Smoking induces sex-specific changes in the small airway proteome.

INTRODUCTION: Cigarette smoke triggers many cellular and signaling responses in the lung and the resulting inflammation plays a central role in smoke-related lung diseases, such as COPD. We explored the effects of smoking on the small airway proteome in samples obtained by collection of exhaled particles with the aim to identify specific proteins dysregulated by smoking. METHODS: Exhaled particles were obtained from 38 current smokers, 47 former smokers and 22 healthy controls with the PExA method. 120 ng of sample was collected from individual subjects and analyzed with the SOMAscan proteomics platform. General linear model-based statistics were performed. RESULTS: Two hundred and three proteins were detected in at least half of 107 total samples. Active smoking exerted a significant impact on the protein composition of respiratory tract lining fluid (RTLF), with 81 proteins altered in current smokers compared to never smokers (p < 0.05, q < 0.124). Among the proteins most clearly discriminating between current and never smokers were sRAGE, FSTL3, SPOCK2 and protein S, all of them being less abundant in current smokers. Analysis stratified for sex unveiled sex differences with more pronounced proteomic alterations due to active smoking in females than males. Proteins whose abundance was altered by active smoking in women were to a larger extent related to the complement system. The small airway protein profile of former smokers appeared to be more similar to that observed in never smokers. CONCLUSIONS: The study shows that smoking has a strong impact on protein expression in the small airways, and that smoking affects men and women differently, suggesting PExA sampling combined with high sensitivity protein analysis offers a promising platform for early detection of COPD and identification of novel COPD drug targets.

Potential of Cell Surface Engineering with Biocompatible Polymers for Biomedical Applications.

The regulation of the cellular surface with biomaterials can contribute to the progress of biomedical applications. In particular, the cell surface is exposed to immunological surveillance and reactions in transplantation therapy, and modulation of cell surface properties might improve transplantation outcomes. The transplantation of therapeutic cells, tissue, and organs is an effective and fundamental treatment and has contributed to saving lives and improving quality of life. Because of shortages, donor cells, tissues, and organs are carefully transplanted with the goal of retaining activity and viability. However, some issues remain to be resolved in terms of reducing side effects, improving graft survival, managing innate and adaptive immune responses, and improving transplant storage and procedures. Given that the transplantation process involves multiple steps and is technically complicated, an engineering approach together with medical approaches to resolving these issues could enhance success. In particular, cell surface engineering with biocompatible polymers looks promising for improving transplantation therapy and has potential for other biomedical applications. Here we review the significance of polymer-based surface modification of cells and organs for biomedical applications, focusing on the following three topics: Cell protection: cellular protection through local immune regulation using cell surface modification with biocompatible polymers. This protection could extend to preventing attack by the host immune system, freeing recipients from taking immunosuppressive drugs, and avoiding a second transplantation. Cell attachment: cell manipulation, which is an important technique for delivery of therapeutic cells and their alignment for recellularization of decellularized tissues and organs in regenerative therapy. Cell fusion: fusion of different cells, which can lead to the formation of new functional cells that could be useful for generating, e.g., immunologically competent or metabolically active cells.

Complement activation in individuals with previous subclinical Lyme borreliosis and patients with previous Lyme neuroborreliosis.

Lyme borreliosis (LB) is caused by Borrelia burgdorferi and infection may lead to not only a large variety of clinical manifestations but also a subclinical outcome. The aim of the present study was to investigate if there is a constitutional difference in complement activation between individuals with previous subclinical Lyme borreliosis (SB) and patients previously diagnosed with Lyme neuroborreliosis (LNB).Lepirudin plasma for activation studies was collected from 60 SB individuals and from 22 patients pre-diagnosed with LNB. The plasma was incubated with live Borrelia spirochetes of two strains (complement sensitive B. garinii Lu59 and complement resistant B. afzelii ACA1).Complement factor C3 was measured in non-activated lepirudin plasma with immune-nephelometry and C3a and sC5b-9 generated during complement activation were measured by enzyme-linked immunosorbent assay.We found that the complement sensitive Lu59 induced higher complement activation than the complement resistant ACA1 when measuring activation products C3a and sC5b-9 in SB and LNB patients, p < 0.0001. No significant difference was found between SB and LNB patients in systemic levels of C3. Furthermore, SB individuals generated a higher activation of C3 cleavage to C3a (C3a/C3 ratio) than LNB patients after activation with ACA1, p < 0.001, but no significant differences were found in response to Lu59. In conclusion, Lu59 induced higher complement activation than ACA1 and individuals with previous SB showed increased generation of C3a compared with patients with previous LNB. In our study population, this mechanism could lead to less elimination of spirochetes in LNB patients and thereby be a factor contributing to the clinical outcome.

Celiac disease and complement activation in response to Streptococcus pneumoniae.

Individuals with celiac disease (CD) are at increased risk of invasive pneumococcal disease (IPD). The aim of this study was to explore whether the complement response to Streptococcus pneumoniae differed according to CD status, and could serve as an explanation for the excess risk of IPD in CD. Twenty-two children with CD and 18 controls, born 1999-2008, were included at Kalmar County Hospital, Sweden. The degree of complement activation was evaluated by comparing levels of activation products C3a and sC5b-9 in plasma incubated for 30 min with Streptococcus pneumoniae and in non-incubated plasma. Complement analyses were performed with enzyme-linked immunosorbent assay (ELISA). Pneumococcal stimulation caused a statistically significant increase in C3a as well as sC5b-9 in both children with CD and controls but there was no difference in response between the groups. After incubation, C3a increased on average 4.6 times and sC5b-9 22 times in both the CD and the control group (p = 0.497 and p = 0.724 respectively).Conclusion: Complement response to Streptococcus pneumoniae seems to be similar in children with and without CD and is thus unlikely to contribute to the increased susceptibility to invasive pneumococcal disease in CD.What is Known:• An excess risk of pneumococcal infections has been demonstrated in individuals with celiac disease.• Infectious complications can depend on hyposplenism but alternative mechanisms are sparsely examined.What is New:• Complement activation in response to Streptococcus pneumoniae was examined in children with and without celiac disease but no differences could be demonstrated.

Complement receptor 2 is up regulated in the spinal cord following nerve root injury and modulates the spinal cord response.

BACKGROUND: Activation of the complement system has been implicated in both acute and chronic states of neurodegeneration. However, a detailed understanding of this complex network of interacting components is still lacking. METHODS: Large-scale global expression profiling in a rat F2(DAxPVG) intercross identified a strong cis-regulatory influence on the local expression of complement receptor 2 (Cr2) in the spinal cord after ventral root avulsion (VRA). Expression of Cr2 in the spinal cord was studied in a separate cohort of DA and PVG rats at different time-points after VRA, and also following sciatic nerve transection (SNT) in the same strains. Consequently, Cr2 (-/-) mice and Wt controls were used to further explore the role of Cr2 in the spinal cord following SNT. The in vivo experiments were complemented by astrocyte and microglia cell cultures. RESULTS: Expression of Cr2 in naïve spinal cord was low but strongly up regulated at 5-7 days after both VRA and SNT. Levels of Cr2 expression, as well as astrocyte activation, was higher in PVG rats than DA rats following both VRA and SNT. Subsequent in vitro studies proposed astrocytes as the main source of Cr2 expression. A functional role for Cr2 is suggested by the finding that transgenic mice lacking Cr2 displayed increased loss of synaptic nerve terminals following nerve injury. We also detected increased levels of soluble CR2 (sCR2) in the cerebrospinal fluid of rats following VRA. CONCLUSIONS: These results demonstrate that local expression of Cr2 in the central nervous system is part of the axotomy reaction and is suggested to modulate subsequent complement mediated effects.

Can cells and biomaterials in therapeutic medicine be shielded from innate immune recognition?

Biomaterials (e.g. polymers, metals, or ceramics), cell and cell cluster (e.g. pancreatic islets) transplantation are beginning to offer novel treatment modalities for some otherwise intractable diseases. The innate immune system is involved in incompatibility reactions that occur when biomaterials or cells are introduced into the blood circulation. In particular, the complement, coagulation and contact systems are involved in the recognition of biomaterials and cells, eliciting activation of platelets and leukocytes. Such treatments are associated with anaphylactoid and thrombotic reactions, inflammation, and rejection of biomaterials and cells, leading to treatment failures and adverse reactions. We discuss here the new technologies that are being developed to shield the biomaterial and cell surfaces from recognition by the innate immune system.

Complement diagnostics: concepts, indications, and practical guidelines.

Aberrations in the complement system have been shown to be direct or indirect pathophysiological mechanisms in a number of diseases and pathological conditions such as autoimmune disease, infections, cancer, allogeneic and xenogeneic transplantation, and inflammation. Complement analyses have been performed on these conditions in both prospective and retrospective studies and significant differences have been found between groups of patients, but in many diseases, it has not been possible to make predictions for individual patients because of the lack of sensitivity and specificity of many of the assays used. The basic indications for serological diagnostic complement analysis today may be divided into three major categories: (a) acquired and inherited complement deficiencies; (b) disorders with complement activation; (c) inherited and acquired C1INH deficiencies. Here, we summarize indications, techniques, and interpretations for basic complement analyses and present an algorithm, which we follow in our routine laboratory.

Complement activation by CpG in a human whole blood loop system: mechanisms and immunomodulatory effects.

Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.

Mapping pro- and antiangiogenic factors on the surface of prostasomes of normal and malignant cell origin.

BACKGROUND: Angiogenesis is the formation of new blood vessels by capillary sprouting from pre-existing vessels. Tumor growth is angiogenesis-dependent and the formation of new blood vessels is associated with the increased expression of angiogenic factors. Prostasomes are secretory granules produced, stored and released by the glandular epithelial cells of the prostate. We investigated the expression of selected angiogenic and anti-angiogenic factors on the surface of prostasomes of different origins as well as the direct effect of prostasomes on angiogenesis. METHODS: VEGF, endothelin-1, endostatin, and thrombospondin-1 were determined on prostasomes from seminal fluid and human prostate cancer cell lines (DU145,PC-3,LNCaP) using different immunochemical techniques. Human dermal microvascular endothelial cells were incubated with seminal and DU145 cell-prostasomes and with radioactive thymidine. The effect of prostasomes on angiogenesis was judged by measuring the uptake of labeled thymidine. The presence of any deleterious effects of prostasomes on the endothelial cells was investigated using thymidine assay and confocal laser microscopy. RESULTS: VEGF and endothelin-1 were determined on malignant cell-prostasomes (no difference between cell lines) but not determined on seminal prostasomes. The same applies for the expression of endostatin but with much higher expression on malignant cell-prostasomes with obvious differences between them. Seminal and DU145 cell-prostasomes were found to have anti-angiogenic effect which was more expressed by DU145 cell-prostasomes. No deleterious effect of prostasomes on endothelial function was detected using either thymidine assay or microscopy. CONCLUSIONS: Prostasomes contain pro- and anti-angiogenic factors that function to counteract each other unless the impact from one side exceeds the other to bring about dysequilibrium.

Activated human platelets induce factor XIIa-mediated contact activation.

Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.

Mannose-Binding Lectin is Associated with Thrombosis and Coagulopathy in Critically Ill COVID-19 Patients.

The ongoing COVID-19 pandemic has caused significant morbidity and mortality worldwide, as well as profound effects on society. COVID-19 patients have an increased risk of thromboembolic (TE) complications, which develop despite pharmacological thromboprophylaxis. The mechanism behind COVID-19-associated coagulopathy remains unclear. Mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation, has been suggested as a potential amplifier of blood coagulation during thromboinflammation. Here we describe data from a cohort of critically ill COVID-19 patients (n = 65) treated at a tertiary hospital center intensive care unit (ICU). A subset of patients had strongly elevated MBL plasma levels, and activity upon ICU admission, and patients who developed symptomatic TE (14%) had significantly higher MBL levels than patients without TE. MBL was strongly correlated to plasma D-dimer levels, a marker of COVID-19 coagulopathy, but showed no relationship to degree of inflammation or other organ dysfunction. In conclusion, we have identified complement activation through the MBL pathway as a novel amplification mechanism that contributes to pathological thrombosis in critically ill COVID-19 patients. Pharmacological targeting of the MBL pathway could be a novel treatment option for thrombosis in COVID-19. Laboratory testing of MBL levels could be of value for identifying COVID-19 patients at risk for TE events.

Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation.

BACKGROUND: A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non-human primates. The detrimental instant blood-mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen. MATERIALS AND METHODS: Low molecular weight dextran sulfate (LMW-DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non-human primate model of pancreatic islet transplantation. Adult porcine islets (10,000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW-DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW-DS and Compstatin. RESULTS: Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T-cell infiltration observed in the control (heparin-treated) animals were abrogated in the LMW-DS-treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW-DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro. CONCLUSIONS: The xenogeneic IBMIR in this non-human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW-DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig-human clinical islet transplantation.

The role of complement in biomaterial-induced inflammation.

Biomaterials are regularly used in various types of artificial tissues and organs, such as oxygenators, plasmapheresis equipment, hemodialysers, catheters, prostheses, stents, vascular grafts, miniature pumps, sensors and heart aids. Although progress has been made regarding bioincompatibility, many materials and procedures are associated with side effects, in particular bioincompatibility-induced inflammation, infections and subsequent loss of function. After cardiopulmonary bypass, coagulopathies can occur and lead to cognitive disturbances, stroke and extended hospitalization. Hemodialysis is associated with anaphylatoid reactions that cause whole-body inflammation and may contribute to accelerated arteriosclerosis. Stents cause restenosis and, in severe cases, thrombotic reactions. This situation indicates that there is still a need to try to understand the mechanisms involved in these incompatibility reactions in order to be able to improve the biomaterials and to develop treatments that attenuate the reactions and thereby reduce patients' discomfort, treatment time and cost. This overview deals with the role of complement in the incompatibility reactions that occur when biomaterials come in contact with blood and other body fluids.

Author Correction: Dispersion of TiO2 nanoparticles improves burn wound healing and tissue regeneration through specific interaction with blood serum proteins.

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Dispersion of TiO2 nanoparticles improves burn wound healing and tissue regeneration through specific interaction with blood serum proteins.

Burn wounds are one of the most important causes of mortality and especially morbidity around the world. Burn wound healing and skin tissue regeneration remain thus one of the most important challenges facing the mankind. In the present study we have addressed this challenge, applying a solution-stabilized dispersion TiO2 nanoparticles, hypothesizing that their ability to adsorb proteins will render them a strong capacity in inducing body fluid coagulation and create a protective hybrid material coating. The in vitro study of interaction between human blood and titania resulted at enhanced TiO2 concentrations in formation of rather dense gel composite materials and even at lower content revealed specific adsorption pattern initiating the cascade response, promising to facilitate the regrowth of the skin. The subsequent in vivo study of the healing of burn wounds in rats demonstrated formation of a strongly adherent crust of a nanocomposite, preventing infection and inflammation with quicker reduction of wound area compared to untreated control. The most important result in applying the TiO2 dispersion was the apparently improved regeneration of damaged tissues with appreciable decrease in scar formation and skin color anomalies.

Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation.

There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low-molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15-60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets.

Surface-attached PEO in the form of activated Pluronic with immobilized factor H reduces both coagulation and complement activation in a whole-blood model.

In the present work we have bound Pluronic, a class of triblock copolymers consisting of a block of polypropylene oxide (PPO) surrounded on each side by polyethylene oxide (PEO) blocks, to polystyrene surfaces and investigated the thrombogenicity and complement activation of this construct upon exposure to whole blood. The surface was highly inert towards coagulation, unfortunately at the expense of increased complement activation. We, therefore, as an alternative approach, used End-Group Activated Pluronic to conjugate factor H, a regulator of complement activation (RCA), to the surface. The bound factor H did not detach from the surface upon incubation with human serum. Furthermore, factor H bound in a physiological conformation could to a significant degree attenuate complement activation at the Pluronic surface. Thus, we have created a hybrid surface in which the coagulation-inert properties of the original Pluronic are supplemented with a specific complement-inhibitory effect. Medical device technology includes numerous potential applications for crosslinkers that are capable of specifically binding biomolecules to surfaces with retained activity. These applications include coupling of functional biomolecules to biomedical devices such as stents and grafts. The biomolecule may be an RCA, antibody, or other beneficial ligand.

Inhibition of thrombin abrogates the instant blood-mediated inflammatory reaction triggered by isolated human islets: possible application of the thrombin inhibitor melagatran in clinical islet transplantation.

A thrombotic/inflammatory reaction is elicited when isolated islets of Langerhans come in contact with ABO-compatible blood. The detrimental effects of this instant blood-mediated inflammatory reaction (IBMIR) provide a reasonable explanation for the observation that an unexpectedly high number of islets, from several donors, are needed to produce normoglycemia in transplant patients with type 1 diabetes. In this study, the hypothesis that a specific thrombin inhibitor, Melagatran, could reduce IBMIR in an in vitro model in which human islets are exposed to ABO-compatible blood was tested. The administration of Melagatran abrogated IBMIR dose-dependently. Islets exposed to blood, in the absence or presence of 0.4 micromol/l Melagatran, exhibited a loss of integrity and were found to be trapped in macroscopic clots containing platelets and CD11b(+) leukocytes. At concentrations from 1 to 10 micromol/l, Melagatran inhibited both coagulation and complement activation. Also, platelet and leukocyte activation and consumption were decreased. Islet morphology was maintained with almost no platelets adhering to the surface, and infiltration by CD11b(+) leukocytes was considerably reduced. In conclusion, Melagatran significantly reduced IBMIR in this model system. This protective effect indicates that thrombin plays a pivotal role in IBMIR and suggests that thrombin inhibition can improve the outcome of clinical islet transplantation.

Binding of C3 fragments on top of adsorbed plasma proteins during complement activation on a model biomaterial surface.

In the present study we investigate whether complement activation in blood in contact with a model biomaterial surface (polystyrene) occurs directly on the material surface or on top of an adsorbed plasma protein layer. Quartz crystal microbalance-dissipation analysis (QCM-D) complemented with enzyme immunoassays and Western blotting were used. QCM-D showed that the surface was immediately covered with a plasma protein film of approximately 8 nm. Complement activation that started concomitantly with the adsorption of the protein film was triggered by a self-limiting classical pathway activation. After adsorption of the protein film, alternative pathway activation provided the bulk of the C3b deposition that added 25% more mass to the surface. The build up of alternative pathway convertase complexes using purified C3 and factors B and D on different protein films as monitored by QCM-D showed that only adsorbed albumin, IgG, but not fibrinogen, allowed C3b binding, convertase assembly and amplification. Western blotting of eluted proteins from the material surface demonstrated that the C3 fragments were covalently bound to other proteins. This is consistent with a model in which the activation is triggered by initiating convertases formed by means of the initially adsorbed proteins and the main C3b binding is mediated by the alternative pathway on top of the adsorbed protein film.