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 , CalininaRESUMO
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 , SuínosRESUMO
In 2007, a multifaceted syndrome, associated with anti-NMDA receptor autoantibodies (NMDAR-AB) of immunoglobulin-G isotype, has been described, which variably consists of psychosis, epilepsy, cognitive decline and extrapyramidal symptoms. Prevalence and significance of NMDAR-AB in complex neuropsychiatric disease versus health, however, have remained unclear. We tested sera of 2817 subjects (1325 healthy, 1081 schizophrenic, 263 Parkinson and 148 affective-disorder subjects) for presence of NMDAR-AB, conducted a genome-wide genetic association study, comparing AB carriers versus non-carriers, and assessed their influenza AB status. For mechanistic insight and documentation of AB functionality, in vivo experiments involving mice with deficient blood-brain barrier (ApoE(-/-)) and in vitro endocytosis assays in primary cortical neurons were performed. In 10.5% of subjects, NMDAR-AB (NR1 subunit) of any immunoglobulin isotype were detected, with no difference in seroprevalence, titer or in vitro functionality between patients and healthy controls. Administration of extracted human serum to mice influenced basal and MK-801-induced activity in the open field only in ApoE(-/-) mice injected with NMDAR-AB-positive serum but not in respective controls. Seropositive schizophrenic patients with a history of neurotrauma or birth complications, indicating an at least temporarily compromised blood-brain barrier, had more neurological abnormalities than seronegative patients with comparable history. A common genetic variant (rs524991, P=6.15E-08) as well as past influenza A (P=0.024) or B (P=0.006) infection were identified as predisposing factors for NMDAR-AB seropositivity. The >10% overall seroprevalence of NMDAR-AB of both healthy individuals and patients is unexpectedly high. Clinical significance, however, apparently depends on association with past or present perturbations of blood-brain barrier function.
Assuntos
Autoanticorpos/sangue , Barreira Hematoencefálica/metabolismo , Transtornos do Humor/metabolismo , Doença de Parkinson/metabolismo , Receptores de N-Metil-D-Aspartato/imunologia , Esquizofrenia/metabolismo , Adulto , Idoso , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Córtex Cerebral/metabolismo , Endocitose/fisiologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Influenza Humana/genética , Influenza Humana/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Transtornos do Humor/genética , Neurônios/metabolismo , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único , Receptores de N-Metil-D-Aspartato/genética , Esquizofrenia/genéticaRESUMO
Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies.
Assuntos
Anticorpos Antinucleares/análise , Sistemas Computacionais , Técnica Indireta de Fluorescência para Anticorpo/métodos , Interpretação de Imagem Assistida por Computador/instrumentação , Microscopia de Fluorescência/métodos , Automação , Biomarcadores , Humanos , Armazenamento e Recuperação da Informação , Padrões de Referência , Reprodutibilidade dos Testes , SoftwareRESUMO
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/imunologiaRESUMO
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 RecombinantesRESUMO
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/imunologiaRESUMO
BACKGROUND: Antineuronal antibodies can be associated with both gastrointestinal (GI) and brain disorders. For example, antibodies against the potassium channel subunit dipeptidyl-peptidase-like protein-6 (DPPX) bind to neurons in the central nervous system (CNS) and myenteric plexus and cause encephalitis, commonly preceded by severe unspecific GI symptoms. We therefore investigated the prevalence of antineuronal antibodies indicative of treatable autoimmune CNS etiologies in GI patients. METHODS: Serum samples of 107 patients (Crohn's disease n = 42, ulcerative colitis n = 16, irritable bowel syndrome n = 13, others n = 36) and 44 healthy controls were screened for anti-DPPX and further antineuronal antibodies using immunofluorescence on rat brain and intestine and cell-based assays. Functional effects of high-titer reactive sera were assessed in organ bath and Ussing chamber experiments and compared to non-reactive patient sera. KEY RESULTS: Twenty-one of 107 patients (19.6%) had antibodies against the enteric nervous system, and 22 (20.6%) had anti-CNS antibodies, thus significantly exceeding frequencies in healthy controls (4.5% each). Screening on cell-based assays excluded established antienteric antibodies. Antibody-positive sera were not associated with motility effects in organ bath experiments. However, they induced significant, tetrodotoxin (TTX)-insensitive secretion in Ussing chambers compared to antibody-negative sera. CONCLUSIONS & INFERENCES: Antineuronal antibodies were significantly more frequent in GI patients and associated with functional effects on bowel secretion. Future studies will determine whether such antibodies indicate patients who might benefit from additional antibody-directed therapies. However, well-characterized encephalitis-related autoantibodies such as against DPPX were not detected, underlining their rarity in routine cohorts.
Assuntos
Autoanticorpos/sangue , Gastroenteropatias/sangue , Gastroenteropatias/epidemiologia , Neurônios/metabolismo , Adulto , Idoso , Animais , Biomarcadores/sangue , Feminino , Gastroenteropatias/diagnóstico , Cobaias , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Prevalência , Ratos , Ratos WistarAssuntos
Autoanticorpos/sangue , Laminina/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Lúpus Eritematoso Cutâneo/sangue , Lúpus Eritematoso Sistêmico/sangue , Estrutura Terciária de ProteínaRESUMO
A large number of zinc endopeptidases contain an HEXXHXXGXXH consensus motif in their catalytic site (single letter code; X is any amino acid residue). These enzymes can be grouped into four distinct families, the astacins, the adamalysins, the serralysins and the matrix metalloproteinases (matrixins). Despite a low degree of sequence similarity, their catalytic modules are topologically similar. A topology derived sequence alignment suggests that the four families form a superfamily, called the metzincins because of a perfectly superimposable methionine residue close to the zinc-binding active site. Topological similarity to the thermolysin-like enzymes indicates that these enzymes may have had a common ancestor.
Assuntos
Metaloendopeptidases/química , Zinco/química , Sequência de Aminoácidos , Animais , Humanos , Metaloendopeptidases/classificação , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Análise de SequênciaRESUMO
Astacin, a 200 residue digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype of the "astacin family", which comprises several membrane-bound mammalian endopeptidases and developmentally implicated regulatory proteins. Large trigonal crystals of astacin were grown, and X-ray reflection data to 1.8 A resolution were collected. The astacin structure has been solved by multiple isomorphous replacement using six heavy-atom derivatives, and refined to a crystallographic R-value of 0.158 applying stringent constraints. All 200 residues are clearly defined by electron density; 181 solvent molecules have been localized. Besides the native structure, the structures of Hg-astacin (with a mercury ion replacing the zinc) and of the apoenzyme were also refined. The astacin molecule exhibits a kidney-like shape. It consists of an amino-terminal and a carboxy-terminal domain, with a deep active-site cleft in between. The zinc ion, located at the bottom of this cleft, is co-ordinated in a novel trigonal-bipyramidal geometry by three histidine residues, a tyrosine and by a water molecule, which is also bound to the carboxylate side-chain of Glu93. The amino-terminal domain of astacin consists mainly of two long alpha-helices, one centrally located and one more peripheral, and of a five-stranded pleated beta-sheet. The amino terminus protrudes into an internal, water-filled cavity of the lower domain and forms a buried salt bridge with Glu103; amino-terminally extended pro-forms of astacin are thus not compatible with this structure. The carboxy-terminal domain of astacin is mainly organized in several turns and irregular structures. Because they share sequence identity of about 35%, the structures of the proteolytic domains of the other "astacin" members must be quite similar to astacin. Only a few very short deletions and insertions quite distant from the active-site distinguish their structures from astacin. The five-stranded beta-sheet and the two helices of the amino-terminal domain of astacin are topologically similar to the structure observed in the archetypal zinc-endopeptidase thermolysin; the rest of the structures are, in contrast, completely unrelated in astacin and thermolysin. The zinc ion, the central alpha-helix and the zinc-liganding residues His92, Glu93 and His96 of astacin are nearly superimposable with the respective groups of thermolysin, namely with the zinc ion, the "active-site helix", and His142TL, Glu143TL and His146TL of the zinc-binding consensus motif His-Glu-Xaa-Xaa-His (where Xaa is any amino acid residue).(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Metaloendopeptidases/química , Termolisina/química , Sequência de Aminoácidos , Animais , Apoenzimas/metabolismo , Astacoidea , Sítios de Ligação , Simulação por Computador , Sequência Consenso , Mercúrio , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Solventes , Temperatura , Difração de Raios XRESUMO
BACKGROUND: Antibodies to the Rho GTPase-activating protein 26 (ARHGAP26, GRAF1) (also termed anti-Ca) were first described in patients with cerebellar ataxia. However, ARHGAP26 is also expressed in some hippocampal neurons. Moreover, some of the previously reported patients showed cognitive and affective symptoms. It is unknown whether those symptoms reflected involvement of the limbic system or were part of the so-called cerebellar cognitive/affective syndrome. CASE REPORT: Here, we report a newly diagnosed anti-Ca/ARHGAP26-IgG-positive patient who presented with recurrent psychotic symptoms but no cerebellar ataxia. In addition, low-titer acetylcholine receptor antibodies, voltage-gated potassium channel complex antibodies (but no LGI1 or CASPR2 antibodies) and anti-nuclear antibodies of unknown specificity were detected, suggesting a general autoimmune predisposition. Thymectomy revealed mild thymic nodular hyperplasia. CONCLUSION: This case indicates that the clinical spectrum of ARHGAP26-related autoimmunity might be broader than expected. Studies on the prevalence of anti-Ca/ARHGAP26 in patients with suspected limbic encephalitis seem warranted.
Assuntos
Anticorpos/metabolismo , Proteínas Ativadoras de GTPase/imunologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/imunologia , Transtornos Psicóticos/genética , Adulto , Antipsicóticos/uso terapêutico , Cálcio/metabolismo , Cerebelo/metabolismo , Diazepam/uso terapêutico , Feminino , Hipocampo/metabolismo , Humanos , Imunoglobulinas/uso terapêutico , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/metabolismo , Transtornos Psicóticos/patologia , Risperidona/uso terapêuticoRESUMO
The three-dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin II from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and astacin from crayfish are topologically similar, with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable "Met-turn." This structural relationship is supported by a sequence alignment performed on the basis of topological equivalence showing faint but distinct sequential similarity. The alkaline proteinase is about equally distant (26% sequence identity) to both human neutrophil collagenase and astacin and a little further away from adamalysin II (17% identity). The pairs astacin/adamalysin II, astacin/human neutrophil collagenase, and adamalysin II/human neutrophil collagenase exhibit sequence identities of 16%, 14%, and 13%, respectively. Therefore, the corresponding four distinct families of zinc peptidases, the astacins, the matrix metalloproteinases (matrixins, collagenases), the adamalysins/reprolysins (snake venom proteinases/reproductive tract proteins), and the serralysins (large bacterial proteases from Serratia, Erwinia, and Pseudomonas) appear to have originated by divergent evolution from a common ancestor and form a superfamily of proteolytic enzymes for which the designation "metzincins" has been proposed. There is also a faint but significant structural relationship of the metzincins to the thermolysin-like enzymes, which share the truncated zinc-binding motif HEXXH and, moreover, similar topologies in their N-terminal domains.
Assuntos
Colagenases/química , Metaloendopeptidases/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Sítios de Ligação , Evolução Biológica , Colagenases/metabolismo , Metaloproteinase 8 da Matriz , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Serina Endopeptidases/metabolismoRESUMO
The catalytic zinc of astacin, a prototype of the astacin family and the metzincin superfamily of metalloproteinases is coordinated by three histidines, a glutamate bound water and a tyrosine. In order to assess the roles of active site key residues, two mutants, Glu93Ala-astacin and Tyr149Phe-astacin, were expressed in Escherichia coli, affinity-purified and renatured. While the Glu93Ala mutant was inactive, the Tyr149Phe mutant retained about 2. 5% residual activity toward Dns-Pro-Lys-Arg*Ala-Pro-Trp-Val, based on the k(cat)/K(m) value for recombinant wild-type astacin. These results support a model in which Glu93 is the general base in substrate hydrolysis, whereas Tyr149 contributes to transition state binding.
Assuntos
Ácido Glutâmico , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Tirosina , Zinco/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Escherichia coli , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Desnaturação Proteica , Renaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Zinco/químicaRESUMO
Meprins are astacin-like metalloproteases of renal and intestinal epithelia and embryonic neuroepithelial cells. The full length cDNA of the human meprin alpha subunit has been overexpressed in baculovirus-infected insect cells yielding the tetrameric proprotein which could be proteolytically activated and affinity-purified to homogeneity. Recombinant meprin alpha hydrolyzes the synthetic substrate N-benzoyl-tyrosyl-p-aminobenzoic acid (PABA-peptide) and cleaves by limited proteolysis the basement membrane constituents laminin 1 and laminin 5. This supports a concept that meprin alpha, when basolaterally secreted by human colon carcinoma epithelial cells, increases the proteolytic capacity for tumor progression in the stroma.
Assuntos
Precursores Enzimáticos/genética , Metaloendopeptidases/genética , Animais , Baculoviridae/genética , Membrana Basal/metabolismo , Linhagem Celular , Cromatografia de Afinidade , DNA Complementar/isolamento & purificação , Ativação Enzimática , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/metabolismo , Expressão Gênica , Insetos , Laminina/metabolismo , Metaloendopeptidases/biossíntese , Metaloendopeptidases/metabolismo , TransfecçãoRESUMO
A 12-year-old girl developed a severe inflammatory fungal infection of the scalp caused by Trichophyton verrucosm. The infection resulted in scarring alopecia. The natural history, differential diagnosis, pathogenesis, and treatment of this infection of the skin are reviewed.
Assuntos
Tinha do Couro Cabeludo , Alopecia/etiologia , Criança , Diagnóstico Diferencial , Feminino , Griseofulvina/uso terapêutico , Humanos , Tinha do Couro Cabeludo/complicações , Tinha do Couro Cabeludo/patologiaRESUMO
To investigate the possibility of an autoimmune mechanism in idiopathic bilateral vestibulopathy (IBV), we screened patients' sera for antibodies against inner ear structures. IgG antibodies against membranous labyrinth (ampulla, semicircular canals, saccule and utricle) were detected in 8 of 12 patients by immunofluorescence on rat inner ear cryosections. All but one serum of 22 healthy controls and the sera of 6 patients with known autoimmune disorders showed only background staining. Low-titre anti-nuclear IgM antibodies were present in three control sera and one IBV serum. High-titre anti-nuclear IgM was found in a patient with lupus erythematosus and in one with scleroderma. Anti-nuclear IgM was not organ-specific. No human serum used contained detectable anti-vascular preformed antibodies. Cross-reactivity to sections of liver, kidney, cornea, brain and skeletal muscle was absent. Double-staining for IgG and F-actin, the primary constituent of hair cell cilia, did not show predominant Ig-coating of sensory hair cells. Immunosuppressive therapy in 3 IBV patients did not improve the disorder, probably owing to irreversible loss of sensory and neural structures. These data suggest that the bulk of anti-labyrinthine autoantibodies may be an epiphenomenon, yet a small subgroup of organ-specific autoantibodies may synergize with a cellular response in the development of vestibular lesions.
Assuntos
Autoanticorpos/imunologia , Orelha Interna/imunologia , Doenças Vestibulares/etiologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Doenças Vestibulares/sangue , Doenças Vestibulares/imunologiaRESUMO
Two young women had painful ecchymoses of their extremities. In one, this purpuric syndrome followed a chronic course and resulted in severe joint disability. In the other woman, the problem was transient and relatively benign. Emotional factors in their disease were of major importance. No abnormalities of hemostasis or immune function were found. These cases demonstrate the many problems encountered in the diagnosis and management of such patients.
Assuntos
Transtornos Psicofisiológicos , Púrpura/etiologia , Adulto , Feminino , Humanos , Simulação de Doença , Testes Cutâneos/métodos , Estresse PsicológicoRESUMO
14C-Dopamine at a dose between 0.16 and 400 nmol per kg body weight was injected locally into the renal artery and urinary excretion of the label was followed for a period of up to 75 min. During the first renal passage the injected kidney excreted 28.2+/-8.3% (n = 8) of the activity applied. As shown by column chromatography the 14C-activity in urine was mainly present as 3,4-dihydroxyphenyl acetic acid (40%), homovanillic acid (15%) and dopamine (app. 20%). Excretion rate and the pattern of dopamine metabolites in urine was independent of the administered dose. Thus, the excretion of dopamine by the cat kidney is linked to an inactivation by the kidney enzymes MAO and COMT. From the literature it is known that in dog and chicken kidney catecholamines are not metabolized to such a large extent during renal excretion.
Assuntos
Dopamina/metabolismo , Rim/metabolismo , Animais , Biotransformação , Gatos , Dopamina/urina , Feminino , Rim/enzimologia , Masculino , Monoaminoxidase/metabolismoRESUMO
We report on a 33-year-old male patient with generalized acquired lipodystrophy, insulin resistant diabetes mellitus and acanthosis nigricans (Lawrence Syndrome). First probable symptoms of lipodystrophy (weight loss, shrinkage of subcutaneous fatty tissue, and loss of muscular strength) became evident three years ago, with the onset of diabetes mellitus occurring about six months later. The patient suffered from the following clinical symptoms: IDDM with increasing insulin-requirement, extreme reduction of fatty tissue, fatty liver hepatitis with elevated liver enzymes, glomerulopathy, muscular and neuropathic pains, as well as hypertriglyceridaemia. A basal C-peptide concentration is rather high. Definitely, the endogenous insulin secretion is increased. In other words, insulin resistance is documented. In an effort to identify the pathogenetic mechanisms of lipoatrophic diabetes mellitus in this patient and to develop a therapeutic strategy, antibodies against different tissues and endocrinologic regulation were investigated. It was possible to demonstrate the presence of serum autoantibodies against lipocytes of the subcutis and other tissues, against hepatic stellate cells, together with autoantibodies against different endocrine organs. By studying the basis of diabetic abnormalities relating to the growth hormone (GH), the insulin-like growth factor (IGF) dynamics in this patient, i.e. reductions of GH, IGF-I, IGF-II, IGF-Binding protein (IGF-BP) 2 and IGF-BP 3, were detected. An immunosuppressive treatment strategy was not beneficial.