Cobra Venom Factor Research Articles

Acute depletion of complement C3 with cobra venom factor attenuates memory deficits induced by status epilepticus.

Status epilepticus (SE) significantly increases the risk for the development of unprovoked seizures, memory loss, and temporal lobe epilepsy. Our prior studies showed that SE increases complement C3 signaling in the hippocampus, which parallels memory deficits. Additionally, C3 knockout (KO) mice were protected against SE-induced memory impairments, suggesting a mechanistic role for C3 in this pathophysiology. In this study, we utilized cobra venom factor (CVF), a structural analog of C3 that results in its depletion, to investigate the protective effects of post-SE C3 ablation on memory deficits that develop during epileptogenesis. SE was induced in male rats using the chemoconvulsant pilocarpine. Two weeks later, control (C) and SE rats were treated with either vehicle (V) or CVF. Recognition memory was assessed using the novel object recognition (NOR) test in four groups (C + V, C + CVF, SE + V, and SE + CVF). Immunoblotting was used to measure hippocampal protein levels of C3 along with synaptic, astrocyte, and blood-brain barrier (BBB) stability markers, Psd95, GFAP, and albumin, respectively. In the NOR test, rats in the C + V and C + CVF groups spent more time exploring the novel object. SE + V rats explored both objects equally, while SE + CVF rats spent significantly more on the novel object, suggesting a rescue of cognitive performance by CVF. While CVF-mediated C3 depletion did not restore normal protein levels of Psd95 or GFAP in hippocampi of SE + CVF rats, CVF treatment attenuated SE-induced extravasation of albumin, suggesting potential rescue of BBB stability. Our findings indicate that acute C3 depletion with CVF can attenuate memory deficits that develop during SE-induced epileptogenesis. This finding further suggests that targeting C3 could hold promise in addressing cognitive comorbidities associated with SE and acquired epilepsy. A long-lasting seizure, known as status epilepticus (SE), can raise the chance of having more seizures in the future and can lead to memory problems. While the exact reasons for these issues are not completely known, it is believed that brain inflammation might be involved. In this study, we show that the activation of the body's immune response, especially a part called the C3 component, plays a role in the memory problems that occur after an episode of SE.

Baicalin relieves complement alternative pathway activation-induced lung inflammation through inhibition of NF-κB pathway

IntroductionAcute lung injury (ALI) as one kind of acute pulmonary inflammatory disorder, manifests primarily as damage to alveolar epithelial cells and microvascular endothelial cells. Activation of the complement system is a common pathological mechanism in ALI induced by diverse factors, with the complement alternative pathway assuming a pivotal role. Baicalin, a flavonoid derived from the root of Scutellaria baicalensis Georgi, exhibits noteworthy biological activities. The present study attempted the interventional effects and underlying mechanisms of baicalin in microangiopathy in ALI induced by complement alternative pathway activation.MethodsActivation of the complement alternative pathway by cobra venom factor (CVF). HMEC cells were pretreated with baicalin and then exposed to complement activation products. The expression of inflammatory mediators was detected by ELISA, and the intranuclear transcriptional activity of NF-κB was assessed by a dual fluorescent kinase reporter gene assay kit. Before establishing the ALI mouse model, baicalin or PDTC was gavaged for 7 d. CVF was injected into the tail vein to establish the ALI model. The levels of inflammatory mediators in BALF and serum were determined by ELISA. HE staining and immunohistochemistry evaluated pathological changes, complement activation product deposition, and NF-κB p65 phosphorylation in lung tissue.ResultsBaicalin reduced complement alternative activation product-induced expression of HMEC cells adhesion molecules (ICAM-1, VCAM-1, E-selectin) and cytokines (IL-6, TNF-α) as well as upregulation of NF-κB intranuclear transcriptional activity. Baicalin intervention reduced the number of inflammatory cells and protein content in the BALF and decreased the levels of IL-6, TNF-α, and ICAM-1 in serum and IL-6, TNF-α, ICAM-1, and P-selectin in BLAF. In addition, baicalin attenuated inflammatory cell infiltration in the lung of ALI mice and reduced the deposition of complement activation products (C5a, C5b-9) and phosphorylation of NF-κB p65 in lung tissue.ConclusionBaicalin relieves complement alternative pathway activation-induced lung inflammation by inhibition of NF-κB pathway, delaying the progression of ALI.

THSD7A-associated membranous nephropathy involves both complement-mediated and autonomous podocyte injury.

Membranous nephropathy (MN) continues to be a leading cause of nephrotic syndrome in non-diabetic adults. As a unique subtype in the serology-based classification of MN, thrombospondin type 1 domain containing 7A (THSD7A)-associated MN has attracted increasing interest, because, unlike other autoantigens, THSD7A is also expressed in preclinical species, facilitating the study of its role in MN. A heterologous mouse model of THSD7A-associated MN was previously established using a proprietary in-house antibody that was unfortunately not available to the research community. Here, we developed a mouse model of THSD7A-associated MN by administering a commercially available antibody targeting the most N-terminal part of THSD7A. Our model was characterized by heavy proteinuria and pathological features of human MN without sex differences. Complement depletion with cobra venom factor only partially attenuated proteinuria and glomerular injury in this model, entailing that complement-independent pathomechanisms also contribute. Consistently, in vitro in primary podocytes, exposure to the anti-THSD7A antibody caused evident podocytopathic changes, including disruption of actin cytoskeleton integrity, podocyte hypermobility, oxidative stress, and apoptotic cell death. These signs of podocytopathy were preserved, albeit to a lesser extent, after complement inactivation, indicating autonomous podocyte injury. Furthermore, as the first FDA-approved treatment for primary MN, adrenocorticotropic hormone therapy with repository corticotropin injection (Purified Cortrophin Gel®) appeared to be beneficial and significantly attenuated proteinuria and glomerular injury, suggesting that this model may be useful for developing novel treatments or understanding the pathogenesis of MN. Collectively, our model, based on the use of a commercially available anti-THSD7A antibody, will be an important tool for MN research.

Complement activation in wasp venom-induced acute kidney injury

Previous studies have highlighted the significant role of complement activation in kidney injuries induced by rhabdomyolysis, intravascular hemolysis, sepsis, and ischemia-reperfusion. Nevertheless, the specific role and mechanism of complement activation in acute kidney injury (AKI) caused by wasp venom remain unclear. The aim of this study was to elucidate the specific complement pathway activated and investigate complement activation in AKI induced by wasp venom. In this study, a complement-depleted mouse model was used to investigate the role of complement in wasp venom-induced AKI. Mice were randomly categorized into control, cobra venom factor (CVF), AKI, and CVF + AKI groups. Compared to the AKI group, the CVF + AKI group showed improved pathological changes in kidneys and reduced blood urea nitrogen (BUN) levels. The expression levels of renal complement 3 (C3), complement 5 (C5), complement 1q (C1q), factor B (FB), mannose-binding lectin (MBL), and C5b-9 in AKI group were upregulated compared with the control group. Conversely, the renal tissue expression levels of C3, C5, C1q, FB, MBL, and C5b-9 were decreased in the CVF + AKI group compared to those in the AKI group. Complement activation occurs through all three pathways in AKI induced by wasp venom. Furthermore, complement depletion by CVF attenuates wasp venom-induced nephrotoxicity, suggesting that complement activation plays a primary role in the pathogenesis of wasp venom-induced AKI.

Treatment with Cobra Venom Factor Decreases Ischemic Tissue Damage in Mice.

Tissue ischemia, caused by the blockage of blood vessels, can result in substantial damage and impaired tissue performance. Information regarding the functional contribution of the complement system in the context of ischemia and angiogenesis is lacking. To investigate the influence of complement activation and depletion upon femoral artery ligation (FAL), Cobra venom factor (CVF) (that functionally resembles C3b, the activated form of complement component C3) was applied in mice in comparison to control mice. Seven days after induction of muscle ischemia through FAL, gastrocnemius muscles of mice were excised and subjected to (immuno-)histological analyses. H&E and apoptotic cell staining (TUNEL) staining revealed a significant reduction in ischemic tissue damage in CVF-treated mice compared to controls. The control mice, however, exhibited a significantly higher capillary-to-muscle fiber ratio and a higher number of proliferating endothelial cells (CD31+/CD45-/BrdU+). The total number of leukocytes (CD45+) substantially decreased in CVF-treated mice versus control mice. Moreover, the CVF-treated group displayed a shift towards the M2-like anti-inflammatory and regenerative macrophage phenotype (CD68+/MRC1+). In conclusion, our findings suggest that treatment with CVF leads to reduced ischemic tissue damage along with decreased leukocyte recruitment but increased numbers of M2-like polarized macrophages, thereby enhancing tissue regeneration, repair, and healing.

Proteomic Investigation of Cape Cobra (Naja nivea) Venom Reveals First Evidence of Quaternary Protein Structures.

Naja nivea (N. nivea) is classed as a category one snake by the World Health Organization since its envenomation causes high levels of mortality and disability annually. Despite this, there has been little research into the venom composition of N. nivea, with only one full venom proteome published to date. Our current study separated N. nivea venom using size exclusion chromatography before utilizing a traditional bottom-up proteomics approach to unravel the composition of the venom proteome. As expected by its clinical presentation, N. nivea venom was found to consist mainly of neurotoxins, with three-finger toxins (3FTx), making up 76.01% of the total venom proteome. Additionally, cysteine-rich secretory proteins (CRISPs), vespryns (VESPs), cobra venom factors (CVFs), 5'-nucleotidases (5'NUCs), nerve growth factors (NGFs), phospholipase A2s (PLA2), acetylcholinesterases (AChEs), Kunitz-type serine protease inhibitor (KUN), phosphodiesterases (PDEs), L-amino acid oxidases (LAAOs), hydrolases (HYDs), snake venom metalloproteinases (SVMPs), and snake venom serine protease (SVSP) toxins were also identified in decreasing order of abundance. Interestingly, contrary to previous reports, we find PLA2 toxins in N. nivea venom. This highlights the importance of repeatedly profiling the venom of the same species to account for intra-species variation. Additionally, we report the first evidence of covalent protein complexes in N. nivea venom, which likely contribute to the potency of this venom.

Complement and platelets: prothrombotic cell activation requires membrane attack complex–induced release of danger signals

AbstractComplement activation in the diseases paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) results in cytolysis and fatal thrombotic events, which are largely refractory to anticoagulation and/or antiplatelet therapy. Anticomplement therapy, however, efficiently prevents thrombotic events in PNH and aHUS, but the underlying mechanisms remain unresolved. We show that complement-mediated hemolysis in whole blood induces platelet activation similarly to activation by adenosine 5′-diphosphate (ADP). Blockage of C3 or C5 abolished platelet activation. We found that human platelets failed to respond functionally to the anaphylatoxins C3a and C5a. Instead, complement activation did lead to prothrombotic cell activation in the whole blood when membrane attack complex (MAC)-mediated cytolysis occurred. Consequently, we demonstrate that ADP receptor antagonists efficiently inhibited platelet activation, although full complement activation, which causes hemolysis, occurred. By using an established model of mismatched erythrocyte transfusions in rats, we crossvalidated these findings in vivo using the complement inhibitor OmCI and cobra venom factor. Consumptive complement activation in this animal model only led to a thrombotic phenotype when MAC-mediated cytolysis occurred. In conclusion, complement activation only induces substantial prothrombotic cell activation if terminal pathway activation culminates in MAC-mediated release of intracellular ADP. These results explain why anticomplement therapy efficiently prevents thromboembolisms without interfering negatively with hemostasis.

Decomplexation of Venom Proteome of Pakistani Cobra (Naja naja naja)

The venom proteome of Naja naja from Sindh, Pakistan was decomplexed utilizing reverse phase HPLC and SDS PAGE. The results were compared with already reported Naja naja species of the region. The banding pattern represents all the major families of proteins including three-finger toxins, phospholipase A2, snake venom metalloproteases, L-amino acid oxidases, phosphodiesterase, nucleotidases, cysteine-rich secretory proteins, serine proteases, nerve growth factor, cobra venom factor, acetylcholinesterases, Kunitz-type serine protease inhibitors and C-type lectin proteins. The decomplexation of the venom showed the best possible separation through RP-HPLC elution of venom components containing small peptides, small and large proteins based on hydrophobicity. The SDS PAGE under reducing and non-reducing conditions of HPLC fractions highlighted the presence of several proteins.

Technique for xenogeneic cross-circulation to support human donor lungs ex vivo.

Xenogeneic cross-circulation (XC) is an experimental method for ex vivo organ support and recovery that could expand the pool of donor lungs suitable for transplantation. The objective of this study was to establish and validate a standardized, reproducible, and broadly applicable technique for performing xenogeneic XC to support and recover injured human donor lungs ex vivo. Human donor lungs (n=9) declined for transplantation were procured, cannulated, and subjected to 24 hours of xenogeneic XC with anesthetized xeno-support swine (Yorkshire/Landrace) treated with standard immunosuppression (methylprednisolone, mycophenolate mofetil, tacrolimus) and complement-depleting cobra venom factor. Standard lung-protective perfusion and ventilation strategies, including periodic lung recruitment maneuvers, were used throughout xenogeneic XC. Every 6 hours, ex vivo donor lung function (gas exchange, compliance, airway pressures, pulmonary vascular dynamics, lung weight) was evaluated. At the experimental endpoint, comprehensive assessments of the lungs were performed by bronchoscopy, histology, and electron microscopy. Student's t-test and 1-way analysis of variance with Dunnett's post-hoc test was performed, and p < 0.05 was considered significant. After 24 hours of xenogeneic XC, gas exchange (PaO2/FiO2) increased by 158% (endpoint: 364 ± 142 mm Hg; p=0.06), and dynamic compliance increased by 127% (endpoint: 46 ± 20 ml/cmH2O; p=0.04). Airway pressures, pulmonary vascular pressures, and lung weight remained stable (p > 0.05) and within normal ranges. Over 24 hours of xenogeneic XC, gross and microscopic lung architecture were preserved: airway bronchoscopy and parenchymal histomorphology appeared normal, with intact blood-gas barrier. Xenogeneic cross-circulation is a robust method for ex vivo support, evaluation, and improvement of injured human donor lungs declined for transplantation.

Complement Drives Acute Chest Syndrome in a Model of Sickle Cell Disease

Background: Despite being the first genetic disease described, sickle cell disease (SCD) continues to afflict millions of individuals worldwide. Patients with SCD can experience severe complications, including vasoocclusive crisis (VOC), which can progress to acute chest syndrome (ACS), a form of acute lung injury. While VOC can cause significant morbidity, ACS is the leading cause of mortality in patients with SCD. Despite the devastating consequences of ACS, no interventions exist that directly treat this complication. As a result, when patients present with ACS, options often remain limited to supportive care. The lack of optimal treatment options for patients with SCD experiencing ACS directly contributes to their morbidity and mortality. Using a combined approach of prospective observational studies in patients with SCD that experienced ACS and a novel preclinical model specifically designed to define the role of complement in SCD pathophysiology, we tested the hypothesis that complement plays a central role in the pathophysiology of ACS. Methods: Samples from patients with SCD experiencing ACS were compared to baseline measurements for the same patient obtained at outpatient follow up 4-6 weeks later. ELISA based assays were used to measure Ba, Bb, C3a, C5a, C5b-9 (membrane attack complex (MAC)) in each sample. SCD mice (HbSS) and control mice (HbAA) were injected with 7.5 units of cobra venom factor (CVF), a potent complement activator, followed by evaluation of hematocrit (Hct) measured by a Sysmex veterinary hematology analyzer, free heme by calorimetric assay, and complement deposition on red blood cells (RBCs) by flow cytometry or pulmonary endothelial cells by confocal analysis. Respiratory rate and O2 saturation were measured using a MouseOx pulse oximeter. HbSS or HbAA mice were crossed with complement component 3 (C3) knock out (KO) mice to generate HbSS x C3 KO and HbAA x C3 KO mice. Hemin (70 to 210 mmol/kg concentration) or CVF (7.5 units/mouse) were injected into HbSS, HbAA, HbSS x C3 KO or HbAA x C3 KO followed by evaluation of hemolysis, C3 deposition and pulmonary function. A one-way ANOVA with a Tukey's post hoc with a p value <0.05 was considered significant. Results: During ACS in patients, significant elevations in Ba and Bb, complement activation products unique to the alternative pathway, and downstream activation of complement, including C3a, C5a and MAC, were observed (p<0.0001). Changes in complement activation were accompanied by a drop in hemoglobin during ACS when compared to baseline (p<0.001), suggesting that complement activation results in hemolysis. To formally test this, the impact of complement activation in a preclinical model of SCD was defined. Injection of CVF or hemin resulted in rapid C3 deposition on RBCs and pulmonary endothelium in HbSS, but not HbAA, recipients. In addition, CVF or hemin injection was accompanied by increased hemolysis and ACS manifested by increased respiratory rate, drop in oxygen saturation and death within 2 hours in the majority of recipients (p<0.001). In contrast, control Hb AA mice were unaffected by CVF or hemin injection. Given the role of free heme in the development of ACS, we next defined the role of complement in hemin-induced lung injury. To accomplish this, SCD mice were crossed with C3 KO mice to generate HbSS x C3 KO. HbSS x C3 KO mice displayed a mild increase in Hb values at baseline (p<0.01), suggesting a role of C3 even in steady-state SCD-related hemolysis. Equally important, injection of HbSS x C3 KO mice with CVF or hemin failed to result in the same levels of C3 deposition, hemolysis, compromised pulmonary function, or mortality (p<0.001) as HbSS recipients. Discussion: These results demonstrate a critical role for C3 in the development of acute lung injury in SCD. The sensitivity of HbSS RBCs to complement-induced hemolysis and C3 deposition observed following hemin injection in SCD recipients suggests that C3 plays a key role in decreased hematocrits and pulmonary injury that accompanies ACS. As treatment of ACS largely relies on supportive care, these results suggest that approaches aimed at targeting complement activation may represent a useful prophylactic or treatment strategy for ACS. In doing so, these results hold promise in providing a more direct approach at reducing one of the most severe complications of patients with SCD.

Molecular Determinants of Human Red Blood Cell Survival in Murine Circulation

Introduction: Transfusion of human red blood cells (RBCs) is an irreplaceable life-saving medical intervention. However, as the expression of surface proteins on donor RBCs can regulate their circulatory lifespan, these proteins can affect the efficiency of transfusion. A murine preclinical transfusion model was used to better understand the impact of these proteins on human RBC survival. Methods: Human peripheral blood RBCs were obtained from qualified blood donors as aliquots of leukoreduced, packed CP2D/AS1 RBCs, or separately collected from healthy research donors. RBCs were biotinylated at densities ≤18 mg/ml following established protocols. Unlabeled or biotinylated human RBCs were transfused into immunodeficient NCG mice treated with chlodronate and cobra venom factor before and after transfusion. Human RBCs in murine circulation were identified with FITC-conjugated anti-human CD235a antibodies and/or PE-conjugated streptavidin. Aliquots of transfused RBCs were analyzed by flow cytometry for expression levels of the surface molecules CD35, CD36, CD38, CD44, CD47, CD55, CD59, CD147, CD233 and CD235a before transfusion, and also in peripheral blood samples collected from mice after transfusion. In some studies, human RBCs were treated with antibodies against selected surface molecules before transfusion. Results: Samples collected from transfused mice at serial timepoints showed significantly decreased expressions over time of several human RBC surface molecules: CD47, CD55, CD59, CD147, CD233 and CD235a. In contrast, there were no significant changes in RBC expression of CD35, CD36, CD38 and CD44 in the samples. Among the surface molecules showing lower levels over time, there was a significant positive correlation between CD47 or CD59 expression levels on human RBCs and circulatory survival of those RBCs in the mice. When mixtures of RBCs from two donors were transfused simultaneously, those RBCs with higher CD47 or CD59 expression demonstrated significantly longer survival in murine peripheral blood in comparison with the co-transfused RBCs. Furthermore, pre-incubation of human RBCs with antibodies against CD47 or CD59 prior to transfusion markedly reduced their survival in murine circulation. In contrast, antibodies against CD55, CD147, CD233 or CD235a had no effect on human RBC circulation. Conclusions: Our data show that CD47 and CD59 expression on human RBCs increases their survival after transfusion, and suggest that pre-transfusion profiling of CD47 and CD59 molecules could help predict the efficiency of human RBC transfusion.

Acteoside attenuates acute lung injury following administration of cobra venom factor to mice

BackgroundActeoside, a water-soluble active constituent of diverse valuable medicinal vegetation, has shown strong anti-inflammatory property. However, studies on the anti-inflammatory property of acteoside in complement-induced acute lung injury (ALI) are limited. Therefore, this study aims to evaluate the anti-inflammatory activity of acteoside in cobra venom factor (CVF)-stimulated human microvascular endothelial cells (HMEC) and in ALI mice model. MethodsIn this study, we investigated the effects of acteoside (20, 10, and 5 μg/mL) in vitro in CVF induced HMECs and the activity of acteoside (100, 50, and 20 mg/kg/day bodyweight) in vivo in CVF induced ALI mice. Each eight male mice were orally administered acteoside or the positive drug PDTC (100 mg/kg/day) for 7 days before CVF (35 μg/kg) injection. After injection for 1 h, the pharmacological effects of acteoside were investigated by spectrophotometry, pathological examination, enzyme-linked immunosorbent assay, and immunohistochemistry. ResultsIn vitro, acteoside (20, 10, and 5 μg/mL) reduced the protein expression of adhesion molecules and pro-inflammatory cytokines and transcriptional activity of NF-κB (P < 0.01). In vivo studies showed that acteoside dose-dependently alleviated lung histopathologic lesion, inhibited the production of the protein content of BALF, leukocyte cell number, lung MPO activity, and expression levels of IL-6, TNF-α, and ICAM-1, and suppressed the C5b-9 deposition and NF-κB activation in CVF-induced acute lung inflammation in mice (P < 0.05, 0.01). ConclusionThis study demonstrates that acteoside exerts strong anti-inflammatory activities in the CVF-induced acute lung inflammation model and suggests that acteoside is a potential therapeutic agent for complement-related inflammatory diseases.

The validity of animal models to explore the pathogenic role of the complement system in multiple sclerosis: A review.

Animal models of multiple sclerosis (MS) have been extensively used to characterize the disease mechanisms in MS, as well as to identify potential pharmacologic targets for this condition. In recent years, the immune complement system has gained increased attention as an important effector in the pathogenesis of MS. Evidence from histological, serum, and CSF studies of patients supports an involvement of complement in both relapsing-remitting and progressive MS. In this review, we discuss the history and advances made on the use of MS animal models to profile the effects of the complement system in this condition. The first studies that explored the complement system in the context of MS used cobra venom factor (CVF) as a complement depleting agent in experimental autoimmune encephalomyelitis (EAE) Lewis rats. Since then, multiple mice and rat models of MS have revealed a role of C3 and the alternative complement cascade in the opsonization and phagocytosis of myelin by microglia and myeloid cells. Studies using viral vectors, genetic knockouts and pharmacologic complement inhibitors have also shown an effect of complement in synaptic loss. Antibody-mediated EAE models have revealed an involvement of the C1 complex and the classical complement as an effector of the humoral response in this disease. C1q itself may also be involved in modulating microglia activation and oligodendrocyte differentiation in these animals. In addition, animal and in vitro models have revealed that multiple complement factors may act as modulators of both the innate and adaptive immune responses. Finally, evidence gathered from mice models suggests that the membrane attack complex (MAC) may even exert protective roles in the chronic stages of EAE. Overall, this review summarizes the importance of MS animal models to better characterize the role of the complement system and guide future therapeutic approaches in this condition.

Contribution of animal models to the mechanistic understanding of Alternative Pathway and Amplification Loop (AP/AL)-driven Complement-mediated Diseases.

This review aimed to capture the key findings that animal models have provided around the role of the alternative pathway and amplification loop (AP/AL) in disease. Animal models, particularly mouse models, have been incredibly useful to define the role of complement and the alternative pathway in health and disease; for instance, the use of cobra venom factor and depletion of C3 provided the initial insight that complement was essential to generate an appropriate adaptive immune response. The development of knockout mice have further underlined the importance of the AP/AL in disease, with the FH knockout mouse paving the way for the first anti-complement drugs. The impact from the development of FB, properdin, and C3 knockout mice closely follows this in terms of mechanistic understanding in disease. Indeed, our current understanding that complement plays a role in most conditions at one level or another is rooted in many of these in vivo studies. That C3, in particular, has roles beyond the obvious in innate and adaptive immunity, normal physiology, and cellular functions, with or without other recognized AP components, we would argue, only extends the reach of this arm of the complement system. Humanized mouse models also continue to play their part. Here, we argue that the animal models developed over the last few decades have truly helped define the role of the AP/AL in disease.

Venom Variation of Neonate and Adult Chinese Cobras in Captivity Concerning Their Foraging Strategies.

The venom and transcriptome profile of the captive Chinese cobra (Naja atra) is not characterized until now. Here, LC-MS/MS and illumine technology were used to unveil the venom and trascriptome of neonates and adults N. atra specimens. In captive Chinese cobra, 98 co-existing transcripts for venom-related proteins was contained. A total of 127 proteins belong to 21 protein families were found in the profile of venom. The main components of snake venom were three finger toxins (3-FTx), snake venom metalloproteinase (SVMP), cysteine-rich secretory protein (CRISP), cobra venom factor (CVF), and phosphodiesterase (PDE). During the ontogenesis of captive Chinese cobra, the rearrangement of snake venom composition occurred and with obscure gender difference. CVF, 3-FTx, PDE, phospholipase A2 (PLA2) in adults were more abundant than neonates, while SVMP and CRISP in the neonates was richer than the adults. Ontogenetic changes in the proteome of Chinese cobra venom reveals different strategies for handling prey. The levels of different types of toxin families were dramatically altered in the wild and captive specimens. Therefore, we speculate that the captive process could reshape the snake venom composition vigorously. The clear comprehension of the composition of Chinese cobra venom facilitates the understanding of the mechanism of snakebite intoxication and guides the preparation and administration of traditional antivenom and next-generation drugs for snakebite.

Cobra Venom Factor Boosts Arteriogenesis in Mice.

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

The Hypertensive Effect of Amphotericin B-Containing Liposomes (Abelcet) in Mice: Dissecting the Roles of C3a and C5a Anaphylatoxins, Macrophages and Thromboxane.

Liposomal amphotericin B (Abelcet) can cause infusion (anaphylactoid) reactions in patients whose mechanism is poorly understood. Here, we used mice to investigate the role of complement (C) receptors and the cellular sources of vasoactive mediators in these reactions. Anesthetized male NMRI and thromboxane prostanoid receptor (TP) or cyclooxygenase-1 (COX-1)-deficient and wild type C57Bl6/N mice were intravenously injected with Abelcet at 30 mg/kg. Mean arterial blood pressure (MABP) and heart rate (HR) were measured. In untreated mice, Abelcet caused a short (15 min) but large (30%) increase in MABP. C depletion with cobra venom factor (CVF) and inhibition of C5a receptors with DF2593A considerably prolonged, while C3aR inhibition with SB290157 significantly decreased the hypertensive effect. Likewise, the hypertensive response was abolished in COX-1- and TP-deficient mice. CVF caused a late hypertension in TP-deficient mice. Both macrophage depletion with liposomal clodronate and blockade of platelet GPIIb/IIIa receptors with eptifibatide prolonged the hypertensive effect. The early phase of the hypertensive effect is COX-1- and TP-receptor-dependent, partly mediated by C3aR. In contrast, the late phase is under the control of vasoactive mediators released from platelets and macrophages subsequent to complement activation and C5a binding to its receptor.

POS-042 C3d-Directed Factor H Targeting Delivers Potent and Durable Complement Inhibition and Disease-Modifying Efficacy In Kidney and Skin Without Inhibiting Systemic Complement

POS-042 C3d-Directed Factor H Targeting Delivers Potent and Durable Complement Inhibition and Disease-Modifying Efficacy In Kidney and Skin Without Inhibiting Systemic Complement

Immune Characterization of a Xenogeneic Cross Circulation System for Ex Vivo Human Lung Support

<h3>Purpose</h3> Rehabilitation of injured donor lungs can reduce the shortage of organs suitable for transplantation. We previously established a platform for the maintenance and recovery of extracorporeal human lungs using cross-circulation (XC) with a porcine host. Here, we characterize the human-swine immunologic interactions in this xenogeneic system. <h3>Methods</h3> Human donor lungs declined for clinical transplantation were placed on XC with immunosuppressed (methylprednisolone, mycophenolate, tacrolimus), complement-depleted (Cobra Venom Factor) porcine hosts. Longitudinal analyses of the extracorporeal lung and porcine host were performed over 24 hours. For one of these lung (n = 1), single-cell lung suspensions and paraffin-embedded slides were prepared from biopsies of the lung collected at pre-XC baseline, 1h, and 24h. Infiltration of swine CD45+ immune cells and deposition of swine IgG and IgM in the human lung were assessed by immunohistochemistry and flow cytometry. <h3>Results</h3> The extracorporeal lung demonstrated improved PaO2/FiO2 (602%), dynamic compliance (238%), and gas exchange (O2, 974%; CO2, 198%) over the course of XC. Populations of human and porcine CD45+ leukocytes were readily identified within the post-XC lung. Human immune cells represented 1.48% and porcine immune cells represented 0.99% of isolated live single cells (Figure 1A). Infiltration of CD45+ porcine leukocytes into the lung increased over time, such that porcine neutrophils were visualized in the alveolar spaces (Figure 1B). By flow cytometry, porcine IgG was localized to lung epithelial cells (Figure 1C). Diffuse deposition of IgG and IgM was also detected by IHC (Figure 1D). <h3>Conclusion</h3> In the xenogeneic XC system, porcine immune cell and immunoglobulin infiltration of the extracorporeal human lung can be detected. Despite this, XC was sufficient to facilitate physiologic improvement in the human lung. Further work is needed to minimize cross-species immune responses during <i>ex vivo</i> organ support.

Emerging roles of the complement system in host-pathogen interactions.

The complement system has historically been entertained as a fluid-phase, hepatically derived system which protects the intravascular space from encapsulated bacteria. However, there has been an increasing appreciation for its role in protection against non-encapsulated pathogens. Specifically, we have an improved understanding of how pathogens are recognized by specific complement proteins, as well as how they trigger and evade them. Additionally, we have an improved understanding of locally derived complement proteins, many of which promote host defense. Moreover, intracellular complement proteins have been identified that facilitate local protection and barrier function despite pathogen invasion. Our review aims to summarize these advances in the field as well as provide an insight into the pathophysiological changes occurring when the system is dysregulated in infection.