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Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1ß secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA.
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Artritis Experimental , Humanos , Animales , Ratones , Artritis Experimental/prevención & control , Neutrófilos , Colágeno , Autoanticuerpos , EpítoposRESUMEN
Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.
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Artritis Reumatoide , Citrulinación , Humanos , Desiminasas de la Arginina Proteica/genética , Factor XIIa/metabolismo , Calicreína Plasmática/metabolismo , Factor XIa , Proteínas/metabolismo , AutoanticuerposRESUMEN
OBJECTIVE: We aimed to test the non-inferiority of oral versus intravenous hydration in the incidence of contrast-associated acute kidney injury (CA-AKI) in elderly outpatients undergoing a contrast-enhanced computed tomography (CE-CT) scan. METHODS: PNIC-Na (NCT03476460) is a phase-2, single-center, randomized, open-label, non-inferiority trial. We included outpatients undergoing a CE-CT scan, >65 years having at least one risk factor for CA-AKI, such as diabetes, heart failure, or an estimated glomerular filtration rate (eGFR) of 30-59 mL/min/1.73 m². Participants were randomized (1:1) to oral sodium-chloride capsules or intravenous hydration. The primary outcome was an increase in serum creatinine >0.3 mg/dL or a reduction in eGFR >25% within 48 h. The non-inferiority margin was set at 5%. RESULTS: A total of 271 subjects (mean age 74 years, 66% male) were randomized, and 252 were considered for the main analysis (per-protocol). A total of 123 received oral hydration and 129 intravenous. CA-AKI occurred in 9 (3.6%) of 252 patients and 5/123 (4.1%) in the oral-hydration group vs. 4/129 (3.1%) in the intravenous-hydration group. The absolute difference between the groups was 1.0% (95% CI -4.8% to 7.0%), and the upper limit of the 95% CI exceeded the pre-established non-inferiority margin. No major safety concerns were observed. CONCLUSION: The incidence of CA-AKI was lower than expected. Although both regimens showed similar incidences of CA-AKI, the non-inferiority was not shown.
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Increased platelet destruction is central in the pathogenesis of immune thrombocytopenia. However, impaired platelet production is also relevant and its significance underlies the rationale for treatment with thrombopoietin receptor agonists (TPO-RAs). Previous studies have associated enhanced complement activation with increased disease severity. Additionally, treatment refractoriness has been demonstrated to resolve by the administration of complement-targeted therapeutics in a subset of patients. The association between complement activation and the platelet response to TPO-RA therapy has previously not been investigated. In this study, blood samples from patients with immune thrombocytopenia (n = 15) were prospectively collected before and two, six and 12 weeks after the initiation of TPO-RA therapy. Plasma levels of complement degradation product C4d and soluble terminal complement complexes were assessed. Patients with significantly elevated baseline levels of terminal complement complexes exhibited more often an inadequate platelet response (p = .04), were exclusively subjected to rescue therapy with intravenous immunoglobulin (p = .02), and did not respond with a significant platelet count increase during the study period. C4d showed a significant (p = .01) ability to distinguish samples with significant terminal complement activation, implying engagement of the classical complement pathway. In conclusion, elevated levels of complement biomarkers were associated with a worse TPO-RA treatment response. Larger studies are needed to confirm these results. Biomarkers of complement activation may prove valuable as a prognostic tool to predict which patients that potentially could benefit from complement-inhibiting therapy in the future.
What is the context?Primary immune thrombocytopenia (ITP) is a potentially serious illness associated with an increased risk of bleeds. Manifestations range from confined skin bruising to life-threatening intracranial hemorrhages.It is an acquired immune disorder characterized by increased destruction and impaired production of platelets.Treatments aim at suppressing the destruction and supporting the production of platelets.Thrombopoietin receptor agonists (TPO-RA) are medically approved platelet growth factors that contribute to the generation of new platelets.The complement system is an evolutionary preserved part of innate immunity.Previous studies have indicated that complement activation may be an important contributor to disease and that the administration of complement-inhibiting therapy improves the platelet count in a subset of patients with primary ITP.What is new? The potential association between complement activation and a poor platelet response to TPO-RA therapy in primary ITP has not been previously studied.In fifteen patients with primary ITP starting TPO-RA therapy, we prospectively followed the platelet response and levels of complement biomarkers for 12 weeks.We showed that patients with high levels of complement biomarkers exhibited a worse treatment response during the study period.What is the impact?Our results suggest that levels of complement biomarkers may be valuable to predict which patients with treatment-refractory ITP that potentially could benefit from complement-inhibiting therapy in the futureLarger studies are needed to confirm our results.
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Púrpura Trombocitopénica Idiopática , Trombocitopenia , Humanos , Receptores de Trombopoyetina/agonistas , Estudios Prospectivos , Biomarcadores , Activación de Complemento , Trombopoyetina/farmacología , Trombopoyetina/uso terapéutico , Proteínas Recombinantes de FusiónRESUMEN
INTRODUCTION: The development of neutralising (inhibitors) and non-neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter-laboratory variation, and false-negative as well as false-positive results may affect treatment. Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP-based fluorescence-immunoassay (xFLI). AIM: Validation of our xFLI and comparability with enzyme-linked immunosorbent assay (ELISA) and chromogenic Nijmegen-Bethesda assay (CBA) for anti-FVIII antibodies in haemophilia A (HA) patients. METHODS: The xFLI method was developed with full-length and B-domain deleted factor coupled to magnetic beads, optimised and validated for performance characteristics. Comparability with ELISA and CBA was evaluated in HA patient samples (n = 112), serial samples in six inhibitor patients and reference interval and decision-limits in healthy donors (n = 44). RESULTS: The intra- and inter-assay precision (CV%) for the xFLI method was below 6% and detection limit (LLOQ) .084 ng/mL (NovoEight). All ELISA-positive samples were positive with either Advate or NovoEight. Additionally, 10.7%-14.3% were xFLI-positive and ELISA-negative. All but one CBA-positive sample was above 3SD with xFLI; one was between 2 and 3SD. 29.1% were xFLI-positive and CBA negative. The overall concordance between xFLI and ELISA was 82.1% and xFLI and CBA 77.9%. CONCLUSION: The anti-FVIII antibody xFLI method is adaptable to clinical practice and more sensitive and reproducible than ELISA and CBA. Actual NNA titers are determined to both full-length and B-domain deleted FVIII. The xFLI is thus valuable for confirmation of all anti-FVIII antibodies.
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Hemofilia A , Humanos , Hemofilia A/tratamiento farmacológico , Reproducibilidad de los Resultados , Factor VIII/uso terapéutico , Sensibilidad y Especificidad , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
INTRODUCTION: The development of inhibitory antibodies (inhibitors) in persons with hemophilia B (PwHB) causes significant morbidity. Data on the impact of the F9 variant and immune tolerance induction (ITI) outcome are limited. The aim of this study was to investigate the presence of neutralizing and non-neutralizing antibodies (NNA) in severe hemophilia B (HB) and to evaluate ITI outcome and complications in relation to the pathogenic F9 variant. MATERIALS AND METHODS: Persons with severe HB in the Nordic countries were enrolled and information on F9 variants, inhibitors, ITI and complications were collected. Analyses of anti-FIX antibodies with a fluorescence-immunoassay (xFLI) and an ELISA method were conducted. RESULTS: Seventy-nine PwHB were enrolled. Null variants were seen in 33 (42 %) PwHB and 12 (15 %) had a current or former inhibitor. Eleven (92 %) of the inhibitor patients had experienced allergic manifestations and three (25 %) nephrotic syndrome. Of 10 PwHB with at least one ITI attempt, eight (80 %) were considered tolerant at enrolment. Immunosuppression was included in seven of eight successful or partially successful attempts. Five PwHB had at least one ITI failure before a successful or partially successful ITI. No NNA could be identified. CONCLUSION: A high proportion of severe F9 gene defects among persons with severe HB in the Nordic countries may explain the observed relatively high prevalence of inhibitors. ITI success was independent of the F9 variant and attained despite allergic manifestations and previous ITI failures. Inclusion of immunosuppression tentatively enhances the chances of ITI success. No NNA were observed.
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Hemofilia A , Hemofilia B , Anticuerpos Neutralizantes , Factor IX/genética , Factor VIII , Hemofilia B/genética , Humanos , Tolerancia Inmunológica/genética , Terapia de InmunosupresiónRESUMEN
Fine needle biopsy (FNB) is an effective, minimally invasive and inexpensive diagnostic technique. Under computed tomography (CT)-guidance, lesions that have a difficult approach can be sampled to reach a diagnosis. The aim of this study is to describe the use of CT-guidance to obtain FNB from vertebral and paravertebral lesions in small animals. Ten dogs and one ferret that had undergone CT-guided FNB of vertebral and paravertebral lesions and had a cytological or a histological diagnosis were included in this retrospective study. The FNB samples were taken in four cases from the vertebra, in two cases from the intervertebral disc and in five cases from the intervertebral foramen. Two infectious and nine neoplastic lesions were diagnosed. The percentage of successful FNB was 91%. The percentage of samples with a cytological diagnosis was 80%. The percentage of complications was 9%. Limitations were the small number of animals in the study, the lacking complementary percutaneous biopsies for comparison, the lacking final histological diagnoses in some cases and the intervention of multiple operators. Computed tomography-guided FNB is a useful and safe technique for the diagnosis of vertebral and paravertebral lesions in small animals. However, a degree of expertise is important.
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OBJECTIVE: Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), or myositis. METHODS: Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. RESULTS: A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2-33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7-5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals' capacity to deposit C4b on immune complexes. CONCLUSION: We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.
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Lupus Eritematoso Sistémico , Miositis , Autoanticuerpos/genética , Complemento C4/genética , Complemento C4b/genética , Variaciones en el Número de Copia de ADN , Humanos , Lupus Eritematoso Sistémico/genética , Factores de RiesgoRESUMEN
Platelet transfusion refractoriness is a serious clinical concern that complicates the management of thrombocytopenic patients. Previous studies have suggested a potential role for both complement and platelet activation based on in vitro analyses of platelet concentrates. In this study, the post-transfusion platelet response, as indicated by the corrected count increment at 1 and 24 h after prophylactic platelet transfusions, respectively, was correlated with the 1 h post-transfusion Δconcentration (1 h post-transfusion - pretransfusion) of complement and platelet activation biomarkers. The study was registered as a clinical trial at ClinicalTrials.gov (identifier: NCT02601131) and patients were recruited during inpatient care in the hematological department. Soluble terminal complement complexes, soluble P-selectin and soluble CD40 ligand were analyzed. Confirmed alloimmunized patients were excluded. Included subjects were either given platelet transfusions (n = 43) and categorized into four clinical study groups or included in a non-transfused control group (n = 10). In total, 54 transfusions were included. No transfusion-mediated complement activation was observed. The transfusions were associated with a significant increase in the concentration of soluble P-selectin (p < .001), primarily corresponding to the passive infusion of soluble P-selectin-containing plasma residuals. The Δconcentration of soluble P-selectin was, however, not significantly correlated with the corrected count increments. Thus, significant correlations between biomarkers of complement and platelet activation and the post-transfusion platelet response could not be demonstrated in this study.
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Biomarcadores/metabolismo , Proteínas del Sistema Complemento/fisiología , Activación Plaquetaria/fisiología , Transfusión de Plaquetas/métodos , Adulto , Anciano , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de TiempoRESUMEN
Background: Hereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack. Methods: Sialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays. Results: The proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis. Conclusion: We report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis.
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Plaquetas/metabolismo , Activación de Complemento/inmunología , Heterocigoto , Complejos Multienzimáticos/genética , Mutación , Trombocitopenia/diagnóstico , Trombocitopenia/etiología , Adolescente , Alelos , Plaquetas/inmunología , Plaquetas/ultraestructura , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Hemólisis , Humanos , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Ácido N-Acetilneuramínico/metabolismo , Fenotipo , Pruebas de Función Plaquetaria , Trombocitopenia/sangre , Secuenciación Completa del GenomaRESUMEN
INTRODUCTION: Monitoring replacement therapy with standard and extended half-life (EHL) products is challenging, since one-stage assay (OSA) and chromogenic substrate assay (CSA) results may differ significantly. Recent recommendations include local validation of each new product with recovery within 20-30%, depending on activity level. AIM: To validate factor VIII (FVIII) activity for monitoring products in clinical use on Atellica Coag and to correlate it with thrombin generation. METHODS: Plasma samples spiked with Advate® , Elocta® , Adynovi® , Nuwiq® , NovoEight® and Afstyla® (0.05, 0.20, 0.50 and 0.80 IU/ml) were analysed using Atellica Coag 360 with CSA-1 (Coatest SP) and CSA-2 (FVIII chromogenic), and OSA (Actin FS). Thrombin generation was performed using two thrombin generation assays (TGA-1 (Thrombinoscope) and TGA-2 (Technothrombin). RESULTS: All products at levels above 0.05 IU/ml, except Adynovi, showed acceptable recovery using CSA-1, whereas measurements using CSA-2 gave more results outside the target level. All products, except Afstyla, showed acceptable recovery using OSA. Correlation between CSA-1 and OSA was excellent (r2 =1.0) with biases of 6-3â2%, depending on FVIII product. A clear dose-response was seen for all thrombin generation parameters and products using both methods, except at low levels for lag time using TGA-1. With CSA-1 as an independent variable, the correlations to thrombin peak (measured with TGA-2) were good (r2 = .8-.9). CONCLUSION: Our data revealed good correlation and acceptable bias between CSA and OSA using our sets of reagents, methods and analyser in spiked samples. Thrombin generation gave good correlation to CSA-1 factor activity and is a possible complement to factor activity assays.
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Factor VIII , Hemofilia A , Pruebas de Coagulación Sanguínea , Factor VIII/farmacocinética , Semivida , Hemofilia A/tratamiento farmacológico , Humanos , TrombinaRESUMEN
Background: The techniques described for the identification of the lumbosacral (LS) epidural space in dogs do not guarantee the needle position or an accidental subarachnoid puncture, especially in small size dogs. Aim: To determine the relationship between body weight and the location of the dural sac (DS) using myelography in dogs, and to determine the possibility of subarachnoid puncture during LS epidural based on the position of the DS. Methods: Four masked observers evaluated 70 myelographic studies of dogs, annotating the vertebrae where the DS ended, if it was localized before or after the LS space, and if accidental subarachnoid puncture during LS epidural injection was possible (yes/no). Body weight (kg) was categorized into: less than 10 kg, between 10 and 20 kg, and more than 20 kg and was also converted to body surface area (BSA) as a continuous variable. Results: The DS ended at the LS space or caudally in 50% of dogs. There was a statistically significant difference between the position of the DS and the dog's BSA (p = 0.001). The DS ended caudal to the LS space in 72.7% of dogs weighing <10 kg, in 25% of dogs between 10 and 20 kg and in 15% of dogs in the >20 kg category. The observers considered a possible subarachnoid puncture during LS epidural in 69.7% of patients <10 kg, 16.6% on those between 10 and 20 kg, and in 11.7% of the dogs >20 kg. Conclusion: The DS ended caudal to the LS space in almost 3/4 dogs in the <10 kg category, so accidental subarachnoid puncture during LS epidural is highly possible in this weight range.
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Perros/anatomía & histología , Región Lumbosacra/diagnóstico por imagen , Mielografía/veterinaria , Animales , Espacio Epidural/diagnóstico por imagen , Femenino , Inyecciones Epidurales/veterinaria , Masculino , Agujas/veterinaria , Punciones/veterinaria , Espacio Subaracnoideo/diagnóstico por imagenRESUMEN
Complement-mediated atypical hemolytic uremic syndrome (aHUS) is an ultra-rare renal disease primarily caused by genetic alterations in complement proteins. The genetic work-up required for confirmation of diagnosis is complicated and not always logistically accessible. The aim of the present study was to apply a diagnostic scheme compliant with the American College of Medical Genetics and Genomics guidelines to investigate the prevalence of complement-mediated aHUS among subjects formerly included in a retrospective cohort of clinically suspected aHUS. Clinical outcomes and genetic correlations to complement analyses were assessed. Subjects were investigated with medical record reviewing, inquiries, and laboratory analyses composed of whole genome sequencing; enzyme-linked immunosorbent assays for factor I, factor H, and factor H-specific antibodies; nephelometry for complement components three of four; flow cytometry for CD46 surface expression and immunoblotting for the presence of factor H-related protein 1. In total, 45% (n = 60/134) of the subjects were deceased at the time of study. Twenty of the eligible subjects consented to study participation. Based on genetic sequencing and clinical characteristics, six were categorized as definite/highly suspected complement-mediated aHUS, 10 as non-complement-mediated aHUS and four as having an HUS-like phenotype. In the complement-mediated aHUS group, two subjects had not received an aHUS diagnosis during the routine clinical management. Disease-contributing/likely disease-contributing genetic variants were identified in five subjects, including a novel missense variant in the complement factor H gene (c.3450A>G, p.I1150M). This study illustrates the risk for misdiagnosis in the management of patients with complement-mediated aHUS and the importance of a comprehensive assessment of both phenotype and genotype to reach a diagnosis.
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Síndrome Hemolítico Urémico Atípico/genética , Variación Genética/genética , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Suecia , Adulto JovenRESUMEN
Objective: To examine whether C4d plasma levels correlate with treatment response and C4d kidney deposition in systemic lupus erythematosus (SLE) with lupus nephritis (LN). Methods: C4d plasma levels were analyzed by a unique assay specifically detecting C4d arising from complement activation and C4 plasma levels were quantified with competitive ELISA. SLE patients with LN (71) and active SLE patients without LN (22) plus 145 controls were included. For 52 LN patients samples were available both at baseline and after immunosuppressive treatment. C4d kidney deposition was detected using immunohistochemistry in two matching kidney biopsies of 12 LN patients. Results: In comparison to population-based controls, plasma C4d levels were significantly increased in SLE patients (0.33 mg/L versus 0.94 mg/ml, p < 0.0001) with significantly higher levels in LN patients (1.02 mg/L) than in non-renal SLE patients (0.57 mg/L, p = 0.004). The C4d/C4 ratio was also significantly higher in LN (11.2) than in non-renal SLE patients (2.5, p = 0.0002). According to ROC curve analysis, C4d was found to be an accurate marker to discriminate LN from non-renal SLE patients (p = 0.004). The C4d/C4 ratio displayed even higher specificity, sensitivity and overall accuracy as marker for LN than C4d and C4 alone. At baseline, C4d levels correlated significantly with urine-albumin to creatinine ratio (rs = 0.43, p = 0.011) and with renal activity index (rs = 0.37, p = 0.002). Immunohistochemical staining showed glomerular deposits of C4d in kidney biopsies, which strikingly correlated with plasma C4d levels (rs = 0.7, p = 0.0002). Plasma C4d declined significantly after treatment in patients that experienced favorable clinical and histopathological response (p < 0.0001), while levels remained mainly unchanged in non-responders. Conclusion: Plasma C4d discriminates LN from active non-renal SLE, correlates with C4d kidney deposits and appears valuable in monitoring responsiveness to various treatments. The C4d/C4 ratio might be superior to C4d alone.
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Complejo Antígeno-Anticuerpo/metabolismo , Complemento C4/metabolismo , Terapia de Inmunosupresión , Riñón/metabolismo , Nefritis Lúpica/terapia , Adulto , Anciano , Biomarcadores/sangre , Biomarcadores/metabolismo , Complemento C4/análisis , Progresión de la Enfermedad , Femenino , Humanos , Riñón/patología , Nefritis Lúpica/sangre , Nefritis Lúpica/metabolismo , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Macrophages are a major immune cell type in the tumor microenvironment, where they display a tumor-supporting phenotype. Factor H (FH) is a complement inhibitor that also plays a role in several cellular functions. To date, the phenotype of monocytes stimulated with FH has been unexplored. We discovered that FH is a survival factor for CD14+ primary human monocytes, promoting their differentiation into macrophages in serum-free medium. This activity was localized to the C-terminal domains of FH and it was inhibited in plasma, indicating that the phenomenon may be most relevant in tissues. FH-induced macrophages display characteristics of immunosuppressive cells including expression of CD163 and CD206, release of the anti-inflammatory cytokine IL-10 and changes in metabolism. Furthermore, FH-induced macrophages express low levels of HLA-DR but high levels of co-inhibitory molecule programmed death-ligand 1 (PD-L1), and accordingly, a reduced capacity for T-cell activation. Finally, we show that FH is expressed by human breast cancer cells and that this correlates with the presence of immunosuppressive macrophages, breast cancer recurrence and severity of the disease. We propose that the expression of FH by tumor cells and the promotion of an immunosuppressive cancer microenvironment by this protein should be taken into account when considering the effectiveness of immunotherapies against breast cancer.
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Neoplasias de la Mama , Factor H de Complemento , Inactivadores del Complemento , Femenino , Humanos , Macrófagos , Monocitos , Recurrencia Local de Neoplasia , Microambiente TumoralRESUMEN
BACKGROUND: After a proximal lower limb deep vein thrombosis (DVT; involving popliteal veins or above), up to 40% of patients develop postthrombotic syndrome (PTS) as assessed by the Villalta scale (VS). Poor initial anticoagulant treatment is a known risk factor for PTS. The risk of developing PTS after isolated distal DVT (infra-popliteal DVT without pulmonary embolism), and the impact of anticoagulant treatment on this risk, are uncertain. METHODS: Long-term follow-up of CACTUS double-blind trial comparing 6 weeks of s.c. nadroparin (171 IU/kg/d) versus s.c. placebo for a first symptomatic isolated distal DVT. At least 1 year after randomization, patients had a PTS assessment in clinic or by phone using the VS. RESULTS: After a median follow-up of 6 years, PTS was present in 30% (n = 54) of the 178 patients who had a PTS assessment. PTS was moderate or severe in 24% (n = 13) of cases. There was no statistically significant difference in prevalence of PTS in the nadroparin versus placebo groups (29% versus 32%, P = .6), except in patients without evidence of primary chronic venous insufficiency (9% versus 24%, P = .04). Rates of venous thromboembolism recurrence during follow-up in the nadroparin and placebo groups were, respectively, 8% (n = 7) and 14% (n = 13; P = .2). CONCLUSION: After a first isolated distal DVT, the risk of PTS is substantial but much lower than that reported after proximal DVT. Anticoagulation with nadroparin doesn't provide any clear benefit to prevent PTS, except in patients without preexisting chronic venous insufficiency. Anticoagulation might be associated with a lower risk of venous thromboembolism recurrence.
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Cactaceae , Síndrome Postrombótico , Trombosis de la Vena , Anticoagulantes/efectos adversos , Humanos , Vena Poplítea , Síndrome Postrombótico/diagnóstico , Síndrome Postrombótico/epidemiología , Síndrome Postrombótico/etiología , Factores de Riesgo , Trombosis de la Vena/diagnóstico , Trombosis de la Vena/tratamiento farmacológico , Trombosis de la Vena/epidemiologíaRESUMEN
BACKGROUND: Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simplified flow cytometric platelet activation assay using microtiter plates and 2/ to correlate the outcome to gold standard method LTA, and to clinical bleeding assessment tool scores (BAT score). METHODS: The PACT method was developed in microtiter plates using adenosine diphosphate (ADP), collagen-derived peptide (CRP-XL) and thrombin receptor activator for peptide 6 (TRAP-6) as agonists. Antibodies against GPIIb-IIIa activation epitope (PAC1), P-selectin (CD62P) and lysosome-associated membrane glycoprotein 3 (LAMP3; CD63) were used as platelet activation markers. Sixty-six patients referred to the coagulation unit for bleeding symptoms were included in this single-center observational study. Platelet activation was determined by PACT and LTA. The results of both methods were correlated to BAT score. RESULTS: A two-by-two analysis using Cohen's kappa analysis gave moderate agreement between LTA and PACT (82%, kappa = 0.57), when PACT analysis with ADP and CRP-XL was compared to LTA. Using LTA as reference method, positive predictive value was 70% and negative predictive value was 87%. A substantial number of patients had high BAT score and normal LTA and PACT results. Patients with abnormal LTA or PACT results had higher BAT score than patients with normal results, but the difference was not significant. CONCLUSIONS: The performance in microtiter plates simplified the PACT method and enabled analysis of more patients at the same time. Our results indicate that with modification of the current PACT assay, a higher negative predictive value can be obtained. Furthermore, with comparable result to LTA the PACT could be used as a screening assay for inherited platelet disorders.
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Trastornos de las Plaquetas Sanguíneas , Plaquetas/metabolismo , Citometría de Flujo/métodos , Enfermedades Genéticas Congénitas , Activación Plaquetaria , Trastornos de las Plaquetas Sanguíneas/sangre , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Femenino , Enfermedades Genéticas Congénitas/sangre , Enfermedades Genéticas Congénitas/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Función Plaquetaria/métodosRESUMEN
BACKGROUND: In the present study, we sought to evaluate the complement activation product C4d as a marker for lupus nephritis in systemic lupus erythematosus (SLE). METHODS: C4d levels were determined by enzyme-linked immunosorbent assay in plasma samples of patients with established SLE using a novel approach based on detection of a short linear cleavage neoepitope. Cross-sectional associations were studied in 98 patients with SLE with samples taken at lower or higher respective disease activity. Temporal associations were investigated in 69 patients with SLE who were followed longitudinally for up to 5 years. Plasma samples from 77 healthy donors were included as controls. RESULTS: C4d levels were negligible in healthy control subjects and significantly increased in patients with SLE in the cross-sectional study (p < 0.0001). C4d levels discriminated between higher and lower disease activity according to ROC curve analysis (p < 0.001), exhibiting a positive predictive value of 68%. At higher disease activity, C4d levels correlated with the modified Systemic Lupus Erythematosus Disease Activity Index (p = 0.011) and predominantly with lupus nephritis (p = 0.003), exhibiting a sensitivity of 79% to identify patients with nephritis. High C4d levels together with the presence of anti-dsDNA autoantibodies preceded and thus predicted future lupus nephritis in the longitudinal study (OR 5.4, 95% CI 1.4-21.3). When we considered only patients with renal involvement (19 of 69) during the longitudinal study, we found that high C4d levels alone could forecast recurrence of future lupus nephritis (OR 3.3, 95% CI 1.2-9.6). CONCLUSIONS: C4d appears to be a valuable marker for use in monitoring of patients with SLE, particularly for lupus nephritis. Importantly, C4d levels can predict impending flares of lupus nephritis and may thus be useful for informing treatment.
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Biomarcadores/sangre , Nefritis Lúpica/sangre , Nefritis Lúpica/diagnóstico , Fragmentos de Péptidos/sangre , Adulto , Anciano , Anciano de 80 o más Años , Complemento C4b , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Neutrophil extracellular traps (NETs) can stimulate thrombosis, and their degradation is decreased in several autoimmune disorders. It was recently reported that some patients with haemolytic uraemic syndrome (HUS) also fail to degrade NETs and that neutrophils from Shiga toxin-associated HUS are primed to form NETs. METHOD: We used a well-characterized cohort of 74 thrombotic microangiopathy (TMA) patients, with a subset also providing follow-up samples, and 112 age-matched controls to investigate NET degradation and serum nuclease activity in TMA before, during and after treatment. RESULTS: We identified that in the cohort of TMA patients, 50% of patients with Shiga toxin-associated HUS displayed a decreased ability to degrade NETs. NET degradation correlated with serum nuclease activity, but not with autoantibodies against double-stranded DNA, which has been previously observed in some autoimmune disorders. Further, NET degradation negatively correlated with serum creatinine levels, suggesting that kidney function was negatively impacted by the low NET degradation ability. CONCLUSIONS: We revealed that decreased NET degradation is a common feature of Shiga toxin-associated HUS and that it is associated with decreased kidney function in these patients. It remains to be clarified whether improving NET degradation would be beneficial for the patient.
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Trampas Extracelulares/metabolismo , Síndrome Hemolítico-Urémico/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Microangiopatías Trombóticas/inmunología , Adolescente , Adulto , Anciano , Células Cultivadas , Niño , Preescolar , Estudios de Cohortes , Desoxirribonucleasas/metabolismo , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Toxina Shiga/inmunología , Adulto JovenRESUMEN
BACKGROUND: The efficacy and safety of anticoagulant treatment is not established for patients with acute symptomatic deep vein thrombosis (DVT) of the calf. We aimed to assess whether therapeutic anticoagulation is superior to placebo in patients with symptomatic calf DVT. METHODS: In this randomised, double-blind, placebo-controlled trial, we enrolled low-risk outpatients (without active cancer or previous venous thromboembolic disease) with a first acute symptomatic DVT in the calf from 23 university medical centres or community medical clinics in Canada, France, and Switzerland. We randomly assigned (1:1) patients to receive either the low-molecular-weight heparin nadroparin (171 UI/kg, subcutaneously, once a day) or placebo (saline 0·9%, subcutaneously, once a day) for 6 weeks (42 days). Central randomisation was done using a computer-generated randomisation list, stratified by study centre. Random allocation sequences of variable block size were centrally determined by an independent research clinical centre. Study staff, patients, and outcome assessors (central adjudication committee) were masked to group assignment. Numbered boxes of active drug or placebo were provided to pharmacies in identical packaging. All patients were prescribed compression stockings and followed up for 90 days. The primary efficacy outcome was a composite measure of extension of calf DVT to proximal veins, contralateral proximal DVT, and symptomatic pulmonary embolism at day 42 in the modified intention-to-treat population. The primary safety outcome was major or clinically relevant non-major bleeding at day 42. The trial was registered with ClinicalTrials.gov, number NCT00421538. FINDINGS: Between Feb 1, 2008, and Nov 30, 2014, we screened 746 patients, enrolling 259 patients (50% of the prespecified sample size), before the trial steering committee terminated the trial because of expiry of study drug and slow recruitment. The intention-to-treat analysis population comprised 122 patients in the nadroparin group and 130 in the placebo group. There was no significant difference between the groups in the composite primary outcome, which occurred in four patients (3%) in the nadroparin group and in seven (5%) in the placebo group (risk difference -2·1%, 95% CI -7·8 to 3·5; p=0·54). Bleeding occurred in five patients (4%) in the nadroparin group and no patients in the placebo group (risk difference 4·1, 95% CI 0·4 to 9·2; p=0·0255). In the nadroparin group one patient died from metastatic pancreatic cancer and one patient was diagnosed with heparin-induced thrombocytopenia type 2. INTERPRETATION: Nadroparin was not superior to placebo in reducing the risk of proximal extension or venous thromboembolic events in low-risk outpatients with symptomatic calf DVT, but did increase the risk of bleeding. Avoidance of systematic anticoagulation for calf DVT could have a substantial impact on individual patients and from a public health perspective. FUNDING: Swiss National Science Foundation, the Programme Hospitalier de Recherche Clinique in France, and the Canadian Institutes of Health Research.