/- 3.4 yr; baseline FEV1 91.2 +/- 10.4% pred; PC20 1.0 mg/ml methacholine +/- 1.7 doubling dose) entered a methacholine and a hypertonic saline period in random order. In each period doubling doses of either methacholine (0.03 to 256 mg/ml) or hypertonic saline (0.9 to 14.4% NaCl) were inhaled on two occasions 7 d apart, using standardized tidal breathing methods. The response was obtained by FEV1 and, in order to assess volume history effects on airway caliber, by the ratio of flows obtained from volume history standardized maximal and partial expiratory flow-volume curves (M/P ratio). Breathlessness was measured by a visual analogue scale (VAS), which ranged from 0 (none) to 100% (most severe experienced). The subjects were blinded to the response in lung function. The changes from baseline in VAS scores at intervals of 5% fall in FEV1 (delta VAS) and the changes in M/P ratios (delta M/P ratio) were calculated by linear interpolation. The results were analyzed by MANOVA. There were no differences in baseline FEV1 or baseline VAS scores between the methacholine and hypertonic saline periods (p > 0.40).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Asma/fisiopatologia , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição/fisiologia , Dispneia/fisiopatologia , Adulto , Asma/diagnóstico , Testes de Provocação Brônquica , Estudos Cross-Over , Feminino , Volume Expiratório Forçado/fisiologia , Humanos , Masculino , Cloreto de Metacolina , Solução Salina Hipertônica , Índice de Gravidade de DoençaRESUMO
OBJECTIVES: To investigate if sequence alterations of the excitatory amino acid transporter gene EAAT2 (GLT-1) may be a contributory factor to the pathogenesis of motor system degeneration. EAAT2 serves as a candidate gene as its reduced expression was reported in patients with amyotrophic lateral sclerosis (ALS). Furthermore, neurolathyrism, a motor neuron disease clinically related to hereditary spastic paraplegia (HSP), has been associated with an exogenous excitotoxin. METHODS: Sequence alterations were screened for in the coding region of EAAT2 in 55 patients with ALS and one family with autosomal dominant HSP (AD-HSP). RESULTS: In ALS, no sequence alteration in the EAAT2 gene have been found. Interestingly, a heterozygous A79G mutation of the EAAT2 gene was detected in two of seven affected patients with AD-HSP in the same kindred. The absence of cosegregation with the familial disease showed that the detected variant was not the cause of disease. The A79G sequence variant was not found in 55 patients with ALS or in 50 non-neurological controls. CONCLUSION: The allelic variant of the EAAT2 gene in conjunction with the primary gene defect may be a modifying factor for the highly variable AD-HSP phenotype.
Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação Puntual/genética , Receptores de Neurotransmissores/genética , Paraplegia Espástica Hereditária/genética , Adulto , Alelos , Cromossomos Humanos Par 14/genética , Transportador 2 de Aminoácido Excitatório , Heterozigoto , Humanos , Linhagem , Fenótipo , Reação em Cadeia da Polimerase/métodosRESUMO
Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)-product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a "GC-clamp" is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.
Assuntos
DNA/análise , Eletroforese/métodos , Mutação , Reação em Cadeia da Polimerase/métodos , Fibrilinas , Proteínas dos Microfilamentos/genética , Neurofibromina 1 , Proteínas/genética , Sensibilidade e Especificidade , TemperaturaRESUMO
Cysteinyl leukotrienes are potent inflammatory mediators that are considered to play a role in the pathophysiology of asthma. It can be postulated that leukotrienes exert their bronchoconstricting effects, in part, through secondary release of endogenous neuropeptides. We examined the effect of inhaled thiorphan, an inhibitor of a neuropeptide degrading enzyme, on the concentration-response curve to leukotriene D4 (LTD4) in a two-period, double-blind, cross-over and placebo-controlled study, in 16 nonasthmatic and 12 asthmatic subjects. Thiorphan or placebo were aerosolized and administered in two 0.5 ml doses of 1.25 mg.ml-1 each, 10 min prior to LTD4 inhalation. The airway response was measured by forced expiratory volume in one second (FEV1) and partial expiratory flow-volume curves (expiratory flow at 40% of forced vital capacity; V40p), and expressed as % fall from baseline. Complete concentration-response curves to inhaled LTD4 were recorded and characterized by their position (provocative concentration producing a 20% fall in FEV1 and a 40% fall in V40p; PC20FEV1 and PC40 V40p) and, in the nonasthmatics, also by the maximal-response plateau (MFEV1, MV40p). Post-pretreatment baseline values of FEV1 and V40p were not different between thiorphan and placebo pretreatment. In both groups of subjects, there was no significant difference in lnPC40V40p or lnPC20FEV1 to LTD4 between the two pretreatments mean difference +/- SD (in doubling concentrations): 0.12 +/- 0.73 and -0.19 +/- 1.23, respectively, in asthmatics; and 0.17 +/- 0.95 and -0.99 +/- 1.95, respectively, in nonasthmatics. The maximal-response plateau could not be obtained in the majority of the asthmatic subjects.(ABSTRACT TRUNCATED AT 250 WORDS)