Serum PRO-C3 is useful for risk prediction and fibrosis assessment in MAFLD with chronic kidney disease in an Asian cohort.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is an emerging risk factor for chronic kidney disease (CKD). N-terminal propeptide of collagen type 3 (PRO-C3) is a biomarker of advanced fibrosis in MAFLD and PRO-C3 may be involved in renal fibrosis. We aimed to use PRO-C3 measurements to generate a new algorithmic score to test the prediction of MAFLD with chronic kidney disease (MAFLD-CKD). METHODS: A derivation and independent validation cohort of 750 and 129 Asian patients with biopsy-confirmed MAFLD were included. Serum PRO-C3 concentration was measured and regression analyses were performed to examine associations with MAFLD-CKD. A derivative algorithm for MAFLD-CKD risk prediction was evaluated with receiver operator characteristic (ROC) curve analysis. RESULTS: The study included two Asian cohorts (n = 180 with MAFLD-CKD; mean-eGFR: 94.93 mL/min/1.73 m2; median-urinary albumin-to-creatinine ratio: 6.58 mg/mmol). PRO-C3 was associated with the severity of MAFLD-CKD and independently associated with MAFLD-CKD (adjusted odds ratio = 1.16, 95% confidence interval [CI]: 1.08-1.23, p < .001). A new non-invasive score (termed PERIOD) including PRO-C3 efficiently predicted MAFLD-CKD (AUROC = .842, 95% CI: .805-.875). Accuracy, specificity and negative predictive values were 80.2%, 85.1% and 88.4%, respectively. In the validation cohort, the PERIOD score had good diagnostic performance (AUROC = .807, 95% CI: .691-.893) with similar results in all patient subgroups. In the MAFLD-CKD subgroup, the accuracy for identifying advanced fibrosis was further improved by combining the PRO-C3-based ADAPT with the Agile 3+ scores (AUROC = .90, 95% CI: .836-.964). CONCLUSIONS: The PERIOD score is helpful for accurately predicting the risk of MAFLD-CKD. PRO-C3 can also be used to assess liver fibrosis in people with MAFLD-CKD.

Pegcetacoplan (EMPAVELI®) para la hemoglobinuria paroxística nocturna / Pegcetacoplan (EMPAVELI®) for Paroxysmal Nocturnal Hemoglobinuria

INTRODUCCIÓN: La hemoglobinuria paroxística nocturna (HPN) es una enfermedad clonal no maligna de la hemopoyesis que se origina a partir de una mutación del gen PIG-A en una célula madre hemopoyética.1 Esta mutación, impide la síntesis del ancla glicosil-fosfatidil-inositol (GPI) que mantiene unidas a la membrana celular a múltiples proteínas. Entre dichas proteínas están el CD55 y el CD59 que constituyen defensas celulares contra componentes del complemento. La hemólisis intravascular crónica y/o paroxística, y la predilección por la trombosis, son causadas por la pérdida de los inhibidores del complemento CD55 y CD59 en la superficie de los glóbulos rojos. La HPN se clasifica según los antecedentes de enfermedad hematológica previa, la clínica y los hallazgos de los estudios complementarios, en dos grupos fisiopatológicos (con o sin hemólisis intravascular) y tres categorías clínicas (clásica, en el contexto de otra enfermedad medular o subclínica). Las cuatro manifestaciones clásicas de la HPN son la anemia por hemólisis intravascular, los episodios de hemoglobinuria, la leucopenia y/o plaquetopenia acompañantes de grado variable, y las trombosis con frecuencia en sitios inusuales. También, una serie de síntomas y signos deterioran la calidad de vida en estas personas como son la disnea, la fatiga, la disfagia, los episodios de dolor abdominal y la disfunción eréctil en varones.1 Por su valor pronóstico, los compromisos más importantes y potencialmente mortales son las trombosis, el progreso del fallo medular, el daño renal, la hipertensión pulmonar y la evolución clonal. Una fracción de pacientes también puede desarrollar anemia aplásica clínicamente grave o síndrome mielodisplásico, así como médula ósea hipocelular o displásica. TECNOLOGÍA: Pegcetacoplan (EMPAVELI®) es una fármaco que se une a la proteína del complemento C3 y su fragmento de activación C3b, regulando así la escisión de C3 y la generación de la activación del complemento. En la HPN, la hemólisis extravascular se ve facilitada por la opsonización C3b, mientras que la hemólisis intravascular está mediada por el complejo de ataque de membrana cascada abajo. Pegcetacoplan actúa proximalmente en la cascada del complemento controlando tanto la hemólisis extravascular mediada por C3b como la hemólisis intravascular terminal mediada por el complemento. OBJETIVO: El objetivo del presente informe es evaluar rápidamente los parámetros de eficacia, seguridad, costos y recomendaciones disponibles acerca del empleo de pegcetacoplan (EMPAVELI®) para el tratamiento de personas con la hemoglobinuria paroxística nocturna. MÉTODOS: Se realizó una búsqueda bibliográfica en las principales bases de datos tales como PUBMED, LILACS, BRISA, COCHRANE, SCIELO, EMBASE, TRIPDATABASE como así también en sociedades científicas, agencias reguladoras, financiadores de salud y agencias de evaluación de tecnologías sanitarias. Se priorizó la inclusión de revisiones sistemáticas, ensayos clínicos controlados aleatorizados, evaluación de tecnología sanitaria y guías de práctica clínica de alta calidad metodológica. La fecha de búsqueda de información fue hasta el 27 de mayo de 2022. RECOMENDACIONES: No se hallaron guías de práctica clínica actualizadas en Argentina y en el Mundo que mencionen la tecnología en la indicación evaluada. La Sociedad Argentina de Hematología en sus Guías de Diagnóstico y Tratamiento publicada en 2021 no menciona al fármaco, sin embargo, destaca que el eculizumab a logrado en tres ensayos clínicos beneficios importantes como el bloqueo de la hemólisis intravascular, la mejoría de la fatiga y de la disnea, la reducción de los requerimientos transfusionales, el aumento de los niveles de hemoglobina, una reducción mayor al 80% de eventos tromboembólicos, una mejoría o estabilización de la función renal en pacientes con deterioro de la misma, una reducción de los niveles del péptido natriurético cerebral, un marcado descenso de la presión arterial pulmonar, y un aumento de la sobrevida de los pacientes sin modificación de la evolución clonal a mielodisplasia o a leucemia mieloide aguda. Instituto Nacional para la Excelencia en Salud y Atención (NICE, su sigla del inglés National Institute for Health and Care Excellence) de Reino Unido recomienda al pegcetacoplan, como una opción para el tratamiento de la HPN, en adultos que presentan anemia después de al menos tres meses de tratamiento con un inhibidor de C5 (eculizumab y ravulizumab) solo si la empresa cumple con el acuerdo comercial estipulado. CONCLUSIONES: La evidencia que sustenta la aprobación de comercialización de pegcetacoplan (EMPAVELI®) en adultos con hemoglobinuria paroxística nocturna por parte de las agencias regulatorias relevadas se basa en un solo estudio de Fase III controlado, abierto y multicéntrico frente a eculizumab (única alternativa autorizada en nuestro país), en pacientes con anemia a pesar del tratamiento con eculizumab. Este estudio demuestra que pegcetacoplan es mejor al eculizumab para el cambio en el nivel de hemoglobina y para la menor necesidad de transfusiones al mediano plazo. No hay evidencia sobre el efecto del tratamiento en otros desenlaces importantes demostrados por el eculizumab o para seguimientos más largos. Las agencias regulatorias relevadas han autorizado recientemente la comercialización de pegcetacoplan bajo condiciones especiales. Estados Unidos la ha autorizado en programa restringido bajo la Estrategia de Evaluación y Mitigación de Riesgos, mientras que Europa solo en pacientes que presenten anemia después del tratamiento con un inhibidor de C5 (eculizumab y ravulizumab) durante al menos tres meses. Las guías de practica clínicas relevadas no mencionan el fármaco, mientras que el Instituto Nacional para la Excelencia en Salud y Atención de Reino Unido lo recomienda como una opción para el tratamiento de adultos con hemoglobinuria paroxística nocturna que presenten anemia después de al menos tres meses de tratamiento con eculizumab o ravulizumab, y se cumpla el acuerdo comercial con el productor de la tecnología. El costo del tratamiento es elevado y se desconoce el precio de la bomba de infusión específica para la administración de este fármaco.

Serum Interleukin-26 Is a New Biomarker for Disease Activity Assessment in Systemic Lupus Erythematosus.

Objective: Interleukin-26 (IL-26) has a unique ability to activate innate immune cells due to its binding to circulating double-stranded DNA. High levels of IL-26 have been reported in patients with chronic inflammation. We aimed to investigate IL-26 levels in patients with systemic lupus erythematosus (SLE). Methods: IL-26 serum levels were quantified by ELISA for 47 healthy controls and 109 SLE patients previously enrolled in the PLUS study. Performance of IL-26 levels and classical markers (autoantibodies or complement consumption) to identify an active SLE disease (SLE disease activity index (SLEDAI) score > 4) were compared. Results: IL-26 levels were significantly higher in SLE patients than in controls (4.04 ± 11.66 and 0.74 ± 2.02 ng/mL; p = 0.005). IL-26 levels were also significantly higher in patients with active disease than those with inactive disease (33.08 ± 21.06 vs 1.10 ± 3.80 ng/mL, p < 0.0001). IL-26 levels correlated with SLEDAI score and the urine protein to creatinine ratio (uPCR) (p < 0.001). Patients with high IL-26 levels had higher SLEDAI score, anti-DNA antibodies levels, and uPCR (p < 0.05). They presented more frequently with C3 or C4 complement consumption. Lastly, IL-26 showed stronger performance than classical markers (complement consumption or autoantibodies) for active disease identification. Conclusions: Our results suggest that, in addition to classical SLE serological markers, the measurement of IL-26 levels may be a useful biomarker for active disease identification in SLE patients.

The utility of immunoglobulin A/complement 3 and immunoglobulin G/immunoglobulin M ratios in the assessment of disease activation in patients with Behçet disease.

OBJECTIVE: Pathogenesis of Behçet disease (BD) has not yet been clearly revealed and there is no ideal test for the estimation of disease activation at present. This study aimed to assess the efficiencies of IgG/IgM and IgA/C3 ratios in determining activation of BD. METHOD: This retrospective cohort study consisted of 140 patients with BD. Patients were divided into two groups: (1) active BD (n = 89) and (2) inactive BD (n = 51) and were compared in terms of demographic features, clinical characteristics and laboratory test results. IgA/C3 and IgG/IgM ratios were compared according to organ system involvement; receiver operating characteristic (ROC) curve analysis was performed in order to assess the performance of IgA/C3 and IgG/IgM ratios in determining patient disease status. RESULTS: Significantly higher levels of erythrocyte sedimentation rate, C-reactive protein, IgA, G, C4, IgA/C3, IgG/IgM ratios (p = .007 for IgA and p < .001 for others) and significantly lower levels of IgM and C3 were observed in patients with active BD (p < .001). The IgG/IgM ratio was significantly higher in patients with vascular involvement (p = .017) and the IgA/C3 ratio was significantly higher in patients with arthritis (p = .007). Cut-off values of 0.019 (70.8% sensitivity, 62% specificity) and 7.08 (84.3% sensitivity, 80% specificity) were determined for IgA/C3 and IgG/IgM ratios, respectively. CONCLUSION: IgA/C3 and IgG/IgM ratios may be used as additional parameters for the assessment of BD status.

In vivo and in vitro assessment of host-parasite interaction between the parasitic copepod Brachiella thynni (Copepoda; Lernaeopodidae) and the farmed Atlantic bluefin tuna (Thunnus thynnus).

The Atlantic bluefin tuna (ABFT; Thunnus thynnus) today represents one of the economically most important species for Croatian fisheries industry. Although the most diverse and abundant parasitofauna is usually found in the largest specimens of wild ABFT, the opposite was observed in captivity where parasite populations significantly decline by the end of the farming cycle. Copepod Brachiella thynni, is a skin parasite frequently parasitizing tuna, whose population also decreases in number throughout the rearing process. In order to better understand the immunity mechanisms underlying ABFT reaction to B. thynni infection, we studied expression profiles of immunity related genes; interleukin 1ß (il1ß), tumour necrosis factors (tnfα1, tnfα2), complement component 4 (c4) and caspase 3 (casp3), in peripheral blood leukocytes (PBLs) during in vitro stimulation by B. thynni protein extracts (i.e. antigens) and in infected tissues at B. thynni parasitation site. Finally, a histopathological analysis of semi-thin and ultra-thin sections of tissues surrounding B. thynii attachment site was performed to evaluate the severity of parasite-induced lesions and identify involved cell lineages. In vitro stimulation of ABFT PBLs with B. thynii antigens caused a dose-depended upregulation of selected genes, among which tnfα1 showed the highest induction by both concentrations of B. thynni protein extract. However, targeted genes were not significantly upregulated in the infected tissue. Also, no significant alterations in ultrastructure of epithelial layers surrounding B. thynii attachment site were noticed, except local tissue erosion, necrosis of squamous epithelium and proliferation of rodlet and goblet cells. Our results suggest that B. thynii has evolved strategies to successfully bypass both innate immune response and the connective-tissue proliferation processes. Therefore, the observed disappearance of this copepod by the end of the rearing process is more likely related to its limited lifespan on the host and its inability to complete the life cycle in the rearing cages, rather than host's reaction.

Assessment of the Role of C3(H2O) in the Alternative Pathway.

In this study we investigate the hydrolysis of C3 to C3(H2O) and its ability to initiate activation via the alternative pathway (AP) of the complement system. The internal thioester bond within C3 is hydrolyzed by water in plasma because of its inherent lability. This results in the formation of non-proteolytically activated C3(H2O) which is believed have C3b-like properties and be able to form an active initial fluid phase C3 convertase together with Factor B (FB). The generation of C3(H2O) occurs at a low but constant rate in blood, but the formation can be greatly accelerated by the interaction with various surfaces or nucleophilic and chaotropic agents. In order to more specifically elucidate the relevance of the C3(H2O) for AP activation, formation was induced in solution by repeated freeze/thawing, methylamine or KCSN treatment and named C3(x) where the x can be any of the reactive nucleophilic or chaotropic agents. Isolation and characterization of C3(x) showed that it exists in several forms with varying attributes, where some have more C3b-like properties and can be cleaved by Factor I in the presence of Factor H. However, in common for all these variants is that they are less active partners in initial formation of the AP convertase compared with the corresponding activity of C3b. These observations support the idea that formation of C3(x) in the fluid phase is not a strong initiator of the AP. It is rather likely that the AP mainly acts as an amplification mechanism of complement activation that is triggered by deposition of target-bound C3b molecules generated by other means.

Assessment of serum sialic acid correlated with C3 in children with Mycoplasma pneumoniae pneumonia.

BACKGROUND: Different from the diagnosis of bacterial infections, Mycoplasma pneumoniae pneumonia (MPP) is still lacking of convenient non-specific laboratory parameters. METHOD: A total of 125 children with MPP were included in the MPP group and 89 children with Mycoplasma-negative pneumonia were included in the control group, and the sera were collected from the children at both the acute and recovery stages in the two groups. RESULTS: The sialic acid and C3 in the MPP group were significantly higher than those in the control group both at the acute and at the recovery stage. On the other hand, the sialic acid and C3 at the acute stage were significantly higher than those at the recovery stage in the MPP group. However, in the control group, the sialic acid and C3 demonstrated IgG exhibited no significant change between the acute stage and the recovery stage. Lastly, positive correlations between sialic acid level and C3 level were identified in the MPP group at both acute and recovery stages. CONCLUSION: Our study demonstrated that the serum sialic acid correlated with C3 specifically increased in children with MPP, indicating that it might be the important non-specific parameters in the diagnosis of MPP.

Performance evaluation of 14 specific proteins measurement checked by an External Quality Assessment Scheme.

AIMS OF THE STUDY: To evaluate the state-of-the-art of 14 specific proteins measurement; to evaluate the laboratories' performance and the degree of harmonization in reporting results of participants in the External Quality Assessment Program of the Centre of Biomedical Research (CRB). METHODS: Overall and system-related inter-laboratory analytical variability (mean CVs%) and between-system differences (mean bias%) were evaluated from data of six EQA cycles 2013-2018. Moreover, we evaluated the analytical performance of participants as well as the units used to express proteins results. RESULTS: Overall inter-laboratory variability ranged from 3.8% for haptoglobin (HPT) to 12.5% for α1-antitrypsin (AAT) and decreased for IgA, α2-macroglobulin (A2M) and transferrin (TRF). Mean CVs% were generally higher for Siemens BN and Beckman Immage immunonephelometric systems, but <7.0% for all proteins. Mean bias > 7.0% was observed for BN (IgA, C4, AAT, transthyretin TTR), Siemens Vista (IgA, C4) and Immage (C4), whereas mean bias < -7.0% was found for Immage (AAT), Beckman AU (IgM) and Roche Cobas (C4, TTR, C-reactive protein). The laboratories' performance within the limits ranged from 85.1% of albumin (ALB) to 97.2% of HPT. The census of units employed in 2018, demonstrated that ~ 70% of laboratories still express the results in mg/dL. CONCLUSIONS: Despite a reduction in inter-laboratory variability for some proteins, different analytical systems showed both proportional and constant bias between methods. Units used by participants have not been substantially changed and dL is still largely used. The CRB EQA Program, with its performance data sets, is a valuable resource for laboratories and IVD manufacturers and support the goals of harmonization.

Assessment of Complement Activation by Nanoparticles: Development of a SPR Based Method and Comparison with Current High Throughput Methods.

PURPOSE: A Surface Plasmon Resonance chip (SPR) was developed to study the activation of complement system triggered by nanomaterials in contact with human serum, which is an important concern today to warrant safety of nanomedicines. METHODS: The developed chip was tested for its specificity in complex medium and its longevity of use. It was then employed to assess the release of complement fragments upon incubation of nanoparticles in serum. A comparison was made with other current methods assessing complement activation (µC-IE, ELISA). RESULTS: The SPR chip was found to give a consistent response for C3a release upon activation by nanoparticles. Results were similar to those obtained by µC-IE. However, ELISA detection of iC3b fragments showed an explained high non-specific background. The impact of sample preparation preceding the analysis was assessed with the newly develop SPR method. The removal of nanoparticles before analysis showed an important modification in the obtained response, possibly leading to false negative results. CONCLUSION: The SPR chip developed in this work allows for an automated assessment of complement activation triggered by nanoparticles with possibility of multiplexed analysis. The design of the chip proved to give consistent results of complement activation by nanoparticles.

Evolution of compstatin family as therapeutic complement inhibitors.

INTRODUCTION: Therapeutic modulation of complement activation is considered as a promising approach for the treatment of host tissue damage in several inflammatory and autoimmune diseases. Complement component protein C3 is a particularly attractive drug target for complement inhibitors, due to its central role in three pathways of complement activation cascade. Areas covered: The author provides a comprehensive review on compstatin family peptides which have been discovered and optimized as potent and selective C3 inhibitors via a combination of chemical, biophysical and computational approaches. New generations of the compstatin family with improved potency and therapeutic properties have been developed in recent years. Over two decades, compstatin demonstrated therapeutic potential as a first-of-its-kind complement inhibitor in a series of disease models, with encouraging efforts in clinical trials. Expert opinion: Compstatin holds promise for new therapeutic implications in blocking the effect of the complement cascade in a variety of disease conditions. The development of cost-effective treatment options with suitable dosing route and schedule will be critical for patients with complement mediated chronic diseases.

Complement factor 3 among children with hepatitis A: Assessment of bilirubin levels.

OBJECTIVE: To evaluate the complement factor 3 levels in children with hepatitis A. METHODS: This observational study was conducted at the Infectious Diseases Hospital of Hotan District, China, from September 2014 to January 2015, and comprised children with hepatitis A and controls. The patients were divided into two groups. The ones with total bilirubin less than or equal to 2mg/dl comprised group A, while the ones whose total bilirubin was more than 2mg/dl was named group B. Besides, we enrolled age- and gender-matched healthy children as controls. SPSS 13 was used for data analysis. RESULTS: Of the 100 participants, 41(41%) were in group A, 29(29%) in group B and 30(30%) were controls. The serum level of alanine aminotransferase, aspartate aminotransferase, total bile acid, the incidence of ascites and the incidence of hepatic encephalopathy were significantly increased in patients of group B when compared to group A (p=0.046, p=0.009, p<0.0001, p=0.018 and p=0.026). The levels of prothrombin time activity, total protein and albumin were higher in group A (p<0.0001, p<0.0001, and p <0.0001). Total hepatitis A patients had significantly lower serum complement factor 3 levels compared to normal controls (p =0.018). Group B had significantly lower serum complement factor 3 levels compared to normal controls (p <0.0001) and group A (p<0.0001). In total patients, complement factor 3 levels were negatively correlated with total bilirubin and alanine aminotransferase (p=0.029), while complement factor 3 levels were positively correlated with prothrombin time activity (p=0.001). CONCLUSIONS: Complement factor 3 values were found to be decreased in children hospitalised with hyperbilirubinaemia hepatitis A.

Dietary inflammatory index and inflammatory biomarkers in adolescents from LabMed physical activity study.

BACKGROUND/OBJECTIVES: The dietary inflammatory index (DII) is a tool to measure the diet's inflammatory potential and has been used with adults to predict low-grade inflammation. The present study aims to assess whether this dietary score predicts low-grade inflammation in adolescents. SUBJECTS/METHODS: The sample comprises 329 adolescents (55.9% girls), aged 12-18 years, from LabMed Physical Activity Study. DII score was calculated based on a food-frequency questionnaire and categorized into tertiles. We collected blood samples to determine the follow inflammatory biomarkers: C-reactive protein (CRP), interleukin-6 (IL-6), complement component 3 (C3), and 4 (C4). In addition we calculated an overall inflammatory biomarker score. Odds ratios (OR) and 95% confidence intervals (95%CI) were computed from binary logistic regression models. RESULTS: DII score, comparing first with third tertile, was positively associated with IL-6 in crude model (OR = 1.88, 95%CI:1.09-3.24, p trend = 0.011) and in fully adjusted (for biological and lifestyle variables) (OR = 3.38, 95%CI:1.24-9.20, p trend = 0.023). Also, DII score was positively associated with C4, when fully adjusted (OR = 3.12, 95%CI:1.21-8.10, p trend = 0.016). DII score was negatively associated with C3 in crude model, comparing first with second but not with third tertile, and no significant associations in fully adjusted model were observed, although a trend was found (OR = 1.71, 95%CI:0.63-4.66, p trend = 0.044). No significant associations were observed between DII score and CRP. However, DII score was positively associated with the overall inflammatory biomarker score, when fully adjusted (OR = 5.61, 95%CI:2.00-15.78, p trend = 0.002). CONCLUSIONS: DII score can be useful to assess the diet's inflammatory potential and its association with low-grade inflammation in adolescents.

Assessment of immune status of yellowfin seabream (Acanthopagrus latus) during short term exposure to phenanthrene.

The aim of the present investigation was to assess the immune status in yellowfin seabream (Acanthopagrus latus) exposed to different concentrations of phenanthrene (Phe) for 14days. In addition, the Phe accumulation in the fish muscle was measured during the experiment. Fish were injected with different concentrations (0, 2, 20 and 40mg/kg) of Phe and samples were taken from tissue and blood of fish 1, 4, 7 and 14days after injection. Exposure of fish to Phe caused a significant decrease in white blood cells, C3 and C4 levels, lysosomal membrane stability, lysozyme activity after 4days and antibacterial activity after 7days of the experiment. In contrast, cortisol level significantly increased after 4days. The concentration of Phe in fish muscle increased rapidly after 4days. The main tissue changes observed in the head kidney including increase in melanomacrophage centers (MMCs), empty spaces between cells and hemorrhage. The degree of tissue changes ranged from normal to moderate in Phe-treated fish. The size and number of MMCs in treated fish were significantly higher than control. In conclusion, Phe toxicity in yellowfin seabream can induce increased cortisol level, tissue changes and immune suppression.

Targeted Intraceptor Nanoparticle for Neovascular Macular Degeneration: Preclinical Dose Optimization and Toxicology Assessment.

Neovascular age-related macular degeneration (AMD) is treated with anti-VEGF intravitreal injections, which can cause geographic atrophy, infection, and retinal fibrosis. To minimize these toxicities, we developed a nanoparticle delivery system for recombinant Flt23k intraceptor plasmid (RGD.Flt23k.NP) to suppress VEGF intracellularly within choroidal neovascular (CNV) lesions in a laser-induced CNV mouse model through intravenous administration. In the current study, we examined the efficacy and safety of RGD.Flt23k.NP in mice. The effect of various doses was determined using fluorescein angiography and optical coherence tomography to evaluate CNV leakage and volume. Efficacy was determined by the rate of inhibition of CNV volume at 2 weeks post-treatment. RGD.Flt23k.NP had peak efficacy at a dose range of 30-60 µg pFlt23k/mouse. Using the lower dose (30 µg pFlt23k/mouse), RGD.Flt23k.NP safety was determined both in single-dose groups and in repeat-dose (three times) groups by measuring body weight, organ weight, hemoglobin levels, complement C3 levels, and histological changes in vital organs. Neither toxicity nor inflammation from RGD.Flt23k.NP was detected. No side effect was detected on visual function. Thus, systemic RGD.Flt23k.NP may be an alternative to standard intravitreal anti-VEGF therapy for the treatment of neovascular AMD.

Assessment of Relationship between Wilms' Tumor Gene (WT1) Expression in Peripheral Blood of Acute Leukemia Patients and Serum IL-12 and C3 Levels.

BACKGROUND: Leukemia is a common cancer among children and adolescents. Wilms' tumor gene (WT1) is highly expressed in patients with acute leukemia. It is found as a tumor associated antigen (TAA) in various types of hematopoietic malignancies and can be employed as a useful marker for targeted immunotherapy and monitoring of minimal residual disease (MRD). In this regard, WT1 is a transcription factor that promotes gene activation or repression depending on cellular and promoter context. The purpose of this study was assessment of WT1 gene expression in patients with acute leukemia, measurement of IL-12 and C3 levels in serum and evaluation of the relationship between them. MATERIALS AND METHODS: We evaluated the expression of WT1 mRNA using real-time quantitative RT-PCR and serum levels of IL-12 and C3 using ELISA and nephelometry in peripheral blood of 12 newly diagnosed patients with acute leukemia and 12 controls. RESULTS: The results of our study showed that the average wT1 gene expression in patients was 7.7 times higher than in healthy controls (P <0.05). In addition, IL-12 (P = 0.003) and C3 (P <0.0001) were significantly decreased in the test group compared to controls. CONCLUSIONS: WT1 expression levels are significantly higher in patients compared with control subjects whereas serum levels of interleukin-12 and C3 are significantly lower in patients. Wt1 expression levels in patients are inversely related with serum levels of IL-12 and C3.

In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.

Complement C3 depletion links to the expansion of regulatory T cells and compromises T-cell immunity in human abdominal sepsis: a prospective pilot study.

PURPOSE: Complement depletion commonly occurred in septic patients, but this problem was often underestimated during the treatment process. This study was designed to determine the association between complement depletion and T-cell immunosuppression. METHODS: From November 2011 to March 2012, patients with severe abdominal sepsis were prospectively enrolled in a single center. The baseline levels of complement C3 were used to stratify subjects into 2 groups. Plasma levels of complement components, percentage of regulatory T cells (Tregs), and T-cell immunity indexes were monitored at times after admission. The relationship between C3 depletion and T-cell response was investigated, with clinical outcomes explored meanwhile. RESULTS: A total of 60 patients aged 43.9 ± 11.3 years were included within the period. C3 depletion, occurring in 65% of enrolled subjects, was strongly correlated with Treg expansion (P = .001) and decreased CD4(+)/CD8(+) ratio (P = .008). This depletion was also related to prolonged hospital stay (P = .001), delayed time to operation (P < .001), increased postoperative complications (P = .036), and hospital expenditure (P < .001). CONCLUSIONS: Complement C3 depletion was found to be linked to the expansion of Tregs during abdominal sepsis. Such depletion and associated immunosuppression should be paid close attention in the critical care.

Risk assessment model for development of advanced age-related macular degeneration.

OBJECTIVE: To design a risk assessment model for development of advanced age-related macular degeneration (AMD) incorporating phenotypic, demographic, environmental, and genetic risk factors. METHODS: We evaluated longitudinal data from 2846 participants in the Age-Related Eye Disease Study. At baseline, these individuals had all levels of AMD, ranging from none to unilateral advanced AMD (neovascular or geographic atrophy). Follow-up averaged 9.3 years. We performed a Cox proportional hazards analysis with demographic, environmental, phenotypic, and genetic covariates and constructed a risk assessment model for development of advanced AMD. Performance of the model was evaluated using the C statistic and the Brier score and externally validated in participants in the Complications of Age-Related Macular Degeneration Prevention Trial. RESULTS: The final model included the following independent variables: age, smoking history, family history of AMD (first-degree member), phenotype based on a modified Age-Related Eye Disease Study simple scale score, and genetic variants CFH Y402H and ARMS2 A69S. The model did well on performance measures, with very good discrimination (C statistic = 0.872) and excellent calibration and overall performance (Brier score at 5 years = 0.08). Successful external validation was performed, and a risk assessment tool was designed for use with or without the genetic component. CONCLUSIONS: We constructed a risk assessment model for development of advanced AMD. The model performed well on measures of discrimination, calibration, and overall performance and was successfully externally validated. This risk assessment tool is available for online use.

Alternative complement pathway assessment in patients with atypical HUS.

The atypical Hemolytic Uremic Syndrome (aHUS) is a rare thrombotic microangiopathy leading to end stage renal disease in approximately 60% of patients. Over the last decade, a clear link has been demonstrated between this disease and defective complement regulation. The hallmark of the aHUS is the association with mutations in complement alternative pathway genes. Endothelial damage is related to complement dysregulation, but the exact mechanism is just starting to be elucidated. Screening for and characterization of mutations in the components of the C3 convertase (C3 and FB) or its regulators (FH, FI, MCP, and Thrombomodulin) or anti-FH antibodies has become an indispensable part of the disease's diagnostic. This review will initially summarize current knowledge on the understanding of complement activation and regulation, followed by a description on the genetic analysis as well as the methods used for complement protein quantification. Another part of this review will focus on the mechanisms of action of aHUS-associated mutations. We will emphasize on when and why some mutations lead to protein deficiency, while others result in - to dysfunctional but normally expressed proteins. Finally, we will discuss how the therapy of aHUS patients can be modified according to the functional consequences of each particular genetic defect.

Assessment of the transcription levels for the complement activation control system in eutopic endometrium in women with two or more consecutive miscarriages of unknown etiology.

Human endometrium, deciuda and placenta have been shown to express factors that inhibit the complement activation cascade - decay-accelerating factor (DAF), membrane cofactor protein (MCP) and the C3 complement component. In the following study we have analyzed the transcripts levels for DAF, MCP and heparin-binding epidermal growth factor-like growth factor (HB-EGF), the C3 complement component and receptor for vascular endothelial growth factor (VEGFR1) as markers of endometrial unbalance between factors activating the complement system in women with consecutive miscarriages. Study enrolled 30 women with at least two consecutive miscarriages, and 19 healthly women, that comprised the control group. RNA was isolated from endometrial samples. Transcripts levels of DAF and MCP was higher in women with consecutive miscarriages compared to controls, 0.78 vs 5.08 (p<0.001) and 0.25 vs 0.17 (p=0.001) respectively. In consecutive miscarriages group, DAF and MCP expression was correlated with the C3 expression, with r=0.60; p<0.001 and r= 0.40; p=0.03 respectively. Correlation between DAF and C3 was also noted in controls, 0.70; p=0.001. In women with two or more consecutive miscarriages the analysis proved higher expression of genes that encode proteins that inhibit the complement cascade. Further studies are needed to confirm that this might be a reaction to increased presence of the complement factors, which like C3 that are synthesized in the endometrium.