Storage of Transfusion Platelet Concentrates Is Associated with Complement Activation and Reduced Ability of Platelets to Respond to Protease-Activated Receptor-1 and Thromboxane A2 Receptor.

Platelet activation and the complement system are mutually dependent. Here, we investigated the effects of storage time on complement activation and platelet function in routinely produced platelet concentrates. The platelet concentrates (n = 10) were stored at 22 °C for seven days and assessed daily for complement and platelet activation markers. Additionally, platelet function was analyzed in terms of their responsiveness to protease-activated receptor-1 (PAR-1) and thromboxane A2 receptor (TXA2R) activation and their capacity to adhere to collagen. Complement activation increased over the storage period for all analyzed markers, including the C1rs/C1-INH complex (fold change (FC) = 1.9; p < 0.001), MASP-1/C1-INH complex (FC = 2.0; p < 0.001), C4c (FC = 1.8, p < 0.001), C3bc (FC = 4.0; p < 0.01), and soluble C5b-9 (FC = 1.7, p < 0.001). Furthermore, the levels of soluble platelet activation markers increased in the concentrates over the seven-day period, including neutrophil-activating peptide-2 (FC = 2.5; p < 0.0001), transforming growth factor beta 1 (FC = 1.9; p < 0.001) and platelet factor 4 (FC = 2.1; p < 0.0001). The ability of platelets to respond to activation, as measured by surface expression of CD62P and CD63, decreased by 19% and 24% (p < 0.05) for PAR-1 and 69-72% (p < 0.05) for TXA2R activation, respectively, on Day 7 compared to Day 1. The extent of platelet binding to collagen was not significantly impaired during storage. In conclusion, we demonstrated that complement activation increased during the storage of platelets, and this correlated with increased platelet activation and a reduced ability of the platelets to respond to, primarily, TXA2R activation.

A novel selective leukocyte depletion human whole blood model reveals the specific roles of monocytes and granulocytes in the cytokine response to Escherichia coli.

The lepirudin-based human whole blood model is a well-established ex vivo system to characterize inflammatory responses. However, the contribution of individual cell populations to cytokine release has not been investigated. Thus, we modified the model by selectively removing leukocyte subpopulations to elucidate their contribution to the inflammatory response. Lepirudin-anticoagulated whole blood was depleted from monocytes or granulocytes using StraightFrom Whole Blood MicroBeads. Reconstituted blood was incubated with Escherichia coli (108/mL) for 2 hours at 37 °C. CD11b, CD62P, and CD63 were detected by flow cytometry. Complement (C3bc, sC5b-9) and platelet activation (platelet factor 4, NAP-2) were measured by enzyme-linked immunosorbent assay. Cytokines were quantified by multiplex assay. A significant (P < 0.05) specific depletion of the monocyte (mean = 86%; 95% confidence interval = 71%-92%) and granulocyte (mean = 97%; 95% confidence interval = 96%-98%) population was obtained. Background activation induced by the depletion protocol was negligible for complement (C3bc and sC5b-9), leukocytes (CD11b), and platelets (NAP-2). Upon Escherichia coli incubation, release of 10 of the 24 cytokines was solely dependent on monocytes (interleukin [IL]-1ß, IL-2, IL-4, IL-5, IL-17A, interferon-γ, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-1α, and fibroblast growth factor-basic), whereas 8 were dependent on both monocytes and granulocytes (IL-1ra, IL-6, IL-8, IL-9, IL-10, macrophage inflammatory protein-1ß, tumor necrosis factor, and eotaxin). Six cytokines were not monocyte or granulocyte dependent, of which platelet-derived growth factor and RANTES were mainly platelet dependent. We document an effective model for selective depletion of leukocyte subpopulations from whole blood, without causing background activation, allowing in-depth cellular characterization. The results are in accordance with monocytes playing a major role in cytokine release and expand our knowledge of the significant role of granulocytes in the response to E. coli.

Quartz Crystal Microbalance Platform for SARS-CoV-2 Immuno-Diagnostics.

Rapid and accurate serological analysis of SARS-CoV-2 antibodies is important for assessing immune protection from vaccination or infection of individuals and for projecting virus spread within a population. The quartz crystal microbalance (QCM) is a label-free flow-based sensor platform that offers an opportunity to detect the binding of a fluid-phase ligand to an immobilized target molecule in real time. A QCM-based assay was developed for the detection of SARS-CoV-2 antibody binding and evaluated for assay reproducibility. The assay was cross-compared to the Roche electrochemiluminescence assay (ECLIA) Elecsys® Anti-SARS-CoV-2 serology test kit and YHLO's chemiluminescence immunoassay (CLIA). The day-to-day reproducibility of the assay had a correlation of r2 = 0.99, p < 0.001. The assay linearity was r2 = 0.96, p < 0.001, for dilution in both serum and buffer. In the cross-comparison analysis of 119 human serum samples, 59 were positive in the Roche, 52 in the YHLO, and 48 in the QCM immunoassay. Despite differences in the detection method and antigen used for antibody capture, there was good coherence between the assays, 80-100% for positive and 96-100% for negative test results. In summation, the QCM-based SARS-CoV-2 IgG immunoassay showed high reproducibility and linearity, along with good coherence with the ELISA-based assays. Still, factors including antibody titer and antigen-binding affinity may differentially affect the various assays' responses.

Functional evaluation of complement factor I variants by immunoassays and SDS-PAGE.

Factor I (FI) is an essential regulator of the complement system. Together with co-factors, FI degrades C3b, which inhibits further complement activation. Genetic mutations in FI are associated with pathological conditions like age-related macular degeneration and atypical hemolytic uremic syndome. Here, we evaluated eight recombinant FI genetic variants found in patients. We assessed FI's co-factor activity in the presence of two co-factors; Factor H and soluble CR1. Different analytical assays were employed; SDS-PAGE to evaluate the degradation of C3b, ELISA to measure the generation of fluid phase iC3b and the degradation of surface-bound C3b using a novel Luminex bead-based assay. We demonstrate that mutations in the FIMAC and SP domains of FI led to significantly reduced protease activity, whereas the two analyzed mutations in the LDLRA2 domain did not result in any profound changes in FI's function. The different assays employed displayed a strong positive correlation, but differences in the activity of the genetic variants Ile55Phe and Gly261Asp could only be observed by combining different methods and co-factors for evaluating FI activity. In conclusion, our results provide a new perspective regarding available diagnostic tools for assessing the impact of mutations in FI.

In vitro evaluation of iron oxide nanoparticle-induced thromboinflammatory response using a combined human whole blood and endothelial cell model.

Iron oxide nanoparticles (IONPs) are widely used in diagnostic and therapeutic settings. Upon systemic administration, however, they are rapidly recognized by components of innate immunity, which limit their therapeutic capacity and can potentially lead to adverse side effects. IONPs were previously found to induce the inflammatory response in human whole blood, including activation of the complement system and increased secretion of cytokines. Here, we investigated the thromboinflammatory response of 10-30 nm IONPs in lepirudin anticoagulated whole blood in interplay with endothelial cells and evaluated the therapeutic effect of applying complement inhibitors to limit adverse effects related to thromboinflammation. We found that IONPs induced complement activation, primarily at the C3-level, in whole blood incubated for up to four hours at 37°C with and without human microvascular endothelial cells. Furthermore, IONPs mediated a strong thromboinflammatory response, as seen by the significantly increased release of 21 of the 27 analyzed cytokines (p<0.05). IONPs also significantly increased cell-activation markers of endothelial cells [ICAM-1 (p<0.0001), P/E-selectin (p<0.05)], monocytes, and granulocytes [CD11b (p<0.001)], and platelets [CD62P (p<0.05), CD63 (p<0.05), NAP-2 (p<0.01), PF4 (p<0.05)], and showed cytotoxic effects, as seen by increased LDH (p<0.001) and heme (p<0.0001) levels. We found that inflammation and endothelial cell activation were partly complement-dependent and inhibition of complement at the level of C3 by compstatin Cp40 significantly attenuated expression of ICAM-1 (p<0.01) and selectins (p<0.05). We show that complement activation plays an important role in the IONPs-induced thromboinflammatory response and that complement inhibition is promising in improving IONPs biocompatibility.

Escherichia coli-induced inflammatory responses are temperature-dependent in human whole blood ex vivo.

Systemic inflammatory conditions are often associated with hypothermia or hyperthermia. Therapeutic hypothermia is used in post-cardiac arrest and some other acute diseases. There is a need for more knowledge concerning the effect of various temperatures on the acute inflammatory response. The complement system plays a crucial role in initiating the inflammatory response. We hypothesized that temperatures above and below the physiologic 37 °C affect complement activation and cytokine production ex vivo. Lepirudin-anticoagulated human whole blood from 10 healthy donors was incubated in the presence or absence of Escherichia coli at different temperatures (4 °C, 12 °C, 20 °C, 33 °C, 37 °C, 39 °C, and 41 °C). Complement activation was assessed by the terminal C5b-9 complement complex (TCC) and the alternative convertase C3bBbP using ELISA. Cytokines were measured using a 27-plex assay. Granulocyte and monocyte activation was evaluated by CD11b surface expression using flow cytometry. A consistent increase in complement activation was observed with rising temperature, reaching a maximum at 41 °C, both in the absence (C3bBbP p < 0.05) and presence (C3bBbP p < 0.05 and TCC p < 0.05) of E. coli. Temperature alone did not affect cytokine production, whereas incubation with E. coli significantly increased cytokine levels of IL-1ß, IL-2, IL-6, IL-8, IFN-γ, and TNF at temperatures > 20 °C. Maximum increase occurred at 39 °C. However, a consistent decrease was observed at 41 °C, significant for IL-1ß (p = 0.003). Granulocyte CD11b displayed the same temperature-dependent pattern as cytokines, with a corresponding increase in endothelial cell apoptosis and necrosis. Thus, blood temperature differentially determines the degree of complement activation and cytokine release.

Pitfalls in complement analysis: A systematic literature review of assessing complement activation.

Background: The complement system is an essential component of our innate defense and plays a vital role in the pathogenesis of many diseases. Assessment of complement activation is critical in monitoring both disease progression and response to therapy. Complement analysis requires accurate and standardized sampling and assay procedures, which has proven to be challenging. Objective: We performed a systematic analysis of the current methods used to assess complement components and reviewed whether the identified studies performed their complement measurements according to the recommended practice regarding pre-analytical sample handling and assay technique. Results are supplemented with own data regarding the assessment of key complement biomarkers to illustrate the importance of accurate sampling and measuring of complement components. Methods: A literature search using the Pubmed/MEDLINE database was performed focusing on studies measuring the key complement components C3, C5 and/or their split products and/or the soluble variant of the terminal C5b-9 complement complex (sTCC) in human blood samples that were published between February 2017 and February 2022. The identified studies were reviewed whether they had used the correct sample type and techniques for their analyses. Results: A total of 92 out of 376 studies were selected for full-text analysis. Forty-five studies (49%) were identified as using the correct sample type and techniques for their complement analyses, while 25 studies (27%) did not use the correct sample type or technique. For 22 studies (24%), it was not specified which sample type was used. Conclusion: A substantial part of the reviewed studies did not use the appropriate sample type for assessing complement activation or did not mention which sample type was used. This deviation from the standardized procedure can lead to misinterpretation of complement biomarker levels and hampers proper comparison of complement measurements between studies. Therefore, this study underlines the necessity of general guidelines for accurate and standardized complement analysis.

Synthetic Oligodeoxynucleotide CpG Motifs Activate Human Complement through Their Backbone Structure and Induce Complement-Dependent Cytokine Release.

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone-dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2-deficient serum. CpG-B increased levels of IL-1ß, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1-dependent cytokine release.

Heme Interferes With Complement Factor I-Dependent Regulation by Enhancing Alternative Pathway Activation.

Hemolysis, as a result of disease or exposure to biomaterials, is characterized by excess amounts of cell-free heme intravascularly and consumption of the protective heme-scavenger proteins in plasma. The liberation of heme has been linked to the activation of inflammatory systems, including the complement system, through alternative pathway activation. Here, we investigated the impact of heme on the regulatory function of the complement system. Heme dose-dependently inhibited factor I-mediated degradation of soluble and surface-bound C3b, when incubated in plasma or buffer with complement regulatory proteins. Inhibition occurred with factor H and soluble complement receptor 1 as co-factors, and the mechanism was linked to the direct heme-interaction with factor I. The heme-scavenger protein hemopexin was the main contaminant in purified factor I preparations. This led us to identify that hemopexin formed a complex with factor I in normal human plasma. These complexes were significantly reduced during acute vasoocclusive pain crisis in patients with sickle cell disease, but the complexes were normalized at their baseline outpatient clinic visit. Hemopexin exposed a protective function of factor I activity in vitro, but only when it was present before the addition of heme. In conclusion, we present a mechanistic explanation of how heme promotes uncontrolled complement alternative pathway amplification by interfering with the regulatory capacity of factor I. Reduced levels of hemopexin and hemopexin-factor I complexes during an acute hemolytic crisis is a risk factor for heme-mediated factor I inhibition.

Thrombin Differentially Modulates the Acute Inflammatory Response to Escherichia coli and Staphylococcus aureus in Human Whole Blood.

Thrombin plays a central role in thromboinflammatory responses, but its activity is blocked in the common ex vivo human whole blood models, making an ex vivo study of thrombin effects on thromboinflammatory responses unfeasible. In this study, we exploited the anticoagulant peptide Gly-Pro-Arg-Pro (GPRP) that blocks fibrin polymerization to study the effects of thrombin on acute inflammation in response to Escherichia coli and Staphylococcus aureus Human blood was anticoagulated with either GPRP or the thrombin inhibitor lepirudin and incubated with either E. coli or S. aureus for up to 4 h at 37°C. In GPRP-anticoagulated blood, there were spontaneous elevations in thrombin levels and platelet activation, which further increased in the presence of bacteria. Complement activation and the expression of activation markers on monocytes and granulocytes increased to the same extent in both blood models in response to bacteria. Most cytokines were not elevated in response to thrombin alone, but thrombin presence substantially and heterogeneously modulated several cytokines that increased in response to bacterial incubations. Bacterial-induced releases of IL-8, MIP-1α, and MIP-1ß were potentiated in the thrombin-active GPRP model, whereas the levels of IP-10, TNF, IL-6, and IL-1ß were elevated in the thrombin-inactive lepirudin model. Complement C5-blockade, combined with CD14 inhibition, reduced the overall cytokine release significantly, both in thrombin-active and thrombin-inactive models. Our data support that thrombin itself marginally induces leukocyte-dependent cytokine release in this isolated human whole blood but is a significant modulator of bacteria-induced inflammation by a differential effect on cytokine patterns.

Application of the C3 inhibitor compstatin in a human whole blood model designed for complement research - 20 years of experience and future perspectives.

The complex molecular and cellular biological systems that maintain host homeostasis undergo continuous crosstalk. Complement, a component of innate immunity, is one such system. Initially regarded as a system to protect the host from infection, complement has more recently been shown to have numerous other functions, including involvement in embryonic development, tissue modeling, and repair. Furthermore, the complement system plays a major role in the pathophysiology of many diseases. Through interactions with other plasma cascades, including hemostasis, complement activation leads to the broad host-protective response known as thromboinflammation. Most complement research has been limited to reductionistic models of purified components and cells and their interactions in vitro. However, to study the pathophysiology of complement-driven diseases, including the interaction between the complement system and other inflammatory systems, holistic models demonstrating only minimal interference with complement activity are needed. Here we describe two such models; whole blood anticoagulated with either the thrombin inhibitor lepirudin or the fibrin polymerization peptide blocker GPRP, both of which retain complement activity and preserve the ability of complement to be mutually reactive with other inflammatory systems. For instance, to examine the relative roles of C3 and C5 in complement activation, it is possible to compare the effects of the C3 inhibitor compstatin effects to those of inhibitors of C5 and C5aR1. We also discuss how complement is activated by both pathogen-associated molecular patterns, inducing infectious inflammation caused by organisms such as Gram-negative and Gram-positive bacteria, and by sterile damage-associated molecular patterns, including cholesterol crystals and artificial materials used in clinical medicine. When C3 is inhibited, it is important to determine the mechanism by which inflammation is attenuated, i.e., whether the attenuation derives directly from C3 activation products or via downstream activation of C5, since the mechanism involved may determine the appropriate choice of inhibitor under various conditions. With some exceptions, most inflammatory responses are dependent on C5 and C5aR1; one exception is venous air embolism, in which air bubbles enter the blood circulation and trigger a mainly C3-dependent thromboembolism, with the formation of an active C3 convertase, without a corresponding C5 activation. Under such conditions, an inhibitor of C3 is needed to attenuate the inflammation. Our holistic blood models will be useful for further studies of the inhibition of any complement target, not just C3 or C5. The focus here will be on targeting the critical complement component, activation product, or receptor that is important for the pathophysiology in a variety of disease conditions.

Platelet-Depletion of Whole Blood Reveals That Platelets Potentiate the Release of IL-8 From Leukocytes Into Plasma in a Thrombin-Dependent Manner.

Objective: In a recent study, we found an elevated level of interleukin 8 (IL-8) in response to bacterial incubation in thrombin-sufficient human whole blood anticoagulated by the fibrin polymerization blocking peptide GPRP. Whether thrombin directly activated leukocytes or mediated the release via thrombin-dependent activation of platelets remains unresolved. Herein, we addressed the role of thrombin and platelets in IL-8 release. Methods: We separated platelets from whole blood using a combination of 0.7% (w/v) citrate and GPRP for attenuating the hemostatic response during the separation of platelets. Cytokine responses were compared in whole blood and platelet-depleted blood upon Escherichia coli incubation. Cytokine responses were also profiled with and without reconstitution of either platelets or the supernatant from activated platelets. Results: Platelets were not activated during the separation process but responded to stimuli upon re-calcification. Plasma levels of IL-1ß, IL-1Ra, IL-6, IL-8, IP-10, MIP-1α, and MIP-1ß were significantly reduced in platelet-depleted blood compared to whole blood, but recovered in the presence of platelets, or with the supernatant of activated platelets. The leukocyte fraction and platelets were each found to contribute to the elevation of IL-8 at around 5 ng/ml; however, if combined, the release of IL-8 increased to 26 ng/ml. This process was dependent on thrombin since the levels of IL-8 remained at 5 ng/ml in whole blood if thrombin was blocked. Intracellular staining revealed that monocytes were the main source for IL-8 expression. Conclusion: Our findings suggest that the release of IL-8 is mediated by the leukocytes, mainly monocytes, but potentiated via thrombin-dependent activation of platelets.

Characterization of the foreign body response of titanium implants modified with polyphenolic coatings.

The foreign body response is dictating the outcome of wound healing around any implanted materials. Patients who suffer from chronic inflammatory diseases and impaired wound healing often face a higher risk for implant failure. Therefore, functional surfaces need to be developed to improve tissue integration. For this purpose, we evaluated the impact of surface coatings made of antioxidant polyphenolic molecules tannic acid (TA) and pyrogallol (PG) on the host response in human blood. Our results showed that although the polyphenolic surface modifications impact the initial blood protein adsorption compared to Ti, the complement and coagulation systems are triggered. Despite complement activation, monocytes and granulocytes remained inactivated, which was manifested in a low pro-inflammatory cytokine expression. Under oxidative stress, both coatings were able to reduce intracellular reactive oxygen species in human gingival fibroblasts (hGFs). However, no anti-inflammatory effects of polyphenolic coatings could be verified in hGFs stimulated with lipopolysaccharide and IL-1ß. Although polyphenols reportedly inhibit the NF-κB signaling pathway, phosphorylation of NF-κB p65 was observed. In conclusion, our results indicated that TA and PG coatings improved the hemocompatibility of titanium surfaces and have the potential to reduce oxidative stress during wound healing.

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood.

Introduction: Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy. Methods: In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy. Results and Discussion: We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.

A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity.

Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.

Tuning gelatin-based hydrogel towards bioadhesive ocular tissue engineering applications.

Gelatin based adhesives have been used in the last decades in different biomedical applications due to the excellent biocompatibility, easy processability, transparency, non-toxicity, and reasonable mechanical properties to mimic the extracellular matrix (ECM). Gelatin adhesives can be easily tuned to gain different viscoelastic and mechanical properties that facilitate its ocular application. We herein grafted glycidyl methacrylate on the gelatin backbone with a simple chemical modification of the precursor, utilizing epoxide ring-opening reactions and visible light-crosslinking. This chemical modification allows the obtaining of an elastic protein-based hydrogel (GELGYM) with excellent biomimetic properties, approaching those of the native tissue. GELGYM can be modulated to be stretched up to 4 times its initial length and withstand high tensile stresses up to 1.95 MPa with compressive strains as high as 80% compared to Gelatin-methacryloyl (GeIMA), the most studied derivative of gelatin used as a bioadhesive. GELGYM is also highly biocompatible and supports cellular adhesion, proliferation, and migration in both 2 and 3-dimensional cell-cultures. These characteristics along with its super adhesion to biological tissues such as cornea, aorta, heart, muscle, kidney, liver, and spleen suggest widespread applications of this hydrogel in many biomedical areas such as transplantation, tissue adhesive, wound dressing, bioprinting, and drug and cell delivery.

Innate Immune Invisible Ultrasmall Gold Nanoparticles-Framework for Synthesis and Evaluation.

Nanomedicine is seen as a potential central player in the delivery of personalized medicine. Biocompatibility issues of nanoparticles have largely been resolved over the past decade. Despite their tremendous progress, less than 1% of applied nanosystems can hit their intended target location, such as a solid tumor, and this remains an obstacle to their full ability and potential with a high translational value. Therefore, achieving immune-tolerable, blood-compatible, and biofriendly nanoparticles remains an unmet need. The translational success of nanoformulations from bench to bedside involves a thorough assessment of their design, compatibility beyond cytotoxicity such as immune toxicity, blood compatibility, and immune-mediated destruction/rejection/clearance profile. Here, we report a one-pot process-engineered synthesis of ultrasmall gold nanoparticles (uGNPs) suitable for better body and renal clearance delivery of their payloads. We have obtained uGNP sizes of as low as 3 nm and have engineered the synthesis to allow them to be accurately sized (almost nanometer by nanometer). The synthesized uGNPs are biocompatible and can easily be functionalized to carry drugs, peptides, antibodies, and other therapeutic molecules. We have performed in vitro cell viability assays, immunotoxicity assays, inflammatory cytokine analysis, a complement activation study, and blood coagulation studies with the uGNPs to confirm their safety. These can help to set up a long-term safety-benefit framework of experimentation to reveal whether any designed nanoparticles are immune-tolerable and can be used as payload carriers for next-generation vaccines, chemotherapeutic drugs, and theranostic agents with better body clearance ability and deep tissue penetration.

Quantification of Porcine Complement Activation Fragment C3a by a Neoepitope-Based Enzyme-Linked Immunosorbent Assay.

Enzyme-linked immunosorbent assay (ELISA) enables fast and simple quantification of analytes in the pico- to nanogram range in complex samples. Here, we describe an ELISA for the detection of porcine C3a as a marker for complement activation. Antibody specificity is critical for a robust assay. This assay is based on a pair of antibodies specific for the porcine C3a molecule and thus does not react with native C3.

Combined blockade of complement C5 and TLR co-receptor CD14 synergistically inhibits pig-to-human corneal xenograft induced innate inflammatory responses.

Inadequate supplies of donor corneas have evoked an escalating interest in corneal xenotransplantation. However, innate immune responses contribute significantly to the mechanism of xenograft rejection. We hypothesized that complement component C5 and TLR co-receptor CD14 inhibition would inhibit porcine cornea induced innate immune responses. Therefore, we measured cytokine release in human blood, induced by three forms of corneal xenografts with or without inhibitors. Native porcine cornea (NPC) induced interleukins (IL-1ß, IL-2, IL-6, IL-8, IL-1ra), chemokines (MCP-1, MIP-1α, MIP-1ß) and other cytokines (TNF, G-CSF, INF-γ, FGF-basic). Decellularized (DPC) and gamma-irradiated cornea (g-DPC) elevated the release of those cytokines. C5-blockade by eculizumab inhibited all the cytokines except G-CSF when induced by NPC. However, C5-blockade failed to reduce DPC and g-DPC induced cytokines. Blockade of CD14 inhibited DPC-induced cytokines except for IL-8, MCP-1, MIP-1α, and G-CSF, while it inhibited all of them when induced by g-DPC. Combined blockade of C5 and CD14 inhibited the maximum number of cytokines regardless of the xenograft type. Finally, by using the TLR4 specific inhibitor Eritoran, we showed that TLR4 activation was the basis for the CD14 effect. Thus, blockade of C5, when combined with TLR4 inhibition, may have therapeutic potential in pig-to-human corneal xenotransplantation. STATEMENT OF SIGNIFICANCE: Bio-engineered corneal xenografts are on the verge of becoming a viable alternative to allogenic human-donor-cornea, but the host's innate immune response is still a critical barrier for graft acceptance. By overruling this barrier, limited graft availability would no longer be an issue for treating corneal diseases. We showed that the xenograft induced inflammation is initiated by the complement system and toll-like receptor activation. Intriguingly, the inflammatory response was efficiently blocked by simultaneously targeting bottleneck molecules in the complement system (C5) and the TLR co-receptor CD14 with pharmaceutical inhibitors. We postulate that a combination of C5 and CD14 inhibition could have a great therapeutic potential to overcome the immunologic barrier in pig-to-human corneal xenotransplantation.