Effect of total flavonoids of Dracocephalum moldavica L. On neuroinflammation in Alzheimer's disease model amyloid-ß (Aß1-42)-peptide-induced astrocyte activation.

One of the main pathological features noted in Alzheimer's disease (AD) is the presence of plagues of aggregated ß-amyloid (Aß1-42)-peptides. Excess deposition of amyloid-ß oligomers (AßO) are known to promote neuroinflammation. Sequentially, following neuroinflammation astrocytes become activated with cellular characteristics to initiate activated astrocytes. The purpose of this study was to determine whether total flavonoids derived from Dracocephalum moldavica L. (TFDM) inhibited Aß1-42-induced damage attributed to activated C8-D1A astrocytes. Western blotting and ELISA were used to determine the expression of glial fibrillary acidic protein (GFAP), and complement C3 to establish the activation status of astrocytes following induction from exposure to Aß1-42. Data demonstrated that stimulation of C8-D1A astrocytes by treatment with 40 µM Aß1-42 for 24 hr produced significant elevation in protein expression and protein levels of acidic protein (GFAP) and complement C3 accompanied by increased expression and levels of inflammatory cytokines. Treatment with TFDM or the clinically employed drug donepezil in AD therapy reduced production of inflammatory cytokines, and toxicity initiated following activation of C8-D1A astrocytes following exposure to Aß1-42. Therefore, TFDM similar to donepezil inhibited inflammatory secretion in reactive astrocytes, suggesting that TFDM may be considered as a potential compound to be utilized in AD therapy.

Inhibition of complement C3 prevents osteoarthritis progression in guinea pigs by blocking STAT1 activation.

Osteoarthritis (OA) is one of the leading causes of disability, affecting over 500 million adults worldwide. Previous studies have found that various inflammatory factors can contribute to the pathogenesis of OA, including complement factors in the synovial fluid of OA patients. However, the pathogenesis of this disease is still not known, and the only therapy of severe OA is total joint replacements. Total joint replacements are invasive, expensive, and affect quality of life. Here we show that when human articular chondrocytes are stimulated with pro-inflammatory mediator interleukin-1ß (IL-1ß) there is an increase in inflammatory factors including complement component 3 (C3). We also found the transcription factor, signal transducer and activator of transcription 1 (STAT1), is responsible for increased C3 expression after IL-1ß stimulation in human articular chondrocytes. A specific STAT1 inhibitor, fludarabine, attenuates the hyper-expression of C3 and delays/prevents spontaneous OA in Dunkin-Hartley guinea pigs. Since fludarabine is already clinically used for chemotherapy, this study has great translational potential as a unique disease-modifying osteoarthritis drug (DMOAD) in treating primary OA.

Normobaric hyperoxia alleviates complement C3-mediated synaptic pruning and brain injury after intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a common cerebrovascular disease, and the complement cascade exacerbates brain injury after ICH. As the most abundant component of the complement system, complement component 3 (C3) plays essential roles in all three complement pathways. However, the effects of C3 on neurological impairment and brain injury in ICH patients and the related mechanism have not been fully elucidated. Normobaric hyperoxia (NBO) is regarded as a treatment for ICH patients, and recent clinical studies also have confirmed the neuroprotective role of NBO against acute ICH-mediated brain damage, but the underlying mechanism still remains elusive. AIMS: In the present study, we investigated the effects of complement C3 on NBO-treated ICH patients and model mice, and the underlying mechanism of NBO therapy in ICH-mediated brain injury. RESULTS: Hemorrhagic injury resulted in the high plasma C3 levels in ICH patients, and the plasma C3 levels were closely related to hemorrhagic severity and clinical outcomes after ICH. BO treatment alleviated neurologic impairments and rescued the hemorrhagic-induced increase in plasma C3 levels in ICH patients and model mice. Moreover, the results indicated that NBO exerted its protective effects of on brain injury after ICH by downregulating the expression of C3 in microglia and alleviating microglia-mediated synaptic pruning. CONCLUSIONS: Our results revealed that NBO exerts its neuroprotective effects by reducing C3-mediated synaptic pruning, which suggested that NBO therapy could be used for the clinical treatment of ICH.

Metabolic syndrome in systemic lupus erythematosus patients under Mediterranean diet.

BACKGROUND AND AIMS: Metabolic syndrome (MetS) is a chronic proinflammatory and prothrombotic condition that exacerbates insulin resistance, oxidative damage, and cardiovascular risk, being more prevalent in patients with systemic lupus erythematosus (SLE), a chronic multisystemic autoimmune disorder. This study aim was to determine the prevalence of MetS and associations with SLE clinical characteristics, cardiovascular risk and dietary pattern in a population of Spanish SLE patients. DESIGN AND METHODS: Cross-sectional study of 293 patients was conducted (90.4% females; mean age 46.8 (12.94)). The diagnosis of MetS was established based on the criteria of the National Cholesterol Education Program Adult Treatment Panel III. SLE Disease Activity Index (SLEDAI-2K) and SDI Damage Index were used to assess disease activity and disease-related damage, respectively. Med Diet adherence was assessed through a 14 items questionnaire on food consumption frequency and habits. RESULTS: MetS was present in 15% SLE patients. Triglycerides, high-density lipoprotein cholesterol, systolic blood pressure and waist circumference were significantly increased (p<0.001) in the group of MetS patients. Patients with MetS showed significantly increased SDI damage index (1.70 (1.69) vs 0.88 (1.12), p<0.001) and complement C3 level (118.70 (32.67) vs 107.55 (26.82), p=0.011). No significant differences were observed according to Med Diet adherence level. CONCLUSION: We observed a lower prevalence of MetS in SLE than that reported in previous studies, which may be a result of the good level of adherence to the MedDiet in our study sample. Additionally, MetS was associated with higher SDI and complement C3 levels but no with medication use.

Effects of empagliflozin on collagen biomarkers in patients with heart failure: Findings from the EMPEROR trials.

AIMS: Extracellular matrix remodelling is one of the key pathways involved in heart failure (HF) progression. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) may have a role in attenuating myocardial fibrosis. The impact of SGLT2i on blood markers of collagen turnover in humans is not fully elucidated. This study aimed to investigate the effect of empagliflozin on serum markers of collagen turnover in patients enrolled in the EMPEROR-Preserved and EMPEROR-Reduced trials. METHODS AND RESULTS: Overall, 1084 patients (545 in empagliflozin and 539 in placebo) were included in the analysis. Procollagen type I carboxy-terminal propeptide (PICP), a fragment of N-terminal type III collagen (PRO-C3), procollagen type I amino-terminal peptide (PINP), a fragment of C-terminal type VIa3 collagen (PRO-C6), a fragment of type I collagen (C1M), and a fragment of type III collagen (C3M) were measured in serum at baseline, 12 and 52 weeks. A mixed model repeated measurements model was used to evaluate the effect of empagliflozin versus placebo on the analysed biomarkers. Higher baseline PICP, PRO-C6 and PINP levels were associated with older age, a more severe HF presentation, higher levels of natriuretic peptides and high-sensitivity troponin T, and the presence of comorbid conditions such as chronic kidney disease and atrial fibrillation. Higher PICP levels were associated with the occurrence of the study primary endpoint (a composite of HF hospitalization or cardiovascular death), and PRO-C6 and PINP were associated with the occurrence of sustained worsening of kidney function. On the other hand, PRO-C3, C1M, and C3M were not associated with worse HF severity or study outcomes. Compared to placebo, empagliflozin reduced PICP at week 12 by 5% and at week 52 by 8% (week 12: geometric mean ratio = 0.95, 95% confidence interval [CI] 0.91-0.99, p = 0.012; week 52: geometric mean ratio = 0.92, 95% CI 0.88-0.97, p = 0.003). Additionally, empagliflozin reduced PRO-C3 at week 52 by 7% (week 12: geometric mean ratio = 0.98, 95% CI 0.95-1.02, p = 0.42; week 52: geometric mean ratio = 0.93, 95% CI 0.89-0.98, p = 0.003), without impact on other collagen markers. CONCLUSION: Our observations are consistent with experimental observations that empagliflozin down-regulates profibrotic signalling. The importance of such an effect for the clinical benefits of SGLT2i in HF remains to be elucidated.

Complement Inhibition in Paroxysmal Nocturnal Hemoglobinuria (PNH): A Systematic Review and Expert Opinion from Central Europe on Special Patient Populations.

INTRODUCTION: Hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) is complement-mediated due to the lack of complement inhibitors in the hemopoietic cell membranes, making complement inhibition the best approach to manage PNH. Three complement inhibitors are approved by the European Medicines Agency as targeted therapy for PNH: eculizumab and ravulizumab, two humanized monoclonal antibodies targeting the same complement 5 (C5) epitope, approved in 2007 and 2019, respectively, and the more recently approved cyclic peptide, the complement 3 (C3) inhibitor pegcetacoplan. Although national and international PNH treatment guidelines exist, they do not take into consideration the latest clinical trial evidence. Given the lack of evidence-based data for some clinical situations encountered in real life, we identified specific populations of patients who may benefit from switching to proximal C3 from terminal C5 inhibition. METHODS: The expert recommendations presented here were created using a Delphi-like process by a group of expert PNH specialists across Central Europe. Based on an initial advisory board meeting discussion, recommendations were prepared and reviewed as part of a Delphi survey to test agreement. RESULTS: Using a systematic approach, literature databases were searched for relevant studies, and 50 articles were reviewed by the experts and included as supporting evidence. CONCLUSION: Implementation of these recommendations uniformly across healthcare institutions will promote the best use of complement inhibition in managing PNH, and has the potential to positively impact patient outcomes in Central Europe and worldwide.

Levels of extracellular matrix metabolites are associated with changes in Ankylosing Spondylitis Disease Activity Score and MRI inflammation scores in patients with axial spondyloarthritis during TNF inhibitor therapy.

BACKGROUND/PURPOSE: In axial spondyloarthritis (axSpA) inflammation of the sacroiliac joints and spine is associated with local extracellular matrix (ECM) remodeling of affected tissues. We aimed to investigate the association of ECM metabolites with treatment response in axSpA patients treated with TNF-α inhibitory therapy for 46 weeks. METHODS: In a prospective clinical study of axSpA patients (n=55) initiating a TNF inhibitor (infliximab, etanercept, or adalimumab), serum concentrations of formation of type I (PRO-C1), type III (PRO-C3), and type VI (PRO-C6) collagen; turnover of type IV collagen (PRO-C4), and matrix-metalloproteinase (MMP)-degraded type III (C3M) collagen, MMP-degraded type IV (C4M), type VI (C6M), and type VII (C7M) collagen, and cathepsin-degraded type X collagen (C10C), MMP-mediated metabolite of C-reactive protein (CRPM), citrullinated vimentin (VICM), and neutrophil elastase-degraded elastin (EL-NE) were measured at baseline, week 2, week 22, and week 46. RESULTS: Patients were mostly males (82%), HLA-B27 positive (84%), with a median age of 40 years (IQR: 32-48), disease duration of 5.5 years (IQR: 2-10), and a baseline Ankylosing Spondylitis Disease Activity Score (ASDAS) of 3.9 (IQR: 3.0-4.5). Compared to baseline, PRO-C1 levels were significantly increased after two weeks of treatment, C6M levels were significantly decreased after two and 22 weeks (repeated measures ANOVA, p=0.0014 and p=0.0015, respectively), EL-NE levels were significantly decreased after 2 weeks (p=0.0008), VICM levels were significantly decreased after two and 22 weeks (p=0.0163 and p=0.0374, respectively), and CRP were significantly decreased after two and 22 weeks (both p=0.0001). Baseline levels of PRO-C1, PRO-C3, C6M, VICM, and CRP were all associated with ASDAS clinically important and major improvement after 22 weeks (ΔASDAS ≥1.1) (Mann-Whitney test, p=0.006, p=0.008, p<0.001, <0.001, <0.001, respectively), while C6M, VICM and CRP levels were associated with ASDAS clinically important and major improvement after 46 weeks (ΔASDAS ≥2.0) (p=0.002, p=0.044, and p<0.001, respectively). PRO-C1 and C6M levels were associated with a Bath AS Disease Activity Score (BASDAI) response to TNF-inhibitory therapy after 22 weeks (Mann-Whitney test, p=0.020 and p=0.049, respectively). Baseline levels of PRO-C4 and C6M were correlated with the total SPARCC MRI Spine and Sacroiliac Joint Inflammation score (Spearman's Rho ρ=0.279, p=0.043 and ρ=0.496, p=0.0002, respectively). CONCLUSIONS: Extracellular matrix metabolites were associated with ASDAS response, MRI inflammation, and clinical treatment response during TNF-inhibitory treatment in patients with axSpA.

Pegcetacoplan treatment for geographic atrophy due to age-related macular degeneration: a plain language summary of the FILLY study.

WHAT IS THIS SUMMARY ABOUT?: This is a summary of a publication about the FILLY study, which was published in Ophthalmology in 2020. The FILLY study looked at an investigational medicine called pegcetacoplan as a possible treatment for geographic atrophy. Geographic atrophy, also known as GA, is the late stage of an eye disease called dry age-related macular degeneration, also known as dry AMD. In people with GA, lesions form on a part of the back of the eye called the retina. GA lesions are patches of thin retina. Growth of GA lesions ultimately causes blindness, which cannot be reversed. There is currently no approved treatment for GA. Pegcetacoplan, also called APL-2, could be a possible treatment for GA. Pegcetacoplan is an investigational medicine, which means it has not yet been approved. It is currently being studied in clinical studies to see how well it works. WHAT HAPPENED IN THE FILLY STUDY?: The FILLY study included participants with GA and tested how well pegcetacoplan worked compared to a sham injection (an injection that looks like the study treatment but does not have any medicine in it). The study also looked at how safe it was in adults with GA. WHAT WERE THE RESULTS?: The main questions the researchers wanted to answer were: Did pegcetacoplan slow the growth of the study participants' GA lesions? ○Yes. Overall, the researchers found that pegcetacoplan did slow the growth of the study participants' GA lesions. Did pegcetacoplan change the participants' vision? ○No. Overall, the researchers found that pegcetacoplan did not change the participants' vision. What medical problems happened after the participants received pegcetacoplan? ○The researchers kept track of any serious medical problems that happened during the study, also called serious adverse events. They also kept track of other medical problems that happened, or got worse, only at some point after the participants received the study treatment. These are called treatment emergent adverse events, also known as TEAEs. The serious adverse events and TEAEs that the participants had are described later in this summary. WHAT DO THE RESULTS OF THE STUDY MEAN?: Overall, results from this study showed that participants who received pegcetacoplan had slower growth of GA lesions than participants who received the sham injection. After the participants had stopped receiving pegcetacaoplan, the effect of the treatment seemed to be reduced. Pegcetacoplan did not change how well the participants could see during their vision tests in this trial. ClinicalTrials.gov NCT number: NCT02503332.

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.

Compstatins: the dawn of clinical C3-targeted complement inhibition.

Despite the growing recognition of the complement system as a major contributor to a variety of clinical conditions, the therapeutic arsenal has remained scarce. The introduction of an anti-C5 antibody in 2007 raised confidence in complement-targeted therapy. However, it became apparent that inhibition of late-stage effector generation might not be sufficient in multifactorial complement disorders. Upstream intervention at the level of C3 activation has therefore been considered promising. The approval of pegcetacoplan, a C3 inhibitor of the compstatin family, in 2021 served as critical validation of C3-targeted treatment. This review delineates the evolution of the compstatin family from its academic origins to the clinic and highlights current and potential future applications of this promising drug class in complement diseases.

Absence of complement component 3 does not prevent classical pathway-mediated hemolysis.

Complement component 3 (C3) is emerging as a potential therapeutic target. We studied complement-mediated hemolysis using normal and C3-depleted human sera, wild-type (WT) and C3-deficient rat sera, and WT and C3 knockout rat models. In all of the in vitro and in vivo experiments, we found that the loss of C3 did not prevent classical pathway-mediated hemolysis, but it did almost abolish alternative pathway-mediated hemolysis. Experiments using preassembled classical pathway C3 convertases confirmed that C4b2a directly activated complement component 5 (C5), leading to membrane attack complex formation and hemolysis. Our results suggest that targeting C3 should effectively inhibit hemolysis and tissue damage mediated by the alternative pathway of complement activation, but this approach might have limited efficacy in treating classical pathway-mediated pathological conditions.

Intracellular activation of complement C3 in Paneth cells improves repair of intestinal epithelia during acute injury.

AIM: To explore whether Paneth cells (PCs) and complement system collaborate in the repair of enteric epithelia during acute gastrointestinal injury (AGI). METHODS: Wild-type C57BL/6 mice were employed to induce AGI by performing colon ascendens stent surgery, with sham-operated as control. Exogenous C3 treatment was applied at 6-h postsurgery. After 48 h, overall survival, intestinal damage severity, and C3 intracellular activation were assessed in both epithelial cells and PCs. RESULTS: AGI caused a high mortality, while C3 therapy significantly attenuated epithelial damages and improved survival. Besides, exogenous C3 in vitro enhanced the proliferation and activity of PCs. Importantly, intracellular C3 activation was observed inside of PCs under C3 co-stimulation in vitro. CONCLUSION: C3 immunotherapy might play a valuable role in turnover of gut epithelia through intracellular activation in PCs.

Complement-dependent cytotoxicity of an antibody to melanin in radioimmunotherapy of metastatic melanoma.

AIM: Novel treatments for metastatic melanoma are urgently needed. MATERIALS & METHODS: We developed radioimmunotherapy of metastatic melanoma using 6D2 monoclonal antibody (mAb) to melanin with encouraging therapeutic results, preclinically and in patients. RESULTS: We observed tumor suppression with the unlabeled 6D2 mAb and investigated its tumoricidal mechanisms. In melanoma tumor-bearing mice, we detected more complement-C3 deposition in the tumors from 188-rhenium-labeled 6D2 mAb-treated mice when compared with untreated controls. 6D2 and isotype-control mAb TEPC caused suppression of tumor growth in A2058 melanoma tumor-bearing mice. Tumors of mice treated with the unlabeled 6D2 mAb were infiltrated with more lymphocytes compared with controls. In vitro antibody-dependent cell-mediated cytotoxicity did not contribute to the tumor-suppressive effect of 6D2 mAb, while 6D2 mAb demonstrated a strong effect on initiating complement-dependent cytotoxicity. CONCLUSION: We concluded that 6D2 mAb mediated complement-dependent cytotoxicity, resulting in killing of the tumor cells and suppression of tumor growth. These observations will help to improve the treatment protocols of radioimmunotherapy, as well as immunotherapy.

The effect of human complement C3 protein applied at different times in treatment of polymicrobial sepsis.

OBJECTIVE: This study was designed to investigate the effect of exogenous complement C3 administration on outcomes of sepsis and identify an optimal time for this therapy. MATERIALS AND METHODS: Colon ascendens stent peritonitis (CASP) was performed to induce sepsis in C57BL/6 mice, with sham-operated mice as control. Human purified C3 (HuC3, 1 mg) was administered via intraperitoneal injection, with 200 µl phosphate-buffered saline as control. Mice were categorized by the initiation time of HuC3 treatment. Survival, bacterial burden, vital organ damages, histology changes, and expression of C3 were compared between the groups. RESULTS: CASP-induced sepsis caused rapid complement C3 depletion and severe organ damage. Vital organs suffered from substantial bacterial loads. Exogenous C3 applied in the early stage of sepsis was associated with attenuated organ injuries, enhanced bacterial clearance, and improved survival. Exogenous C3 application promoted the synthesis of C3 in the early stage of sepsis. It appears that 6 h post-CASP surgery is the optimal time for HuC3 therapy. CONCLUSION: The study confirmed the positive effect of exogenous C3 on treatment of polymicrobial sepsis. C3 supplementation prior to the appearance of complement depletion could protect vital organs and its administration in the early stage of sepsis should be encouraged .

The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of Rho and ROCK inhibitors on neurite outgrowth and myelination.

It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentration-dependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.

C3 peptide promotes axonal regeneration and functional motor recovery after peripheral nerve injury.

Peripheral nerve injuries are frequently seen in trauma patients and due to delayed nerve repair, lifelong disabilities often follow this type of injury. Innovative therapies are needed to facilitate and expedite peripheral nerve regeneration. The purpose of this study was to determine the effects of a 1-time topical application of a 26-amino-acid fragment (C3(156-181)), derived from the Clostridium botulinum C3-exoenzyme, on peripheral nerve regeneration in 2 models of nerve injury and repair in adult rats. After sciatic nerve crush, different dosages of C3(156-181) dissolved in buffer or reference solutions (nerve growth factor or C3(bot)-wild-type protein) or vehicle-only were injected through an epineurial opening into the lesion sites. After 10-mm nerve autotransplantation, either 8.0 nmol/kg C3(156-181) or vehicle were injected into the proximal and distal suture sites. For a period of 3 to 10 postoperative weeks, C3(156-181)-treated animals showed a faster motor recovery than control animals. After crush injury, axonal outgrowth and elongation were activated and consequently resulted in faster motor recovery. The nerve autotransplantation model further elucidated that C3(156-181) treatment accounts for better axonal elongation into motor targets and reduced axonal sprouting, which are followed by enhanced axonal maturation and better axonal functionality. The effects of C3(156-181) are likely caused by a nonenzymatic down-regulation of active RhoA. Our results indicate the potential of C3(156-181) as a therapeutic agent for the topical treatment of peripheral nerve repair sites.

Exogenous C3 postpones complement exhaustion and confers organ protection in murine sepsis.

BACKGROUND: Sepsis in human being is a challenging and life-threatening problem. Complement activation is an essential event in sepsis. The present study observed the dynamic levels of complement components in sepsis and evaluated the role of exogenous complement protein in outcomes. The relationship between complement and inflammatory cytokines was also investigated. MATERIALS AND METHODS: Colon ascendens stent peritonitis (CASP) surgery was performed in wild-type C57BL/6 mice to induce sepsis. After 6 h of CASP, a single intraperitoneal injection of human purified C3 (HuC3, 1 mg) was carried out, with 200 uL phosphate-buffered saline injection for control purpose. Plasma levels of C3, complement factor H (CFH), and inflammatory cytokines at different time points were detected. Bacterial burden and organ damage were evaluated after 24 h of surgical procedure. RESULTS: The plasma C3 levels began to fall at 6 h post CASP, followed by an irreversible process of consumption. A single injection of HuC3 stabilized C3 levels for about 6 h, decreasing the 24 h mortality from 60% to 20%. Administration of exogenous C3 reduced bacterial burden and attenuated organ injury in sepsis. Plasma levels of CFH and TNF-α were correlated with the depletion of C3. CONCLUSION: We demonstrated a consumptive depletion of complement components toward septic peritonitis. Exogenous C3 supplementation in early stage of sepsis is helpful to sustain C3 levels, with enhanced bacterial clearance and improved outcomes.

[An exceptional component C3 deficiency revealed by serum protein electrophoresis]. / Un exceptionnel déficit héréditaire en fraction C3 du complément dépisté par électrophorèse des protéines sériques.

We report the case of a 5-years old child referred to the pediatric clinic due to a prolonged history of recurrent otitis. Initial immunologic investigation was normal but a severe C3 complement deficiency was detected by the absence of beta 2-globulin protein fraction using serum protein capillary electrophoresis. C3 was not detected in serum and total complement haemolytic activity was decreased. His mother and father had half of the C3 normal plasma level and a heterozygous mutation of the C3 gene. The diagnosis of hereditary deficiency of the third complement component (C3) with compound heterozygous mutation of the gene was made. This defect in complement protein C3, described to date in only 20 families in the world, is associated with repeated infections. The child is treated with oracillin with relatively good control of symptoms.

Derivatives of human complement component C3 for therapeutic complement depletion: a novel class of therapeutic agents.

To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with less immunogenicity, we have prepared human C3/CVF hybrid proteins, in which the C-terminus of the alpha-chain of human C3 is exchanged with homologous regions of the C-terminus of the beta-chain of CVF. We show that these hybrid proteins are able to deplete complement, both in vitro and in vivo. One hybrid protein, HC3-1496, is shown to be effective in reducing complement-mediated damage in two disease models in mice, collagen-induced arthritis and myocardial ischemia/reperfusion injury. Human C3/CVF hybrid proteins represent a novel class ofbiologicals as potential therapeutic agents in many diseases where complement is involved in the pathogenesis.