Evidencia de altos niveles de IgA polimérica en el suero de pacientes con GN mensaginal con depósitos de IgA (Enfermedad de Berger)

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 11-06-1979.Se ha evidenciado la presencia de una mayor cantidad de IgA polimérica en el suero de pacientes con glomerulonefritis (GN) mesangial IgA (enfermedad de Berger). La enfermedad de Berger constituye una de las nefropatías más frecuentes en nuestro medio. La fotogenia de esta nefritis es oscura. Inicialmente se la considera una GN por inmunocomplejos, debido al aspecto granular de los depósitos de IgA en el mesangio. La detección de inmunocomplejos por distintas técnicas es positiva en pocos casos, y la frecuencia de pacientes en los que se detectan es baja también. Algunas características de esta GN como son: la presencia de altos niveles de IgA sérica en un porcentaje importante de estos pacientes, la presencia de IgA a nivel del mesangio glomerular, como la inmunoglobina única o dominante depositada en muchos casos, y la recidiva de la nefropatía después del transplante renal hicieron centrar nuestro interés en un estudio más a fondo de algunas características bioquímicas de esta inmunoglobulina. Sueros de enfermos con GN mesangial IgA se pasaron a través de una columna de Sephadex G-200 superfino, observándose la aparición de IgA en el primer pico de la cromatografía. Para caracterizar mejor esta inmunoglobulina se ha realizado un estudio cualitativo de la IgA sérica en estos pacientes mediante ultracentrifugación en gradiente de sacarosa a pH 7.4 y 2.8. La cuantificación de la IgA, presente en cada fracción, se realizó por un radioinmunoensayo de doble anticuerpo. Hemos observado un aumento significativo de los porcentajes de IgA sérica en pacientes con GN mesangial IgA entre (9-21) S a pH 7.4. Este aumento se mantiene tras la centrifugación a pH 2.8, salvo una disminución entre (17.21) S. Se ha realizado aislamiento de la IgA sérica de estos pacientes mediante electroforesis en almidón. La IgA anódica tiene un peso molecular superior al del monómero. Esta IgA de alto peso molecular es sensible a tratamiento reductor, produciéndose fragmentos de peso molecular menor mediante reducción con dietiotreitol y alquilación con iodoacetamida. El mantenimiento de los porcentajes después de ultracentrifugación a pH ácido, y la sensibilidad a tratamiento reductor son compatibles con una estructura covalente para estas formas de IgA de alto peso molecular. La presencia de verdaderos polímeros de IgA fue confirmada por dos criterios adicionales establecidos por algunos autores: 1) Presencia de cadena J. 2) Afinidad por el componente secretorio obtenido mediante cromatografía de afinidad, a partir de un calostro humano. En un grupo de estos pacientes se está realizando un tratamiento con difenilhidantoina (droga que disminuye selectivamente la IgA). Con este medicamento se ha observado en algunos pacientes la normalización de los porcentajes de IgA sérica con distintos coeficientes de sedimentación. Recientemente ha sido rebiopsiado uno de estos pacientes, observando una desaparición de la IgA depositada en el mesangio. La anomalía descrita en estos pacientes, y los resultados del tratamiento con difenilhidantoina abren nuevas perspectivas terapéuticas en el tratamiento de esta nefritis.Fac. de Ciencias QuímicasTRUEProQuestpu

Evidencia de altos niveles de IgA polimérica en el suero de pacientes con GN mensaginal con depósitos de IgA (Enfermedad de Berger)

Se ha evidenciado la presencia de una mayor cantidad de IgA polimérica en el suero de pacientes con glomerulonefritis (GN) mesangial IgA (enfermedad de Berger). La enfermedad de Berger constituye una de las nefropatías más frecuentes en nuestro medio. La fotogenia de esta nefritis es oscura. Inicialmente se la considera una GN por inmunocomplejos, debido al aspecto granular de los depósitos de IgA en el mesangio. La detección de inmunocomplejos por distintas técnicas es positiva en pocos casos, y la frecuencia de pacientes en los que se detectan es baja también. Algunas características de esta GN como son: la presencia de altos niveles de IgA sérica en un porcentaje importante de estos pacientes, la presencia de IgA a nivel del mesangio glomerular, como la inmunoglobina única o dominante depositada en muchos casos, y la recidiva de la nefropatía después del transplante renal hicieron centrar nuestro interés en un estudio más a fondo de algunas características bioquímicas de esta inmunoglobulina. Sueros de enfermos con GN mesangial IgA se pasaron a través de una columna de Sephadex G-200 superfino, observándose la aparición de IgA en el primer pico de la cromatografía. Para caracterizar mejor esta inmunoglobulina se ha realizado un estudio cualitativo de la IgA sérica en estos pacientes mediante ultracentrifugación en gradiente de sacarosa a pH 7.4 y 2.8. La cuantificación de la IgA, presente en cada fracción, se realizó por un radioinmunoensayo de doble anticuerpo. Hemos observado un aumento significativo de los porcentajes de IgA sérica en pacientes con GN mesangial IgA entre (9-21) S a pH 7.4. Este aumento se mantiene tras la centrifugación a pH 2.8, salvo una disminución entre (17.21) S. Se ha realizado aislamiento de la IgA sérica de estos pacientes mediante electroforesis en almidón. La IgA anódica tiene un peso molecular superior al del monómero. Esta IgA de alto peso molecular es sensible a tratamiento reductor, produciéndose fragmentos de peso molecular menor mediante reducción con dietiotreitol y alquilación con iodoacetamida. El mantenimiento de los porcentajes después de ultracentrifugación a pH ácido, y la sensibilidad a tratamiento reductor son compatibles con una estructura covalente para estas formas de IgA de alto peso molecular. La presencia de verdaderos polímeros de IgA fue confirmada por dos criterios adicionales establecidos por algunos autores: 1) Presencia de cadena J. 2) Afinidad por el componente secretorio obtenido mediante cromatografía de afinidad, a partir de un calostro humano. En un grupo de estos pacientes se está realizando un tratamiento con difenilhidantoina (droga que disminuye selectivamente la IgA). Con este medicamento se ha observado en algunos pacientes la normalización de los porcentajes de IgA sérica con distintos coeficientes de sedimentación. Recientemente ha sido rebiopsiado uno de estos pacientes, observando una desaparición de la IgA depositada en el mesangio. La anomalía descrita en estos pacientes, y los resultados del tratamiento con difenilhidantoina abren nuevas perspectivas terapéuticas en el tratamiento de esta nefritis

Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum

In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement

Autoantibodies against perilipin 1 as a cause of acquired generalized lipodystrophy

Acquired generalized lipodystrophy (AGL) is a rare condition characterized by an altered distribution of adipose tissue and predisposition to develop hepatic steatosis and fibrosis, diabetes, and hypertriglyceridemia. Diagnosis of AGL is based on the observation of generalized fat loss, autoimmunity and lack of family history of lipodystrophy. The pathogenic mechanism of fat destruction remains unknown but evidences suggest an autoimmune origin. Anti-adipocyte antibodies have been previously reported in patients with AGL, although their involvement in the pathogenesis has been poorly studied and the autoantibody target/s remain/s to be identified. Using a combination of immunochemical and cellular studies, we investigated the presence of anti-adipocyte autoantibodies in patients with AGL, acquired partial lipodystrophy, localized lipoatrophy due to intradermic insulin injections or systemic lupus erythematosus. Moreover, the impact of anti-adipocyte autoantibodies from AGL patients was assessed in cultured mouse preadipocytes. Following this approach, we identified anti-perilipin 1 IgG autoantibodies in the serum of patients with autoimmune variety-AGL, but in no other lipodystrophies tested. These autoantibodies altered the ability of perilipin 1 to regulate lipolysis in cultured preadipocytes causing abnormal, significantly elevated basal lipolysis. Our data provide strong support for the conclusion that perilipin 1 autoantibodies are a cause of generalized lipodystrophy in these patientsFC, ML-T, AL-L, and SG were supported by grants PI15- 00255 from Instituto de Salud Carlos III (ISCIII, Ministerio de Economía y Competitividad) and Fondos FEDER, Complemento II-CM network (B2017/BMD3673), Acciones Cooperativas y Complementarias Intramurales (ACCI) from CIBERER (ISCIII), and Fundación SENEFRO. MdMwas supported by Roche Farma SA and Foundation Domingo Martínez. VA was financed by Consejería de Educación, Juventud y Deporte of Comunidad de Madrid and by Fondo Social Europeo (Programa Operativo de Empleo Juvenil, and Iniciativa de Empleo Juvenil (YEI), (PEJ15/BIO/AI/0045). DA-V was supported by the intramural research program of the Xunta de Galicia (Programa de Consolidación e Estructuración de Unidades de Investigación Competitivas, grant ED341b 2017/19), by the Instituto de Salud Carlos III (grant number: PI08-1449) and the European Regional Development Fund, FEDER and by the Asociación Española de Familiares y Afectados de Lipodistrofias (AELIP

Extravascular hemolysis and complement consumption in Paroxysmal Nocturnal Hemoglobinuria patients undergoing eculizumab treatment

Los datos asociados con este artículo están disponibles en: http://dx.doi.org/10.1016/j.imbio.2016.09.002.Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis that is effectively treated with eculizumab. However, treatment responses are reported heterogeneous with some patients presenting residual hemolysis and requiring RBC transfusions. Recent reports have shown that both extravascular hemolysis and incomplete C5 blockade can explain these suboptimal hematological responses. Here we have tested our eculizumab-treated PNH patients (n = 12) for signs of hemolysis and assessed complement biomarkers. Patients were also genotyped for complement receptor 1 (CR1, CD35) and C5 polymorphisms and evaluated for free eculizumab in plasma. We report that 10 patients (83%) present parameters suggesting persistent hemolysis, although they did not require additional transfusions. Seven of them (58%) become direct Coombs-test positive as a consequence of treatment, including all patients carrying the low-expression CR1-L allele. CH50 and sC5b-9 assays demonstrate that the persistent low-level hemolysis identified in our treated patients is not a consequence of incomplete C5 blockade, supporting that this hemolysis, as has been suggested previously, results from the extravascular removal of C3 opsonized PNH erythrocytes. We also show that continuous alternative pathway activation in eculizumab-treated individuals carrying the CR1-L allele results in abnormally decreased levels of C3 in plasma that could, potentially, increase their susceptibility to bacterial infections. Finally, we encourage a routine evaluation of free eculizumab levels and terminal pathway activity to personalize eculizumab administrationIn this study S.R de C. is supported by the Spanish “Ministerio de Economía y Competitividad” (SAF2011-26583) and the Autonomous Region of Madrid (S2010/BMD-2316

Immunological features of patients affected by Barraquer-Simons syndrome

Background: C3 hypocomplementemia and the presence of C3 nephritic factor (C3NeF), an autoantibody causing complement system over-activation, are common features among most patients affected by Barraquer-Simons syndrome (BSS), an acquired form of partial lipodystrophy. Moreover, BSS is frequently associated with autoimmune diseases. However, the relationship between complement system dysregulation and BSS remains to be fully elucidated. The aim of this study was to provide a comprehensive immunological analysis of the complement system status, autoantibody signatures and HLA profile in BSS. Thirteen subjects with BSS were recruited for the study. The circulating levels of complement components, C3, C4, Factor B (FB) and Properdin (P), as well as an extended autoantibody profile including autoantibodies targeting complement components and regulators were assessed in serum. Additionally, HLA genotyping was carried out using DNA extracted from peripheral blood mononuclear cells. Results: C3, C4 and FB levels were significantly reduced in patients with BSS as compared with healthy subjects. C3NeF was the most frequently found autoantibody (69.2% of cases), followed by anti-C3 (38.5%), and anti-P and anti- FB (30.8% each). Clinical data showed high prevalence of autoimmune diseases (38.5%), the majority of patients (61.5%) being positive for at least one of the autoantibodies tested. The HLA allele DRB1*11 was present in 54% of BSS patients, and the majority of them (31%) were positive for *11:03 (vs 1.3% allelic frequency in the general population). Conclusions: Our results confirmed the association between BSS, autoimmunity and C3 hypocomplementemia. Moreover, the finding of autoantibodies targeting complement system proteins points to complement dysregulation as a central pathological event in the development of BSS.This work was supported by Instituto de Salud Carlos III (Ministerio de Ciencia, Innovación y Universidades, Gobierno de España) and Fondos FEDER (PI15–00255 to M.L-T. and PI08–1449 to D.A-V.), Complemento II-CM network (B2017/BMD3673 to M.L-T), the intramural research program of the Xunta de Galicia (Programa de Consolidación e Estructuración de Unidades de Investigación Competitivas, grant ED341b 2017/19 to D.A-V.), the Asociación Española de Familiares y Afectados de Lipodistrofias (AELIP) (to D.A-V., to F.C. and to P.N.

Consensus statement on the diagnosis, management, and treatment of angioedema mediated by Bradykinin. Part. II: treatment, follow-up, and special situations

Background: There are no previous Spanish guidelines or consensus statements on bradykinin-induced angioedema. Aim: To draft a consensus statement on the management and treatment of angioedema mediated by bradykinin in light of currently available scientifi c evidence and the experience of experts. This statement will serve as a guideline to health professionals. Methods: The consensus was led by the Spanish Study Group on Bradykinin-Induced Angioedema, a working group of the Spanish Society of Allergology and Clinical Immunology. A review was conducted of scientifi c papers on different types of bradykinin-induced angioedema (hereditary and acquired angioedema due to C1 inhibitor defi ciency, hereditary angioedema related to estrogens, angioedema induced by angiotensin-converting enzyme inhibitors). Several discussion meetings were held to reach the consensus. Results: Treatment approaches are discussed, and the consensus reached is described. Specifi c situations are addressed, namely, pregnancy, contraception, travelling, blood donation, and organ transplantation. Conclusions: A review of and consensus on treatment of bradykinin-induced angioedema is presentedIntroducción: No existen guías previas españolas sobre el manejo del angioedema mediado por bradicinina. Objetivos: Alcanzar un consenso sobre el manejo y tratamiento del angioedema mediado por bradicinina a la luz de la evidencia científi ca disponible y la experiencia de los expertos, que sirva como guía para los profesionales de la salud. Métodos: SGBA/GEAB, un grupo de trabajo de la SEAIC dirigió el consenso. Se realizó una revisión de los documentos científi cos publicados sobre los diferentes tipos de angioedema mediado por bradicinina [angioedema hereditario o adquirido por defi ciencia de inhibidor de la C1 esterasa, angioedema hereditario relacionado con estrógenos (AEH tipo III, AEH-FXII), angioedema inducido por IECA (inhibidores del enzima convertidor de angiotensina]. Hubo varias reuniones del SGBA/GEAB para alcanzar el consenso. Resultados: Se revisan y discuten los diferentes tratamientos disponibles y se describe el consenso alcanzado. Se abordan situaciones específi cas (embarazo, anticoncepción, viajes, hemodonación, trasplante de órganos). Conclusiones: Se presenta una revisión del tratamiento del angioedema mediado por bradicinina y un consenso sobre su tratamiento en EspañaDr. Teresa Caballero is a researcher with the Hospital La Paz Health Research Institute (IdiPaz) program for promoting research activities (2009

Atypical hemolytic uremic syndrome-associated variants and autoantibodies impair binding of factor H and factor H-related protein 1 to pentraxin 3

Atypical hemolytic uremic syndrome (aHUS) is a renal disease associated with complement alternative pathway dysregulation and is characterized by endothelial injury. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule expressed by endothelial cells and upregulated under inflammatory conditions. PTX3 activates complement, but it also binds the complement inhibitor factor H. In this study, we show that native factor H, factor H-like protein 1, and factor H-related protein 1 (CFHR1) bind to PTX3 and that PTX3-bound factor H and factor H-like protein 1 maintain their complement regulatory activities. PTX3, when bound to extracellular matrix, recruited functionally active factor H. Residues within short consensus repeat 20 of factor H that are relevant for PTX3 binding were identified using a peptide array. aHUS-associated factor H mutations within this binding site caused a reduced factor H binding to PTX3. Similarly, seven of nine analyzed anti-factor H autoantibodies isolated from aHUS patients inhibited the interaction between factor H and PTX3, and five autoantibodies also inhibited PTX3 binding to CFHR1. Moreover, the aHUS-associated CFHR1*B variant showed reduced binding to PTX3 in comparison with CFHR1*A. Thus, the interactions of PTX3 with complement regulators are impaired by certain mutations and autoantibodies affecting factor H and CFHR1, which could result in an enhanced local complement-mediated inflammation, endothelial cell activation, and damage in aHUS

The molecular and structural bases for the association of complement C3 mutations with atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) associates with complement dysregulation caused by muta-tions and polymorphisms in complement activators and regulators. However, the reasons why somemutations in complement proteins predispose to aHUS are poorly understood. Here, we have investigatedthe functional consequences of three aHUS-associated mutations in C3, R592W, R161W and I1157T. First,we provide evidence that penetrance and disease severity for these mutations is modulated by inheri-tance of documented “risk” haplotypes as has been observed with mutations in other complement genes.Next, we show that all three mutations markedly reduce the efficiency of factor I-mediated C3b cleavagewhen catalyzed by membrane cofactor protein (MCP), but not when catalyzed by factor H. Biacore anal-ysis showed that each mutant C3b bound sMCP (recombinant soluble MCP; CD46) at reduced affinity,providing a molecular basis for its reduced cofactor activity. Lastly, we show by electron microscopystructural analysis a displacement of the TED domain from the MG ring in C3b in two of the C3 mutantsthat explains these defects in regulation. As a whole our data suggest that aHUS-associated mutations inC3 selectively affect regulation of complement on surfaces and provide a structural framework to predictthe functional consequences of the C3 genetic variants found in patients

Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients