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1.
Br J Dermatol ; 180(5): 1099-1106, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30315657

RESUMO

BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease. Two antigens have been identified as targets of circulating autoantibodies (autoAbs) - BP180 and BP230 - with BP180 being a critical transmembrane adhesion protein of basal keratinocytes of the epidermis. The noncollagenous domain 16A (NC16A) of BP180 is the immunodominant epitope in patients with BP, and anti-BP180-NC16A IgG antibodies (Abs) correlate to disease activity. Routine serological testing and follow-up of BP relies on indirect immunofluorescence (IIF) of serum Abs, commonly performed on monkey oesophagus (ME), and/or enzyme-linked immunosorbent assay (ELISA) testing on recombinantly produced fragments of BP180 and BP230 (BP180-NC16A, BP230-C/N). OBJECTIVES: To determine if NC16A epitopes are well represented on ME substrate. METHODS: Sera from different BP cohorts were tested by IIF on ME and normal human skin (NHS). To confirm findings, affinity-purified anti-BP180-NC16A/BP230 polyclonal Abs and recombinant anti-BP180-NC16A/BP230 monoclonal antibodies (mAbs) were used. RESULTS: For sensitive detection of BP180-NC16A-specific IgG Abs, sections of NHS are superior to the widely used ME. Confirmation comes from polyclonal affinity-purified anti-BP180-NC16A/BP230 Abs, and by mAbs cloned from a patient with active BP. CONCLUSIONS: Use of NHS is preferable over ME in routine IIF testing for BP. These results are of clinical relevance because anti-BP180-NC16A IgG titres are correlated to disease activity and detecting them reliably is important for screening, diagnosis and follow-up of patients with BP.


Assuntos
Autoanticorpos/sangue , Autoantígenos/imunologia , Esôfago/imunologia , Colágenos não Fibrilares/imunologia , Penfigoide Bolhoso/diagnóstico , Pele/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Autoanticorpos/imunologia , Autoanticorpos/isolamento & purificação , Epitopos/imunologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo/métodos , Haplorrinos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Penfigoide Bolhoso/sangue , Penfigoide Bolhoso/imunologia , Domínios Proteicos/imunologia , Colágeno Tipo XVII
2.
Br J Dermatol ; 180(1): 149-156, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30216412

RESUMO

BACKGROUND: Antilaminin 332 mucous membrane pemphigoid (MMP) is an autoimmune subepidermal blistering disease with predominant mucosal involvement and autoantibodies against laminin 332. Malignancies have been associated with this disease; however, no standardized detection system for antilaminin 332 serum antibodies is widely available. OBJECTIVES: Development of a sensitive and specific assay for the detection of antilaminin 332 antibodies. METHODS: An indirect immunofluorescence (IF) assay using recombinant laminin 332 was developed and probed with a large number of antilaminin 332 MMP patient sera (n = 93), as well as sera from patients with antilaminin 332-negative MMP (n = 153), bullous pemphigoid (n = 20), pemphigus vulgaris (n = 20) and noninflammatory dermatoses (n = 22), and healthy blood donors (n = 100). RESULTS: In the novel IF assay, sensitivities with the laminin 332 heterotrimer and the individual α3, ß3 and γ2 chains were 77%, 43%, 41% and 13%, respectively, with specificities of 100% for each substrate. The sensitivity for the heterotrimer increased when an anti-IgG4 enriched antitotal IgG conjugate was applied. Antilaminin 332 reactivity paralleled disease activity and was associated with malignancies in 25% of patients with antilaminin 332 MMP. CONCLUSIONS: The novel IF-based assay will facilitate the serological diagnosis of antilaminin 332 MMP and may help to identify patients at risk of a malignancy.


Assuntos
Autoanticorpos/sangue , Autoantígenos/imunologia , Moléculas de Adesão Celular/imunologia , Penfigoide Mucomembranoso Benigno/diagnóstico , Autoanticorpos/imunologia , Estudos de Coortes , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Penfigoide Mucomembranoso Benigno/sangue , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Calinina
3.
J Neuroinflammation ; 14(1): 123, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28645295

RESUMO

BACKGROUND: Autoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS). METHODS: The target antigen was identified by histo-immunoprecipitation using the patients' sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells. RESULTS: Serum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors. CONCLUSIONS: Autoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1-2% of patients with bona fide MS.


Assuntos
Autoanticorpos/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Esclerose Múltipla/metabolismo , Adulto , Animais , Autoanticorpos/imunologia , Feminino , Células HEK293 , Humanos , Masculino , Microdomínios da Membrana/imunologia , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Ratos , Suínos
4.
BMC Neurol ; 16(1): 172, 2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27623618

RESUMO

BACKGROUND: Myasthenia gravis is an autoimmune neuromuscular disorder, which has only rarely been reported to co-manifest with myositis. The diagnosis of concomitant myositis in patients with myasthenia gravis is clinically challenging, and requires targeted investigations for the differential diagnosis, including EMG, autoantibody assays, muscle biopsy and, importantly, imaging of the mediastinum for thymoma screening. CASE PRESENTATION: This report presents a case-vignette of a 72-year-old woman with progressive proximal muscle weakness and myalgias, diagnosed with thymoma-associated myasthenia and bioptically verified granulomatous myositis, with positive autoantibody status for ryanodine receptor and titin antibodies. CONCLUSIONS: The diagnosis of concurrent myositis and myasthenia gravis, especially in the presence of ryanodine receptor and titin antibodies, should lead neurologists to adopt different treatment strategies compared to those applied in myasthenia or myositis alone. Moreover, further evidence is warranted that titin and, particularly, ryanodine receptor antibodies may co-occur or be pathophysiologically involved in myasthenia-myositis cases.


Assuntos
Autoanticorpos/imunologia , Conectina/imunologia , Miastenia Gravis/complicações , Miastenia Gravis/imunologia , Miosite/complicações , Miosite/imunologia , Canal de Liberação de Cálcio do Receptor de Rianodina/imunologia , Timoma/complicações , Idoso , Feminino , Humanos , Timoma/imunologia
5.
J Neurol ; 271(7): 4660-4671, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38609667

RESUMO

BACKGROUND: Myelin oligodendrocyte glycoprotein antibody-associated encephalomyelitis (MOG-EM; also termed MOG antibody-associated disease, MOGAD) is the most important differential diagnosis of both multiple sclerosis and neuromyelitis optica spectrum disorders. A recent proposal for new diagnostic criteria for MOG-EM/MOGAD explicitly recommends the use of immunoglobulin G subclass 1 (IgG1)- or IgG crystallizable fragment (Fc) region-specific assays and allows the use of heavy-and-light-chain-(H+L) specific assays for detecting MOG-IgG. By contrast, the utility of MOG-IgG3-specific testing has not been systematically evaluated. OBJECTIVE: To assess whether the use of MOG-IgG3-specific testing can improve the sensitivity of MOG-IgG testing. METHODS: Re-testing of 22 patients with a definite diagnosis of MOG-EM/MOGAD and clearly positive MOG-IgG status initially but negative or equivocal results in H+L- or Fc-specific routine assays later in the disease course (i.e. patients with spontaneous or treatment-driven seroreversion). RESULTS: In accordance with previous studies that had used MOG-IgG1-specific assays, IgG subclass-specific testing yielded a higher sensitivity than testing by non-subclass-specific assays. Using subclass-specific secondary antibodies, 26/27 supposedly seroreverted samples were still clearly positive for MOG-IgG, with MOG-IgG1 being the most frequently detected subclass (25/27 [93%] samples). However, also MOG-IgG3 was detected in 14/27 (52%) samples (from 12/22 [55%] patients). Most strikingly, MOG-IgG3 was the predominant subclass in 8/27 (30%) samples (from 7/22 [32%] patients), with no unequivocal MOG-IgG1 signal in 2 and only a very weak concomitant MOG-IgG1 signal in the other six samples. By contrast, no significant MOG-IgG3 reactivity was seen in 60 control samples (from 42 healthy individuals and 18 patients with MS). Of note, MOG-IgG3 was also detected in the only patient in our cohort previously diagnosed with MOG-IgA+/IgG- MOG-EM/MOGAD, a recently described new disease subvariant. MOG-IgA and MOG-IgM were negative in all other patients tested. CONCLUSIONS: In some patients with MOG-EM/MOGAD, MOG-IgG is either exclusively or predominantly MOG-IgG3. Thus, the use of IgG1-specific assays might only partly overcome the current limitations of MOG-IgG testing and-just like H+L- and Fcγ-specific testing-might overlook some genuinely seropositive patients. This would have potentially significant consequences for the management of patients with MOG-EM/MOGAD. Given that IgG3 chiefly detects proteins and is a strong activator of complement and other effector mechanisms, MOG-IgG3 may be involved in the immunopathogenesis of MOG-EM/MOGAD. Studies on the frequency and dynamics as well as the clinical and therapeutic significance of MOG-IgG3 seropositivity are warranted.


Assuntos
Autoanticorpos , Imunoglobulina G , Glicoproteína Mielina-Oligodendrócito , Humanos , Glicoproteína Mielina-Oligodendrócito/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Autoanticorpos/sangue , Sensibilidade e Especificidade , Adulto Jovem , Idoso , Encefalomielite/diagnóstico , Encefalomielite/imunologia , Encefalomielite/sangue
6.
Nervenarzt ; 84(4): 471-6, 2013 Apr.
Artigo em Alemão | MEDLINE | ID: mdl-23568169

RESUMO

Modern diagnostics for the determination of neurologically relevant autoantibodies are based on indirect immunofluorescence using tissue sections of the hippocampus, cerebellum and other tissues. For monospecific detection human embryonic kidney (HEK) cells transfected with different neurological antigens are used. Biochip mosaics are designed to give a quick overview and contain 20 or more substances positioned next to each other on a reaction field, which are incubated with the serum or cerebrospinal fluid (CSF) sample. Western blots based on cerebellum or hippocampus extracts or line blots containing defined recombinant antigens are used additionally. Initial investigations should always comprise the parallel analysis of all major antineural autoantibodies instead of performing only single parameter tests. Up until a few years ago autoantibodies against intracellular neuronal antigens were mainly investigated. Antibodies against structures of the neural cell surface, however, are much more frequently found, especially those against glutamate receptors (type NMDA).


Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes do Sistema Nervoso/diagnóstico , Doenças Autoimunes do Sistema Nervoso/imunologia , Encefalomielite/diagnóstico , Encefalomielite/imunologia , Imunoensaio/tendências , Imunoterapia/tendências , Antígenos/imunologia , Bioensaio/tendências , Biomarcadores/sangue , Humanos , Proteínas Recombinantes/imunologia
7.
Br J Dermatol ; 166(5): 964-70, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22242606

RESUMO

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by circulating autoantibodies against BP180 and BP230. For BP180, the NC16A domain has previously been identified as the main antigenic target in BP, while data about the diagnostic value of epitopes on BP230 were inconclusive. OBJECTIVES: To identify the most appropriate epitopes on BP230 to be applied in a simple, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for routine detection of serum autoantibodies. METHODS: Ten overlapping linear fragments covering the whole length of BP230 were expressed in Escherichia coli. Based on Western blot analysis with sera from patients with BP (n = 49) and healthy controls (n = 94), the diagnostic performance of the fragments was compared by receiver operating characteristics curve analysis. The BP230-C3 fragment comprising the C-terminal portion (amino acids 2326-2649) was subsequently applied in a novel ELISA. The operating characteristics of this ELISA were analysed by probing sera from patients with BP (n = 118), pemphigus vulgaris (n = 50), rheumatoid arthritis and other inflammatory arthritides (n = 170), and systemic lupus erythematosus (n = 56), and from healthy blood donors (n = 483). RESULTS: Among all the fragments, BP230-C3 provided the best efficiency in serologically diagnosing BP by Western blot. An ELISA employing BP230-C3 revealed a diagnostic sensitivity of 56·8% and specificity of 97·6%. Its diagnostic added value amounted to 4·2% compared with the anti-BP180-NC16A-4X ELISA alone. CONCLUSIONS: Recombinant BP230-C3 is a suitable target antigen for the detection of serum autoantibodies against BP230.


Assuntos
Autoanticorpos/metabolismo , Mapeamento de Epitopos/métodos , Glicoproteínas de Membrana/metabolismo , Penfigoide Bolhoso/imunologia , Autoanticorpos/imunologia , Western Blotting , Proteínas de Transporte , Estudos de Casos e Controles , Proteínas do Citoesqueleto , Distonina , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Proteínas do Tecido Nervoso , Penfigoide Bolhoso/diagnóstico , Curva ROC , Proteínas Recombinantes
8.
J Neurol ; 268(10): 3758-3765, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33770235

RESUMO

BACKGROUND: A positive MRZ reaction, as defined by intrathecal IgG production against at least two of its constituents, measles virus (M), rubella virus (R) and varicella zoster virus (Z), is detectable in ~ 63% of patients with multiple sclerosis (MS) and is currently considered the laboratory marker with the highest specificity and positive likelihood ratio for MS. However, M, R and Z are only the most well-established constituents of a broader intrathecal humoral immune response in MS. OBJECTIVE: To identify additional anti-microbial antibodies inclusion of which in the classical MRZ panel may result in increased sensitivity without compromising the marker's high specificity for MS. METHODS: We determined the antibody indices (AIs) for 11 viral and bacterial agents (M, R, Z, herpes simplex virus, Epstein-Barr virus, mumps virus, cytomegalovirus, parvovirus B19, Bordetella pertussis, Corynebacterium diphtheriae, and Clostridium tetani) in paired cerebrospinal fluid and serum samples from patients with MS and disease controls. RESULTS: A positive 'classical' MRZ reaction was found in 17/26 (65.4%) MS patients. The five most frequently positive AIs among patients with MS were M (76.9%), Z (61.5%), R (57.7%), parvovirus B19 (42.3%), and mumps (28%). Addition of parvovirus B19 and mumps virus to the MRZ panel resulted in an increase in sensitivity in the MS group from 65.4% to 73.1%, with 22% of the initially MRZ-negative patients exhibiting a de novo-positive response. The extended MRZ panel ('MRZplus') distinguished sharply between MS (≥ 3 AIs in 90% of all positives) and controls (varying diagnoses, from migraine to vasculitis; 0-1 AIs; p < 0.000001). The highest median AI in the MS group was found for parvovirus B19 (3.97), followed by measles virus (2.79). CONCLUSION: Inclusion of parvovirus B19 and mumps virus in the test panel resulted in an increase in the sensitivity and discriminatory power of MRZ. Our results provide a strong rational for prospective studies investigating the role of extended MRZ panels in the differential diagnosis of MS.


Assuntos
Infecções por Vírus Epstein-Barr , Esclerose Múltipla , Parvovirus B19 Humano , Anticorpos Antivirais , Herpesvirus Humano 4 , Humanos , Imunidade , Esclerose Múltipla/diagnóstico , Vírus da Caxumba , Estudos Prospectivos
9.
Ann Rheum Dis ; 68(2): 228-33, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18375539

RESUMO

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) with a C-ANCA or P-ANCA pattern are detected in ANCA-associated vasculitis (AAV). While in most patients with AAV a C-ANCA pattern is due to reactivity with proteinase-3 (PR3)-ANCA, some C-ANCA-positive sera do not react with PR3. OBJECTIVE: The development and evaluation of a direct enzyme-linked immunosorbent assay (ELISA) for PR3-ANCA with increased sensitivity. METHODS: A mixture of human native (hn) and human recombinant (hr) PR3 was used as antigen coating. The resulting ELISA (anti-PR3-hn-hr) was compared with ELISAs using directly coated hn-PR3 or hr-PR3, as well as with a hn-PR3 capture ELISA. Assay characteristics were determined in patients with AAV (n = 248), with special attention for those patients with C-ANCA (n = 132), as well as disease controls (n = 585) and healthy controls (n = 429). Additionally, for prediction of relapses serial samples of 46 patients with PR3-AAV were analysed. RESULTS: At a predefined specificity of 99% both ELISAs containing hr-PR3 revealed a substantial increase in sensitivity. For the prediction of relapses by rises in PR3-ANCA titres the capture ELISA was most optimal (odds ratio 12.5). With an odds ratio of 8.9 the novel anti-PR3-hn-hr ELISA was second best. CONCLUSIONS: Owing to the very high sensitivity of the novel anti-PR3-hn-hr ELISA for the detection of PR3-ANCA in C-ANCA-positive samples of patients with AAV this assay has an excellent diagnostic performance. This feature is combined with a good predictability of clinical relapses in patients with PR3-AAV. These characteristics challenge the dogma that, for detection of PR3-ANCA, capture ELISAs are superior for diagnosis and follow-up.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/sangue , Doenças Autoimunes/diagnóstico , Mieloblastina/imunologia , Vasculite/diagnóstico , Doenças Autoimunes/imunologia , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Prognóstico , Proteínas Recombinantes/imunologia , Recidiva , Sensibilidade e Especificidade , Vasculite/imunologia
10.
FEBS Lett ; 487(3): 351-5, 2001 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-11163357

RESUMO

The isolation of a fully functional SoxM terminal oxidase supercomplex from the archaeon Sulfolobus acidocaldarius has failed thus far and several of its constituents have only been predicted genetically, such as the small Cu protein sulfocyanin and the subunit II bearing a Cu(A) center. Here we report the recombinant expression of sulfocyanin and prove its transcription in Sulfolobus as well as its presence in the enriched complex. It reveals a redox potential of +300 mV and spectroscopic features that are characteristic of type I copper centers. It is highly thermostable and firmly attached to the complex by one putative transmembrane anchor. Surprisingly, subunit II is completely missing from the isolated complex and behaves as an easily dissociable constituent which is a unique case within the terminal oxidase family. Its loss into the soluble phase upon cell disruption can be considered the reason for the inactivity of the isolated membrane complex.


Assuntos
Proteínas de Bactérias , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Periplásmicas , Sulfolobus acidocaldarius/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cobre/química , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Complexo IV da Cadeia de Transporte de Elétrons/genética , Estabilidade Enzimática , Imunoquímica , Substâncias Macromoleculares , Potenciais da Membrana , Oxirredução , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometria , Sulfolobus acidocaldarius/genética , Temperatura , Transcrição Gênica
11.
J Bioenerg Biomembr ; 33(1): 27-34, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11460923

RESUMO

The branched respiratory chain of the archaeon Sulfolobus acidocaldarius contains a supercomplex, SoxM, consisting of a bc1-like subcomplex and a terminal oxidase moiety, including a subunit II analogous polypeptide, SoxH. However, the latter component has never been identified in preparations of SoxM. We demonstrate the presence of an mRNA transcript by Northern analysis. We succeeded in cloning and expressing the respective gene with truncated N-terminus by deleting a 20 AS membrane anchor, which resulted in a water-soluble purple copper protein, which was further characterized. The recombinant subunit II of the SoxM complex contains a correctly inserted binuclear CuA cluster as revealed by UV/vis and EPR spectroscopy. The protein is highly thermostable and displays a redox potential of +237 mV. In recombinant form, the metal interacts with cytochrome c as an artificial electron donor; the physiological electron donor is still unknown, since S. acidocaldarius does not contain any c-type cytochromes. The purple copper center of SoxM shows an interesting pH dependency with a pKa at 6.4, suggesting protonation of the Cu-ligating histidines. Further lowering the pH causes a reversible transition into another cluster form with concomitant liberation of one copper. It may thus provide a model for the study of cluster rearrangements in response to pH.


Assuntos
Proteínas Arqueais , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Sulfolobus acidocaldarius/enzimologia , Sulfolobus acidocaldarius/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Arqueal/genética , Expressão Gênica , Genes Arqueais , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oxirredução , Estrutura Terciária de Proteína , Subunidades Proteicas , RNA Arqueal/genética , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrofotometria
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