RESUMO
OBJECTIVES: To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated proteins autoantibodies (ACPA) reactive to α36-50Cit38,42 and/or ß60-74Cit60,72,74, two peptides identified as bearing the immunodominant epitopes of their major target, citrullinated fibrin. To analyse the relationships of anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 autoantibodies with autoantibodies reactive to the complete citrullinated human fibrinogen molecule (AhFibA) and with anti-CCP2 antibodies. METHODS: 617 sera from 181 patients with established RA and 436 with non-RA rheumatic diseases were tested by ELISA for AhFibA, anti-CCP2, anti-α36-50Cit38,42, anti-ß60-74Cit60,72,74 autoantibodies, and by nephelometry for rheumatoid factor (RF). Diagnostic indexes, correlations and concordances between tests were analysed. Crossreactivity of anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 autoantibodies was assessed in competition experiments. RESULTS: At a diagnostic specificity of 95%, the diagnostic sensitivity of AhFibA (83%) was significantly higher than that of all other tests. The diagnostic sensitivity of anti-ß60-74Cit60,72,74 (71%) was significantly higher than that of anti-α36-50Cit38,42 autoantibodies (51%) but similar to that of anti-CCP2 (74%). Titres of RF, anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 autoantibodies were weakly correlated with each other, whereas titres of anti-ß60-74Cit60,72,74 were strongly correlated with those of AhFibA (r=0.633) and anti-CCP2 (r=0.634). Anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 mainly corresponded to two non-crossreactive subfamilies of ACPA. More than 90% of AhFibA-positive or anti-CCP2-positive sera recognised the α36-50Cit38,42 and/or the ß60-74Cit60,72,74 peptide. CONCLUSIONS: Autoantibodies reactive to α36-50Cit38,42 and ß60-74Cit60,72,74 form two distinct, non-overlapping subfamilies of ACPA that, together, cover practically all the ACPA reactivity to citrullinated fibrinogen and to CCP2 antigens. In established RA, anti-ß60-74Cit60,72,74 autoantibodies show diagnostic indexes similar to those of anti-CCP2.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Citrulina/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/imunologia , Peptídeos Cíclicos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Epitopos , Feminino , Fibrina/imunologia , Fibrinogênio/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Doenças Reumáticas/imunologia , Fator Reumatoide/imunologia , Adulto JovemRESUMO
B-cell depletion therapy (BCDT) based on rituximab (RTX) induces clinical remission in a majority of seropositive patients with Rheumatoid arthritis (RA). However, all patients eventually relapse. The aim of this study was to determine whether dynamic changes in combinations of serological measures of B-cell activation were associated over up to three cycles of BCDT. We included only RA patients who gave an adequate clinical response, as measured by DAS28. Twenty three patients were studied over 1 cycle, 21 over 2, and 15 over 3 cycles of BCDT. Serum analytes including isotypes of Rheumatoid factors (RhF) and anti-citrullinated protein/peptide antibodies (ACPA), B-cell activating factor (BAFF), serum free light chains (SFLC), soluble CD23 (sCD23), antibodies to tetanus toxoid (TT) and to pneumococcal capsular polysaccharide (PCP) were measured by ELISA at 4 key points in each cycle, namely: Baseline (pre-RTX in each cycle); when B-cell depleted (CD19+B-cells < 5/µl); at B-cell return (CD19+B-cells ≥ 5/µl); and at clinical relapse (ΔDAS28 > 1.2). SFLC were used as a measure of plasmablast activity. As sCD23 is cleaved from naïve B-cells coincident with attaining CD27 expression, levels were used as a novel measure of maturation of B-cells to CD27+. The most consistent changes between baseline and B-cell depletion within all 3 cycles were in SFLC, sCD23 and IgM-RhF which fell and in BAFF levels which rose. After 3 complete cycles of BCDT, both IgM autoantibodies and IgG-CCP had decreased, BAFF levels were higher (all p < 0.05); other analytes remained unchanged compared with baseline. Dynamic changes in λSFLC, sCD23, IgM-RhF and BAFF were also consistently associated with relapse in patients with longer clinical responses after B-cell return. Incremental rises in sCD23 levels in cycles 2 and 3 were correlated with time to relapse. Repopulation of the periphery after BCDT is initiated by naïve B-cells and precedes relapse. Our study showed that differentiation into plasmablasts, attended by sCD23 and SFLC production and IgM-RhF specificity may be required to precipitate relapse in patients experiencing longer responses after RTX. These studies also provide novel information related to the resumption of autoimmune responses and their association with B-cell kinetics following BCDT.
Assuntos
Artrite Reumatoide/terapia , Subpopulações de Linfócitos B/imunologia , Depleção Linfocítica , Plasmócitos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Autoanticorpos/sangue , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/patologia , Biomarcadores/metabolismo , Diferenciação Celular , Expressão Gênica , Humanos , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Ativação Linfocitária , Pessoa de Meia-Idade , Plasmócitos/efeitos dos fármacos , Plasmócitos/patologia , Receptores de IgE/genética , Receptores de IgE/imunologia , Recidiva , Indução de Remissão , Rituximab , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
BACKGROUND: Peeling skin disease (PSD), a generalized inflammatory form of peeling skin syndrome, is caused by autosomal recessive nonsense mutations in the corneodesmosin gene (CDSN). OBJECTIVES: To investigate a novel mutation in CDSN. METHODS: A 50-year-old white woman showed widespread peeling with erythema and elevated serum IgE. DNA sequencing, immunohistochemistry, Western blot and real-time polymerase chain reaction analyses of skin biopsies were performed in order to study the genetics and to characterize the molecular profile of the disease. RESULTS: Histology showed hyperkeratosis and acanthosis of the epidermis, and inflammatory infiltrates in the dermis. DNA sequencing revealed a homozygous mutation leading to a premature termination codon in CDSN: p.Gly142*. Protein analyses showed reduced expression of a 16-kDa corneodesmosin mutant in the upper epidermal layers, whereas the full-length protein was absent. CONCLUSIONS: These results are interesting regarding the genotype-phenotype correlations in diseases caused by CDSN mutations. The PSD-causing CDSN mutations identified heretofore result in total corneodesmosin loss, suggesting that PSD is due to full corneodesmosin deficiency. Here, we show for the first time that a mutant corneodesmosin can be stably expressed in some patients with PSD, and that this truncated protein is very probably nonfunctional.
Assuntos
Códon sem Sentido/genética , Dermatite Esfoliativa/genética , Glicoproteínas/genética , Dermatopatias Genéticas/genética , Feminino , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Different methods exist to demonstrate anti-citrullinated protein/peptide antibodies (ACPA). AIMS: To evaluate discrepancy between four ACPA tests. PATIENTS AND METHODS: Population 1 consisted of patients with a new diagnostic problem, including 86 patients with rheumatoid arthritis (RA) and 450 patients without RA. Population 2 consisted of 155 patients with RA who had long-standing disease. Population 3 consisted of 188 patients with psoriatic arthritis and in population 4 there were 192 patients with systemic lupus erythematosus. Populations 1 and 2 were tested with the anti-human fibrinogen antibody (AhfibA) test, anti-CCP2 from Eurodiagnostica (CCP2-euro), anti-CCP2 from Pharmacia (CCP2-phar) and anti-CCP3 test by Inova (CCP3). Samples were annotated as discrepant if positive in one and negative in at least one other test. Each discrepant sample was re-analysed in a different run. Populations 3 and 4 were analysed in the CCP2-euro and AhFibA test. RESULTS: In population 1, ACPA positivity was found in 17 of 450 (3.8%) patients without RA; 14 (82%) of these 17 samples were discrepant. In contrast, 61 of 86 (70.9%) patients with RA were ACPA positive of whom 18 of 61 (29.5%) were discrepant (70.9% vs. 29.5%, p<0.001). The discrepancies between tests could be partly attributed to borderline results, inter-assay discrepancy and inter-test variability. They were more prevalent in patients with systemic lupus erythematosus who were ACPA positive than in those with psoriatic arthritis who were ACPA positive. CONCLUSIONS: Discrepancy between different ACPA tests was observed attributable to the occurrence of borderline results, inter-assay variability and mainly to inter-test variability. The lowest inter-test discrepancy is observed between tests that use the same substrate.
Assuntos
Artrite Reumatoide/diagnóstico , Autoanticorpos/sangue , Peptídeos Cíclicos/imunologia , Artrite Psoriásica/diagnóstico , Artrite Psoriásica/imunologia , Artrite Reumatoide/imunologia , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Desquamation occurs after proteolysis of corneodesmosomal proteins, including corneodesmosin (CDSN), by proteases of the kallikrein family, particularly KLK7. Impaired desquamation is one of the features of psoriasis, and psoriasis-associated single nucleotide polymorphisms of the CDSN gene may potentially modify the proteolysis of the encoded protein. OBJECTIVES: To test whether the proteolysis of CDSN and other corneodesmosomal components is altered in psoriatic epidermis. METHODS: Total protein extracts obtained by tape-stripping of nonlesional and lesional skin from 11 patients were compared by immunoblotting experiments. RESULTS: An almost intact form of CDSN that has never been observed previously in the normal upper stratum corneum was detected in the lesional skin extracts, showing an altered proteolytic processing of the protein. This form was also observed in the nonlesional skin extracts, but in lower amounts. For most patients, increased amounts of desmoglein 1, plakoglobin and of high molecular weight fragments of desmocollin 1 were detected in the lesional skin. For most of them, similar amounts of KLK7 were immunodetected in both nonlesional and lesional skin extracts. No particular differences were observed related to the psoriasis type, the HLA-Cw6 status of the patients or any particular CDSN polymorphisms. CONCLUSIONS: We detected a near full-length form of CDSN that has not been previously observed in normal stratum corneum. The results suggest a reduced degradation of all corneodesmosomal proteins in psoriatic lesions which probably reflects the persistence of corneodesmosomes.
Assuntos
Epiderme/metabolismo , Glicoproteínas/metabolismo , Psoríase/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Desmogleína 1/metabolismo , Desmossomos/metabolismo , Feminino , Genótipo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Calicreínas/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could contribute to the understanding of the pathogenesis of this disease and, ultimately, to the development of methods for preventing the autoimmune response.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Proteínas de Filamentos Intermediários/imunologia , Aminoácidos/análise , Animais , Especificidade de Anticorpos , Autoantígenos/química , Eletroforese em Gel Bidimensional , Proteínas Filagrinas , Imunofluorescência , Humanos , Proteínas de Filamentos Intermediários/química , Queratinas/imunologia , Peso Molecular , RatosRESUMO
The so-called antikeratin antibodies (AKA) and the antiperinuclear factor (APF) are the most specific serological markers of RA. Using indirect immunofluorescence, AKA label the stratum corneum of various cornified epithelia and APF the keratohyalin granules of human buccal mucosa epithelium. We recently demonstrated that AKA recognize human epidermal filaggrin. Here, we report the identification of the major APF antigen as a diffuse protein band of 200-400 kD. This protein is seen to be closely related to human epidermal (pro) filaggrin since it was recognized by four antifilaggrin mAbs specific for different epitopes, and since the APF titers of RA sera were found to be correlated to their AKA titers and to their immunoblotting reactivities to filaggrin. Immunoabsorption of RA sera on purified epidermal filaggrin abolished their reactivities to the granules of buccal epithelial cells and to the 200-400-kD antigen. Moreover, antifilaggrin autoantibodies, i.e., AKA, affinity purified from RA sera, were shown to immunodetect the 200-400-kD antigen and to stain these granules. These results indicate that AKA and APF are largely the same autoantibodies. They recognize human epidermal filaggrin and (pro) filaggrin-related proteins of buccal epithelial cells. Identification of the epitopes recognized by these autoantibodies, which we propose to name antifilaggrin autoantibodies, will certainly open new paths of research into the pathophysiology of RA.
Assuntos
Anticorpos Antinucleares/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Proteínas de Filamentos Intermediários/imunologia , Queratinas/imunologia , Especificidade de Anticorpos , Biomarcadores , Western Blotting , Proteínas Filagrinas , Imunofluorescência , Humanos , Peso Molecular , Mucosa Bucal/imunologiaRESUMO
Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin.
Assuntos
Autoanticorpos/imunologia , Serina Endopeptidases/imunologia , Trombina/imunologia , Adulto , Sítios de Ligação , Transtornos da Coagulação Sanguínea/etiologia , Epitopos/análise , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/classificação , Imunoglobulina G/isolamento & purificação , Masculino , Conformação Proteica , Trombina/antagonistas & inibidores , Trombina/metabolismoRESUMO
Deimination (or citrullination) is a recently described post-translational modification, but its consequences are not yet well understood. It is catalysed by peptidylarginine deiminases (PADs). These enzymes transform arginyl residues involved in a peptidyl link into citrullyl residues in a calcium-dependent manner. Several PAD substrates have already been identified like filaggrin and keratins K1 and K10 in the epidermis, trichohyalin in hair follicles, but also ubiquitous proteins like histones. PADs act in a large panel of physiological functions as cellular differentiation or gene regulation. It has been suggested that deimination plays a role in many major diseases such as rheumatoid arthritis, multiple sclerosis, Alzheimer's disease and psoriasis. Five human genes (PADIs), encoding five highly conserved paralogous enzymes (PAD1-4 and 6), have been characterized. These genes are clustered in a single locus, at 1p35-36 in man. Only PAD1-3 are expressed in human epidermis. PADs seem to be controlled at transcriptional, translational and activity levels and they present particular substrate specificities. In this review, we shall discuss these main biochemical, genetic and functional aspects of PADs together with their pathophysiological implications.
Assuntos
Dermatite Esfoliativa/genética , Mutação da Fase de Leitura/genética , Glicoproteínas/genética , Transtornos da Pigmentação/genética , Criança , Consanguinidade , Feminino , Glicoproteínas/deficiência , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Polimorfismo de Nucleotídeo Único , Dermatopatias/congênitoRESUMO
Although several infectious agents and particularly Epstein-Barr virus (EBV) have been suspected to be involved in aetiology of rheumatoid arthritis (RA), their role still remains elusive. Almost 80% of RA sera contain antibodies to citrullinated proteins/peptides. Among them, the autoantibodies to citrullinated human fibrinogen (AhFibA) are composed of two non-cross-reactive subsets directed to immunodominant epitopes borne by the α36-50Cit and ß60-74Cit fibrin peptides. RA sera also contain antibodies towards the citrullinated EBNA35-58Cit peptide derived from the EBNA-1 protein of EBV. Here, using a large cohort of RA patients and controls, we showed that for a diagnostic specificity of 98.5%, 47% of the AhFibA-positive patients were anti-EBNA35-58Cit-positive and that almost all (98.5%) the anti-EBNA35-58Cit-positive were AhFibA-positive, whereas 86% were anti-ß60-74Cit-positive and only 43% anti-α36-50Cit-positive. AhFibA, anti-EBNA35-58Cit- and anti-ß60-74Cit-antibody titres were significantly correlated. Competition assays showed that anti-EBNA35-58Cit antibodies are highly cross-reactive with the ß60-74Cit peptide. The demonstration that a citrullinated peptide derived from the EBNA-1 protein of EBV presents a molecular mimicry with human citrullinated fibrin constitutes an additional argument for a possible role of EBV in RA aetiopathogeny.
Assuntos
Anticorpos/imunologia , Artrite Reumatoide/imunologia , Reações Cruzadas/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Fibrina/imunologia , Epitopos Imunodominantes/imunologia , Peptídeos/imunologia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Antígenos Nucleares do Vírus Epstein-Barr/química , Fibrina/química , HumanosRESUMO
Atopic dermatitis is a chronic inflammatory skin disease with defects in the epidermal barrier. In a cohort of African-American children, a FLG2 nonsense mutation has been associated with the disease. In the epidermis of European patients, the expression of filaggrin-2, the filaggrin-related protein encoded by FLG2, is decreased. To describe the function of filaggrin-2 and evaluate the impact of its deficiency, its expression was downregulated using lentivirus-mediated shRNA interference in a three-dimensional reconstructed human epidermis (RHE) model. This resulted in parakeratosis and a compact stratum corneum, presence of abnormal vesicles inside the corneocytes, increased pH and reduced amounts of free amino acids at the RHE surface, leading to increased sensitivity to UVB radiations. The expression of differentiation markers was slightly modified. However, we observed reduced proteolytic processing of corneodesmosin, hornerin and filaggrin in parallel with reduced amounts of caspase-14 and bleomycin hydrolase. Our data demonstrated that filaggrin-2 is important for a proper cornification and a functional stratum corneum. Its downregulation in atopic patients may be involved in the disease-associated epidermis impairment.
Assuntos
Epiderme/metabolismo , Proteínas S100/metabolismo , Western Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Proteínas Filagrinas , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Filamentos Intermediários/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas S100/genéticaRESUMO
This paper presents a study of autoantibodies (autoAB) to keratins and to epidermis by a double approach associating a specific immunoenzymatic technique and immunofluorescence. The existence of natural autoAB to keratins in all normal human sera was asserted and the heterogeneity of natural autoAB to the epidermis explored. By a sensitive enzyme-linked immunosorbent assay we detected natural IgM and IgG autoAB to keratin polypeptides extracted from human plantar stratum corneum (SC) in 60 randomly selected normal human sera. The interindividual variation factors of their titers were about 100X in IgM and 50X in IgG antikeratin (AK) autoAB. The IgM and IgG AK autoAB titers varied independently. By a semiquantitative indirect immunofluorescence assay we detected in these sera IgM and IgG autoAB that labeled normal epidermis according to various morphologic patterns. The IgG autoAB labeled SC and suprabasal layers (SBL) in 57.4% of sera, SC in 20.4% and SBL in 7.4%. The IgM autoAB labeled SC and SBL in 52% of sera, SC in 24%, SC and SBL plus basal layer (BL) in 18%, and SBL in 2%. Like the titers, the patterns of IgM and IgG autoAB to epidermis were found to be unrelated. The IgG AK autoAB titers were found to significantly correlate only with the IgG autoAB directed to SC + SBL; the IgM AK autoAB titers only with the IgM autoAB directed to SC + SBL + BL. This showed that these patterns of labeling are typical for AK autoAB and that autoAB to SC, which could not be related to AK autoAB, exist in some normal sera. Antikeratin and antiepidermis IgM autoAB titers were found to be strongly correlated to total amounts of IgM assayed by radial immunodiffusion, indicating that the synthesis of these natural IgM autoAB vary in the same way as that of general IgM synthesis. For the AK and antiepidermis IgG autoAB, however, the same correlation to total serum IgG was found to be much weaker.
Assuntos
Autoanticorpos/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Queratinas/imunologia , Adulto , Ensaio de Imunoadsorção Enzimática , Epiderme/imunologia , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
In order to determine the kinetics of epidermo-dermal junction (EDJ) regeneration during would healing, we studied the regeneration of five EDJ components during reepidermization. Cutaneous wounds (50-mm length, 2-mm width, and 5-mm depth) were produced on the flank area of two pigs and left unsutured. Daily biopsies from day 1 to day 20 were studied by light microscopy on paraffin-embedded sections and by indirect immunofluorescence on cryostat sections using human sera to bullous pemphigoid antigen (BPA) with specificity previously confirmed by indirect immuno-electron microscopy, rabbit antisera to type IV collagen (Coll IV) and to fibronectin, and the monoclonal antibodies (MoAb) 4C 12-8 to laminin and NP-76 to type VII collagen (Coll VII). Histologically, reepidermization started from day 1 and progressed unidirectionally and exclusively from the wound edges. Up to day 9, the distal tips of the neo-epidermal tongues generally extended between the crust and the granulation tissue (GT). They fused on day 10, restoring epidermal continuity. For each EDJ component, the date of appearance (emergence), the spreading under the neo-epidermis tongue (expression), and the morphologic aspect of the labeling were studied. BPA and Coll IV were detected from day 1 to day 20 and found to be expressed all along the neo-EDJ. Fibronectin and laminin were detected from day 1, were present in the proximal and median zones of the neo-EDJ before day 7, up to the distal tip from day 7 to day 9 and were all along the neo-EDJ from day 10 to day 20. Coll VII was only detected from day 3. It was present in the proximal zone on day 3 and day 4, in the proximal and median zones on day 5 and day 6, than all along the neo-EDJ from day 7 to day 20. From day 10, all the labeling characteristics of the five components were found to be similar in the neo-EDJ and in the normal EDJ. With regard to the neo-epidermis progression, we found a synchronism of emergence and expression for BPA and Coll IV, a synchronism of emergence but a delay of expression for fibronectin and laminin and lastly, a delay of emergence and expression for Coll VII. We concluded that BPA and Coll IV could constitute the framework on which the neo-EDJ is progressively built by adjunction of the other components, restitution being obtained just after epidermal continuity is restored.
Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto , Epiderme/química , Proteínas do Tecido Nervoso , Colágenos não Fibrilares , Regeneração , Pele/química , Cicatrização , Animais , Autoantígenos/análise , Colágeno/análise , Distonina , Epiderme/fisiologia , Epiderme/ultraestrutura , Feminino , Fibronectinas/análise , Imunofluorescência , Imuno-Histoquímica , Laminina/análise , Microscopia Imunoeletrônica , Pele/ultraestrutura , Fenômenos Fisiológicos da Pele , Suínos , Colágeno Tipo XVIIRESUMO
To understand the biochemical abnormalities that underlie the reduced desquamation observed in dry skin, we analyzed corneodesmosome degradation in normal and winter xerosis skin. Western blotting of total proteins from corneocytes obtained by varnish-strippings from the legs of 56 volunteers with normal (26) or xerotic (30) skin was performed using antibodies specific for (corneo)desmosome proteins. In the whole population, the amounts of desmoglein 1 and plakoglobin were found to be correlated, but were not related to the amounts of corneodesmosin. This suggests simultaneous proteolysis for the former proteins differing from that of corneodesmosin. Neither entire desmoplakins nor any proteolysis-derived fragments were detected. The amounts of corneodesmosin, desmoglein 1, and plakoglobin detected were found to be significantly higher in xerotic compared with normal skin extracts. Conventional and freeze-fracture electron microscopy showed the absence of nonperipheral corneodesmosomes in the upper stratum corneum of normal skin but the presence of a significant number of these structures in the same layer of winter xerosis skin. These results provide a more precise description of the proteolysis of corneodesmosome components in the upper cornified layer of the epidermis. They support previous studies demonstrating the importance of corneodesmosome degradation in desquamation and reveal that the nonperipheral corneodesmosomes, which are totally degraded during maturation of the stratum corneum in normal skin, persist in winter xerosis, probably leading to abnormal desquamation.
Assuntos
Desmossomos/ultraestrutura , Dermatopatias/patologia , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas/análise , Estações do Ano , Pele/química , Pele/ultraestruturaRESUMO
Little is known about the process leading to desquamation in cornified epithelia. We describe late differentiation antigens (Ag) specific for human cornified squamous epithelia, defined by two murine monoclonal antibodies (MoAb), G36-19 and B17-21, produced after immunization with plantar stratum corneum (SC). Histologically, in epidermis both Ag are cytoplasmic in the lower stratum granulosum (SG), become pericellular in the upper SG, and progressively disappear in the lower SC. In contrast, they persist up to the desquamating corneocytes in the palmoplantar epidermis and hard palate epithelium, as well as in the three cornified epithelial components of the inner root sheath (IRS) of the hair follicle (HF). Cytologically, both Ag are expressed as surface spots only on rough corneocytes. They are largely preserved on cross-linked envelopes (CLE) of the fragile type. Ultrastructurally, both Ag appear in keratinosome-like cytoplasmic vesicles in the upper stratum spinosum (SS) and the SG keratinocytes, then are found in both the regular and reorganizing desmosomes of the SG keratinocytes, and lastly in the corneocyte-specific reorganized desmosomes we propose to name corneodesmosomes. On CLE, the Ag are located on fibrils gathered over the external side of the envelope. Immunochemically, the G36-19--defined epitope is sequential and shared by five non-cytokeratin protein antigens of molecular weight 33.5, 36.5, 40, 49, and 52 kD, the higher molecular weight polypeptides being possibly precursors of the 33.5-kD protein. In contrast, the B17-21 epitope, unaccessible by immunoblotting, is probably conformational. In long-term cultured keratinocytes, the Ag are only expressed when epidermal sheets are morphologically differentiated. The expression is enhanced in the absence of fetal calf serum (FCS) and of epidermal growth factor (EGF). G36-19 and B17-21 Ag participate in a corneodesmosome-CLE superstructure that is probably involved in corneocyte cohesiveness and partly responsible for the mechanical resistance of the SC. These Ag are relevant markers for studying desmosomal maturation during epidermal differentiation and desquamation.
Assuntos
Antígenos de Diferenciação/análise , Desmossomos/imunologia , Animais , Anticorpos Monoclonais , Desmossomos/ultraestrutura , Epitélio/imunologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Queratinócitos/imunologia , Queratinócitos/ultraestrutura , Camundongos/imunologia , Pele/imunologia , Pele/ultraestruturaRESUMO
To improve understanding of human profilaggrin processing to filaggrin, we produced seven monoclonal antibodies against epidermal filaggrin (AHF1-7). They were characterized on human epidermis by indirect immunofluorescence, immunogold labeling, and immunoblotting and found to be directed against seven different epitopes of (pro)filaggrin. AHF1-5 labeled the keratohyalin granules and the fibrous matrix of the lower corneocytes, and recognized filaggrin and profilaggrin. AHF6 also labeled the keratohyalin granules and the corneocyte matrix, but only recognized filaggrin. In addition to this reactivity within the upper epidermis, AHF4-6 stained the cytoplasm of the basal cells, and cross-reactivity of AHF5 and AHF6 with cytokeratin K14 was revealed on immunoblots. It is interesting that AHF7 recognized filaggrin, but not profilaggrin, and labeled only the corneocyte matrix and not the keratohyalin granules. This indicates that filaggrin and cytokeratins share several antigenic determinants and that filaggrin bears at least one epitope absent from its precursor. The original series of monoclonal antibodies described here appears to be a powerful tool for studying human profilaggrin processing in normal conditions and in the keratinization disorders in which processing is altered.
Assuntos
Anticorpos Monoclonais/imunologia , Epiderme/metabolismo , Proteínas de Filamentos Intermediários/imunologia , Proteínas de Filamentos Intermediários/metabolismo , Precursores de Proteínas/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas Filagrinas , Humanos , Immunoblotting , Proteínas de Filamentos Intermediários/química , Fragmentos de Peptídeos/imunologia , Distribuição TecidualRESUMO
The nature of the aging process has been the subject of considerable speculation. Now, some data indicate that free radical reactions going on continuously in the cells contribute to aging. Considering these data, we have investigated the activity of enzymes (catalase, glutathione peroxidase, superoxidismutase) present physiologically in the cell to limit to tolerable levels, the rate of free radicals or H2O2. These enzymes activities were assayed in Paramecium tetraurelia as clonal age increased. Catalase activity increases slightly during aging of paramecia, i.e. during maturity and senescence phases (20-150 fissions). No significant changes in glutathione peroxidase and superoxidismutase is found. Catalase activity was also assayed as a function of culture conditions. As the cells begin starving and the percentage of autogamous cells increases, catalase activity decreases. After autogamy, a large increase of catalase activity occurs during the sexual immaturity phase, i.e. during the first 20 fissions. By another way, H2O2 added in the culture medium (from 0 to 15 X 10(-5)M) causes an important increase of catalase activity (from 100 U.I. to 250 U.I.). The possible role of O-.2, OH. and H2O2 in aging is discussed.
Assuntos
Catalase/metabolismo , Glutationa Peroxidase/metabolismo , Paramecium/enzimologia , Superóxido Dismutase/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Paramecium/citologia , Fatores de TempoRESUMO
Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.