RESUMEN
BACKGROUND: Differential diagnosis can be a challenge for eosinophilic subtypes of renal cell tumors due to their overlapping histomorphological and immunohistochemical features. We aimed to investigate the frequency of rare variants of renal cell carcinomas (RCCs) such as succinate dehydrogenase-deficient RCC (SDDRCC), hereditary leiomyomatosis and RCC (HLRCC)-associated RCC, and eosinophilic, solid, and cystic RCC (ESCRCC) in our population. MATERIALS AND METHODS: Renal tumors which could be considered in the eosinophilic tumor category were included: 91 conventional clear cell RCCs with eosinophilic cytoplasm, 72 papillary RCCs, 74 chromophobe RCCs, 88 oncocytomas, and 37 other rare subtypes. Using the tissue microarray method, succinate dehydrogenase B (SDHB), fumarate hydratase (FH), and cytokeratin 20 (CK20) antibodies were performed by immunohistochemistry. Immunohistochemistry was repeated on whole block sections for selected cases. The utility of these antibodies in the differential diagnosis was also investigated. RESULTS: Loss of SDHB expression was detected in three tumors, two of which showed typical morphology for SDDRCC. In additional two tumors, SDHB showed weak cytoplasmic expression without a mitochondrial pattern (possible-SDHB deficient). None of the tumors showed loss of FH expression. Heterogeneous reactions were observed with SDHB and FH antibodies. Only one ESCRCC was detected with diffuse CK20 positivity. CONCLUSION: SDDRCCs, HLRCC-associated RCCs, and ESCRCCs are very rare tumors depending on the population. Possible weak staining and focal loss of SDHB and FH expression should be kept in mind and genetic testing must be included for equivocal results.
Asunto(s)
Fumarato Hidratasa/metabolismo , Terapia de Inmunosupresión , Queratina-20/metabolismo , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Succinato Deshidrogenasa/metabolismo , Adulto , Diagnóstico Diferencial , Femenino , Fumarato Hidratasa/efectos de los fármacos , Fumarato Hidratasa/inmunología , Humanos , Terapia de Inmunosupresión/métodos , Queratina-20/inmunología , Neoplasias Renales/diagnóstico , Masculino , Persona de Mediana Edad , Succinato Deshidrogenasa/efectos de los fármacos , Succinato Deshidrogenasa/inmunologíaRESUMEN
Purpose: To evaluate whether baseline titers of anti-fumarase antibody are associated with visual prognosis after anti-VEGF treatment for diabetic macular edema (DME). Methods: In this retrospective study, we investigated 52 eyes of 52 DME patients who received intravitreal injections of anti-VEGF drugs (ranibizumab or aflibercept) after blood sampling at baseline. Optical coherence tomography (OCT) images were obtained at every monthly visit. The serum titer of anti-fumarase antibody at baseline was measured using ELISA. We evaluated the relationship between the titer of anti-fumarase antibody at baseline and visual acuity (VA) improvement at 12 months. Results: The serum titer of anti-fumarase IgG was related to the logMAR visual acuity (VA; R = 0.329, P = 0.017) and the disrupted ellipsoid zone (EZ; R = 0.364, P = 0.008) at baseline. The titer of this autoantibody was not associated with logMAR VA (R = -0.007, P = 0.980) but was associated with VA improvement (R = 0.465, P < 0.001) at 12 months upon anti-VEGF treatment. The transverse length of the disrupted EZ line was shortened at 12 months (P < 0.001), and restoration of the EZ line was correlated to the autoantibody titer (R = 0.396, P = 0.004) compared with the decrease in central subfield (CSF) thickness. Multivariate analysis showed that pretreatment logMAR VA (ß = 0.296, P = 0.045) and the autoantibody titer (ß = 0.328, P = 0.017) were associated with VA improvement after anti-VEGF treatment. In contrast, the titer was not associated with logMAR VA at 12 months. Conclusions: Anti-fumarase antibody is a novel serum biomarker predicting better functional efficacy of anti-VEGF treatment for DME.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Autoanticuerpos/sangre , Biomarcadores/sangre , Retinopatía Diabética/sangre , Fumarato Hidratasa/inmunología , Inmunoglobulina G/sangre , Edema Macular/sangre , Anciano , Retinopatía Diabética/tratamiento farmacológico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inyecciones Intravítreas , Edema Macular/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Ranibizumab/uso terapéutico , Receptores de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Estudios Retrospectivos , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Agudeza Visual/fisiologíaRESUMEN
AIMS/HYPOTHESIS: In diabetic macular oedema (DMO), blood components passing through the disrupted blood-retinal barrier cause neuroinflammation, but the mechanism by which autoantibodies induce neuroglial dysfunction is unknown. The aim of this study was to identify a novel autoantibody and to evaluate its pathological effects on clinically relevant photoreceptor injuries. METHODS: Biochemical purification and subsequent peptide fingerprinting were applied to identify autoantigens. The titres of autoantibodies in DMO sera were quantified and their associations with clinical variables were evaluated. Two animal models (i.e. passive transfer of autoantibodies and active immunisation) were characterised with respect to autoimmune mechanisms underlying photoreceptor injuries. RESULTS: After screening serum IgG from individuals with DMO, fumarase, a Krebs cycle enzyme expressed in inner segments, was identified as an autoantigen. Serum levels of anti-fumarase IgG in participants with DMO were higher than those in diabetic participants without DMO (p < 0.001) and were related to photoreceptor damage and visual dysfunction. Passively transferred fumarase IgG from DMO sera in concert with complement impaired the function and structure of rodent photoreceptors. This was consistent with complement activation in the damaged photoreceptors of mice immunised with fumarase. Fumarase was recruited to the cell surface by complement and reacted to this autoantibody. Subsequently, combined administration of anti-fumarase antibody and complement elicited mitochondrial disruption and caspase-3 activation. CONCLUSIONS/INTERPRETATION: This study has identified anti-fumarase antibody as a serum biomarker and demonstrates that the generation of this autoantibody might be a pathological mechanism of autoimmune photoreceptor injuries in DMO.
Asunto(s)
Autoanticuerpos/inmunología , Retinopatía Diabética/patología , Fumarato Hidratasa/inmunología , Inmunoglobulina G , Edema Macular/patología , Células Fotorreceptoras de Vertebrados/patología , Retinopatía Diabética/inmunología , Femenino , Humanos , Edema Macular/inmunología , MasculinoRESUMEN
Germline mutations in Fumarate Hydratase (FH) cause the development of leiomyomas and leiomyosarcomas in the syndromes Multiple Cutaneous and Uterine Leiomyomata (MCUL1) and Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). There is little evidence, however, that FH mutation plays a role in the development of sporadic leiomyomas or leiomyosarcomas. Such observations do not, however, exclude a role for FH in tumour development outside the context of MCUL1/HLRCC, as it is possible that FH expression could be silenced by epigenetic mechanisms. To explore this possibility we have developed a highly specific antibody to FH and analysed a series of forty-five fresh-frozen uterine leiomyomas and nine leiomyosarcomas for FH expression.
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Epigénesis Genética , Fumarato Hidratasa/genética , Leiomioma/genética , Leiomiosarcoma/genética , Neoplasias Uterinas/genética , Adulto , Animales , Femenino , Fumarato Hidratasa/inmunología , Humanos , Inmunoglobulina G/inmunología , Leiomioma/enzimología , Leiomiosarcoma/enzimología , Persona de Mediana Edad , Mutación/genética , Miometrio/enzimología , Miometrio/patología , Conejos , Neoplasias Uterinas/enzimologíaRESUMEN
Biochemical studies with strains of Escherichia coli that are amplified for the products of the three fumarase genes, fumA (FUMA), fumB (FUMB) and fumC (FUMC), have shown that there are two distinct classes of fumarase. The Class I enzymes include FUMA, FUMB, and the immunologically related fumarase of Euglena gracilis. These are characteristically thermolabile dimeric enzymes containing identical subunits of Mr 60,000. FUMA and FUMB are differentially regulated enzymes that function in the citric acid cycle (FUMA) or to provide fumarate as an anaerobic electron acceptor (FUMB), and their affinities for fumarate and L-malate are consistent with these roles. The Class II enzymes include FUMC, and the fumarases of Bacillus subtilis, Saccharomyces cerevisiae and mammalian sources. They are thermostable tetrameric enzymes containing identical subunits Mr 48,000-50,000. The Class II fumarases share a high degree of sequence identity with each other (approx. 60%) and with aspartase (approx. 38%) and argininosuccinase (approx. 15%), and it would appear that these are all members of a family of structurally related enzymes. It is also suggested that the Class I enzymes may belong to a wider family of iron-dependent carboxylic acid hydro-lyases that includes maleate dehydratase and aconitase. Apart from one region containing a Gly-Ser-X-X-Met-X-X-Lys-X-Asn consensus sequence, no significant homology was detected between the Class I and Class II fumarases.
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Proteínas Bacterianas/clasificación , Escherichia coli/enzimología , Fumarato Hidratasa/clasificación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/fisiología , Escherichia coli/genética , Fumarato Hidratasa/genética , Fumarato Hidratasa/inmunología , Fumarato Hidratasa/fisiología , Genes Bacterianos , Calor , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes , Homología de Secuencia de Ácido Nucleico , Especificidad por SustratoRESUMEN
A rapid three-step procedure utilizing heat treatment, ammonium sulfate fractionation, and affinity chromatography on Matrex gel Orange A purified fumarase (EC 4.2.1.2) 632-fold with an 18% yield from crude extracts of Euglena gracilis var. bacillaris. The apparent molecular weight of the native enzyme was 120,000 as determined by gel filtration on Sephacryl S-300. The preparation was over 95% pure, and the subunit molecular weight was 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. The pH optimum for E. gracilis fumarase was 8.4. The Km values for malate and fumarate were 1.4 and 0.031 mM, respectively. Preparative two-dimensional gel electrophoresis was used to further purify the enzyme for antibody production. On Ouchterlony double-immunodiffusion gels, the antifumarase serum gave a sharp precipitin line against total E. gracilis protein and purified E. gracilis fumarase. It did not cross-react with purified pig heart fumarase. On immunoblots of purified E. gracilis fumarase and crude cell extracts of E. gracilis, the antibody recognized a single polypeptide with a molecular weight of approximately 60,000, indicating that the antibody is monospecific. This polypeptide was found in E. gracilis mitochondria. The antibody cross-reacted with an Escherichia coli protein whose molecular weight was approximately 60,000, the reported molecular weight of the fumA gene product of E. coli, but it failed to cross-react with proteins found in crude mouse cell extracts, Bacillus subtilis extracts, or purified pig heart fumarase.
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Euglena gracilis/enzimología , Fumarato Hidratasa/aislamiento & purificación , Anticuerpos/inmunología , Fraccionamiento Químico , Cromatografía de Afinidad , Reacciones Cruzadas , Escherichia coli/enzimología , Fumarato Hidratasa/inmunología , Fumarato Hidratasa/metabolismo , Concentración de Iones de Hidrógeno , Inmunodifusión , Técnicas Inmunológicas , Cinética , Peso Molecular , TemperaturaAsunto(s)
Fumarato Hidratasa/aislamiento & purificación , Hígado/enzimología , Aminoácidos , Animales , Catálisis , Fraccionamiento Celular , Fenómenos Químicos , Química , Citosol/enzimología , Fumarato Hidratasa/inmunología , Fumarato Hidratasa/metabolismo , Sustancias Macromoleculares , Masculino , Mitocondrias Hepáticas/enzimología , Peso Molecular , RatasRESUMEN
Two major forms of fumarate hydratase have been resolved in extracts prepared from a wide variety of mammalian cells by electrophoresis. Fractionation experiments with human and mouse cells suggest that one form (the slower migrating) is localized in the mitochondria, whereas the other form is predominant in the cytoplasm. Analysis of the segregation of the enzyme forms in human-mouse somatic cell hybrids indicates that a gene(s) necessary for the expression of both forms can be assigned to human chromosome 1(confirmation of a previous assignment by van Someren et al., 1974). Electrophoretic analysis suggests that the two forms may be interrelated. Furthermore, they both exhibit identical reactivity toward anti-fumarate hydratase antiserum. It is suggested that a modification of one form may occur in vivo and that the modification may be important in determining the intracellular localization of the enzyme.