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Objectives: To examine whether urinary tract infection-associated stimuli could regulate heme oxygenase-1 (HO-1) expression and to asses the significance of HO-1 in protecting urinary tract epithelial cells against nitric oxide (NO)-induced damage.Methods: Heme oxygenase-1 expression was investigated in the human renal epithelial cell line A498 in response to the uropathogenic Escherichia coli (UPEC) strain IA2, the NO-donor DETA/NONOate (DETA/NO), and proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) using reverse transcriptase polymerase chain reaction and Western blot analysis. Cell viability was examined by the trypan blue exclusion test and light microscopy.Results: The HO-1 inducer hemin and DETA/NO increased HO-1 expression in A498 cells, and glutathione depletion further increased HO-1 expression in response to DETA/NO and hemin. Stimulation with a UPEC strain or cytokines did not upregulate HO-1 expression. The cytokines induced inducible NO synthase expression and caused an increase in nitrite production. Hemin significantly decreased cytokine-induced NO production (P <0.001). DETA/NO decreased the cell viability by approximately 75%, but hemin was able to attenuate DETA/NO-induced cell damage.Conclusions: The expression of HO-1 increased in human renal epithelial cells in response to NO, and the expression was further enhanced in glutathione-depleted cells. The bacteria per se or proinflammatory cytokines were not able to upregulate HO-1. Heme oxygenase-1 protects the cells against NO by feedback inhibition of NO production and by decreasing cell damage. [ABSTRACT FROM AUTHOR]

A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damage. The porcine retina is a favorable in vitro model for studies of the degenerative processes that follow retinal detachment. Photoreceptor degeneration and synaptic injuries develop rapidly in the cultured porcine retina and correlate with resident microglial cell transition into a reactive phenotype. In this in vitro study, we used retinas cultured for five days and analyzed reactive CD11b and Iba1 immunoreactive microglia that localized close to/within the synaptic outer plexiform layer (OPL) and in the outer nuclear layer (ONL). A subpopulation of the CD11b and Iba1immunoreactive microglia also expressed CD68 immunoreactivity on lysosomal membranes or as a diffuse cytoplasmic stain. Some CD68 immunoreactive microglia were juxtaposed to L/M-opsin immunoreactive cone photoreceptors in the ONL. CD11b and Iba immunoelectron microscopy further suggests the presence of a dark microglial phenotype in the degenerating cultured porcine retina. For immunoelectron microscopy, nickel-enhanced diaminobenzidine (DAB) staining resulted in clearly distinguished reaction products in the cytosol of dark microglia., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

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 (TXA 2 R) 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 TXA 2 R 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, TXA 2 R activation.

Transferrin receptor 1 (TfR) delivers iron across cellular membranes by shuttling the ion carrier protein transferrin. This ability to deliver large protein ligands inside cells is taken advantage of by pathogens to infiltrate human cells. Notably, the receptor's outermost ectodomain, the apical domain, is used as a point of attachment for several viruses including hemorrhagic arenaviruses. To better understand interactions with the receptor it would be advantageous to probe sequence determinants in the apical domain with viral spike proteins. Here, we carried out affinity maturation of our computationally designed apical domain from human TfR to identify underlying driving forces that lead to better binding. The improved variants were confirmed by in vitro surface plasmon resonance measurements with dissociation constants obtained in the lower nanomolar range. It was found that the strong binding affinities for the optimized variants matched the strength of interactions with the native receptor. The structure of the best variant was determined experimentally indicating that the conformational change in the hairpin binding motif at the protein-protein interface plays a crucial role. The experimental methodology can be straightforwardly applied to other arenavirus or pathogens that use the apical domain. It can further be useful to probe host-virus compatibility or therapeutic strategies based on the transferrin receptor decoys., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: TW is an inventor of patents pertaining to complement inhibitors for inflammatory disease. He has acted as a consultant to companies (Visterra Inc, and Iveric Bio) that are commercially developing complement inhibitors and has received an honorarium from Alexion Pharmaceuticals for participation in industry conferences and meetings. JL is the founder of Amyndas Pharmaceuticals, which develops complement inhibitors for therapeutic purposes; is inventor of a broad patent portfolio that describes the therapeutic use of complement inhibitors, some of which are developed by Amyndas Pharmaceuticals, inventor of the compstatin technology licensed to Apellis Pharmaceuticals i.e., 41MeW7W/POT-4/APL-1 and pegylated derivatives such as APL-2/pegcetacoplan and APL-9; and has received consulting fees from Achillion, Baxter, LipimetiX, Ra Pharma, Sanofi, and Viropharma. TM is a member of the scientific advisory board of Ra Pharma/UCB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gerogianni, Bal, Mohlin, Woodruff, Lambris, Mollnes, Sjöström and Nilsson.)

A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damages. Experimental retinal detachment in vivo is an invasive and complicated method performed on anesthetized animals. As retinal detachment may result in visual impairment and blindness, research is of fundamental importance for understanding degenerative processes. Both morphological and ethical issues make the porcine retina a favorable organotypic model for studies of the degenerative processes that follow retinal detachment. In the cultured retina, photoreceptor degeneration and synaptic injuries develop rapidly and correlate with resident microglial cells' transition into a reactive phenotype. In this immunohistochemical study, we have begun to analyze the transition of subsets of reactive microglia which are known to localize close to the outer plexiform layer (OPL) in degenerating in vivo and in vitro retina. Biomarkers for reactive microglia included P2Ry12, CD63 and CD68 and the general microglial markers were CD11b, Iba1 and isolectin B 4 (IB 4 ). The reactive microglia markers labeled microglia subpopulations, suggesting that protective or harmful reactive microglia may be present simultaneously in the injured retina. Our findings support the usage of porcine retina cultures for studies of photoreceptor injuries related to retinal detachment.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gerogianni, Dimitrov, Zarantonello, Poillerat, Chonat, Sandholm, McAdam, Ekdahl, Mollnes, Mohlin, Roumenina and Nilsson.)

Human transferrin receptor 1 (TfR) is necessary for the delivery of the iron carrier protein transferrin into cells and can be utilized for targeted delivery across cellular membranes. Binding of transferrin to the receptor is regulated by hereditary hemochromatosis protein (HFE), an iron regulatory protein that partly shares a binding site with transferrin on TfR. Here, we derived essential binding interactions from HFE and computationally grafted these into a library of small protein scaffolds. One of the designed proteins, TB08, was further optimized computationally and experimentally to identify variants with improved binding to TfR. The optimized variant, TB08 S3.1, expressed well in the E. coli expression system and had an affinity to TfR in the low micromolar range, K d  ≈ 1 μm, as determined by surface plasmon resonance. A binding competition assay with transferrin further confirmed the interaction of the evolved variant to TfR at the shared binding surface. Additionally, the GFP-tagged evolved variant of TB08 demonstrated cellular internalization as determined by fluorescent and confocal microscopy in HeLa cells. The designed protein is small, allows for robust cargo tagging, and interacts specifically with TfR, thus making it a valuable tool for the characterization of TfR-mediated cellular transport mechanisms and for the assessment of engineering strategies for cargo delivery across cell membranes., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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-1β, IL-2, IL-6, IL-8, IL-1ra), chemokines (MCP-1, MIP-1α, MIP-1β) 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Accurate determination of complement component C1q is hampered by the fact that C1q is an immune complex binding protein. Consequently, immunochemical techniques which rely on immune complex formation in fluid phase such as nephelometry and turbidimetry tend to give results which differ from those obtained by, for example, ELISA and other solid phase-based assays. In this chapter, we discuss the pros and cons of different techniques for the quantification of C1q and present a comprehensive protocol for a newly developed magnetic bead-based sandwich immunoassay which has replaced nephelometry in our complement diagnostic laboratory at the University Hospital in Uppsala.

In the adult retina, ramifying microglia interact with the outer plexiform layer (OPL) monitoring the synaptic integrity between photoreceptors and post-synaptic target cells. Microglia are reactive during photoreceptor diseases, but their disease-related function(s) are not fully understood. Retinal explant cultures are model systems used to study degenerative events including photoreceptor degeneration and gliosis. Our culture paradigm, with adult porcine retinas subjected to coculture with human A-retinal pigment epithelia-19 (ARPE) cells, is an experimental approach resulting in improved photoreceptor survival and reduced gliosis. Under the in vitro pathological conditions with photoreceptor degeneration, reactive Iba1-and CD11b-immunoreactive microglia and their processes positioned in proximity with the OPL and among photoreceptor outer segments. Coculture for 3 days with ARPE-cells resulted in a significantly increased density of microglia at the OPL. After 5 days of culture, the density of microglia at the OPL was similar between coculture and control specimens. Electron microscopy revealed the presence of two subtypes of microglia: one exhibiting a dark nucleus and cytosol with dilated endoplasmic reticulum, vacuoles, endosomes and mitochondrial variations. This subtype localized close to synaptic structures in the OPL. The other subtype appeared as pale phagocytic microglia localized among degenerating outer segments. The Iba1-and CD11b-immunoreactive microglia in degenerating retina may be of two separate subtypes, which differ in localization, subcellular morphology and perhaps function., (Copyright © 2020. Published by Elsevier Ltd.)

Aberrations in complement system functions have been identified as either direct or indirect pathophysiological mechanisms in many diseases and pathological conditions, such as infections, autoimmune diseases, inflammation, malignancies, and allogeneic transplantation. Currently available techniques to study complement include quantification of (a) individual complement components, (b) complement activation products, and (c) molecular mechanisms/function. An emerging area of major interest in translational studies aims to study and monitor patients on complement regulatory drugs for efficacy as well as adverse events. This area is progressing rapidly with several anti-complement therapeutics under development, in clinical trials, or already in clinical use. In this review, we summarized the appropriate indications, techniques, and interpretations of basic complement analyses, exemplified by a number of clinical disorders., (Copyright © 2019 Mohebnasab, Eriksson, Persson, Sandholm, Mohlin, Huber-Lang, Keating, Ekdahl and Nilsson.)

In the alternative pathway (AP) an amplification loop is formed, which is strictly controlled by various fluid-phase and cell-bound regulators resulting in a state of homeostasis. Generation of the "C3b-like" C3(H 2 O) has been described as essential for AP activation, since it conveniently explains how the initial fluid-phase AP convertase of the amplification loop is generated. Also, the AP has a status of being an unspecific pathway despite thorough regulation at different surfaces. During complement attack in pathological conditions and inflammation, large amounts of C3b are formed by the classical/lectin pathway (CP/LP) convertases. After the discovery of LP´s recognition molecules and its tight interaction with the AP, it is increasingly likely that the AP acts in vivo mainly as a powerful amplification mechanism of complement activation that is triggered by previously generated C3b molecules initiated by the binding of specific recognition molecules. Also in many pathological conditions caused by a dysregulated AP amplification loop such as paroxysmal nocturnal hemoglobulinuria (PNH) and atypical hemolytic uremic syndrome (aHUS), C3b is available due to minute LP and CP activation and/or generated by non-complement proteases. Therefore, C3(H 2 O) generation in vivo may be less important for AP activation during specific attack or dysregulated homeostasis, but may be an important ligand for C3 receptors in cell-cell interactions and a source of C3 for the intracellular complement reservoir., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Platelets play an essential role in maintaining homeostasis in the circulatory system after an injury by forming a platelet thrombus, but they also occupy a central node in the intravascular innate immune system. This concept is supported by their extensive interactions with immune cells and the cascade systems of the blood. In this review we discuss the close relationship between platelets and the complement system and the role of these interactions during thromboinflammation. Platelets are protected from complement-mediated damage by soluble and membrane-expressed complement regulators, but they bind several complement components on their surfaces and trigger complement activation in the fluid phase. Furthermore, localized complement activation may enhance the procoagulant responses of platelets through the generation of procoagulant microparticles by insertion of sublytic amounts of C5b9 into the platelet membrane. We also highlight the role of post-translational protein modifications in regulating the complement system and the critical role of platelets in driving these reactions. In particular, modification of disulfide bonds by thiol isomerases and protein phosphorylation by extracellular kinases have emerged as important mechanisms to fine-tune complement activity in the platelet microenvironment. Lastly, we describe disorders with perturbed complement activation where part of the clinical presentation includes uncontrolled platelet activation that results in thrombocytopenia, and illustrate how complement-targeting drugs are alleviating the prothrombotic phenotype in these patients. Based on these clinical observations, we discuss the role of limited complement activation in enhancing platelet activation and consider how these drugs may provide opportunities for further dissecting the complex interactions between complement and platelets.

In this review article, we focus on activation of the soluble components of the innate immune system triggered by nonbiological compounds and stress variances in activation due to the difference in size between nanoparticles (NPs) and larger particles or bulk material of the same chemical and physical composition. We then discuss the impact of the so-called protein corona which is formed on the surface of NPs when they come in contact with blood or other body fluids. For example, NPs which bind inert proteins, proteins which are prone to activate the contact system (e.g., factor XII), which may lead to clotting and fibrin formation or the complement system (e.g., IgG or C3), which may result in inflammation and vascular damage. Furthermore, we describe a whole blood model which we have developed to monitor activation and interaction between different components of innate immunity: blood protein cascade systems, platelets, leukocytes, cytokine generation, which are induced by NPs. Finally, we describe our own studies on innate immunity system activation induced by three fundamentally different species of NPs (two types of engineered NPs and diesel NPs) as demonstrator of the utility of an initial determination of the composition of the protein corona formed on NPs exposed to ethylenediaminetetraacetic acid (EDTA) plasma and subsequent analysis in our whole blood model.

Increasing evidence indicates an integral role for the complement system in the deleterious inflammatory reactions that occur during critical phases of the transplantation process, such as brain or cardiac death of the donor, surgical trauma, organ preservation and ischaemia-reperfusion injury, as well as in humoral and cellular immune responses to the allograft. Ischaemia is the most common cause of complement activation in kidney transplantation and in combination with reperfusion is a major cause of inflammation and graft damage. Complement also has a prominent role in antibody-mediated rejection (ABMR) owing to ABO and HLA incompatibility, which leads to devastating damage to the transplanted kidney. Emerging drugs and treatment modalities that inhibit complement activation at various stages in the complement cascade are being developed to ameliorate the damage caused by complement activation in transplantation. These promising new therapies have various potential applications at different stages in the process of transplantation, including inhibiting the destructive effects of ischaemia and/or reperfusion injury, treating ABMR, inducing accommodation and modulating the adaptive immune response.

Complement system aberrations have been identified as pathophysiological mechanisms in a number of diseases and pathological conditions either directly or indirectly. Examples of such conditions include infections, inflammation, autoimmune disease, as well as allogeneic and xenogenic transplantation. Both prospective and retrospective studies have demonstrated significant complement-related differences between patient groups and controls. However, due to the low degree of specificity and sensitivity of some of the assays used, it is not always possible to make predictions regarding the complement status of individual patients. Today, there are three main indications for determination of a patient's complement status: (1) complement deficiencies (acquired or inherited); (2) disorders with aberrant complement activation; and (3) C1 inhibitor deficiencies (acquired or inherited). An additional indication is to monitor patients on complement-regulating drugs, an indication which may be expected to increase in the near future since there is now a number of such drugs either under development, already in clinical trials or in clinical use. Available techniques to study complement include quantification of: (1) individual components; (2) activation products, (3) function, and (4) autoantibodies to complement proteins. In this review, we summarize the appropriate indications, techniques, and interpretations of basic serological complement analyses, exemplified by a number of clinical disorders.

Congo red (CR) is a histological stain used for the detection of extracellular amyloids mediating various neurodegenerative diseases. Given that damaged photoreceptors appear to degenerate similarly to other nerve cells, CR staining was evaluated in experimentally injured porcine retina. CR staining appeared mostly as discrete cytosolic deposits with no obvious plaque formation during the investigated time period. Increases of CR labeling coincided temporally with the known accumulation of mislocalized opsins and increases of cell death. Coculture, either with human retinal pigment epithelium (ARPE) or human neural progenitor (ReN) cells, was accompanied by a significant reduction of CR labeling. Of particular interest was the reduction of CR labeling in cone photoreceptors, which are important for the perception of color and fine details and afflicted in age-related macular degeneration (AMD). Electron microscopy revealed inclusions in the inner segment, cell body, and occasionally synaptic terminals of photoreceptor cells in cultured specimens. Closer examinations indicated the presence of different types of inclusions resembling protein aggregates as well as inclusion bodies. The current results indicate that injury-related response resulted in accumulation of CR deposits in photoreceptor cells, and that trophic and/or structural support attenuated this response.

Iron-oxide nanoparticles (NPs) generated by environmental events are likely to represent health problems. α-Fe 2 O 3 NPs were synthesized, characterized and tested in a model for toxicity utilizing human whole blood without added anticoagulant. MALDI-TOF of the corona was performed and activation markers for plasma cascade systems (complement, contact and coagulation systems), platelet consumption and release of growth factors, MPO, and chemokine/cytokines from blood cells were analyzed. The coronas formed on the pristine α-Fe 2 O 3 NPs contained contact system proteins and they induced massive activation of the contact (kinin/kallikrein) system, as well as thrombin generation, platelet activation, and release of two pro-angiogeneic growth factors: platelet-derived growth factor and vascular endothelial growth factor, whereas complement activation was unaffected. The α-Fe 2 O 3 NPs exhibited a noticeable toxicity, with kinin/kallikrein activation, which may be associated with hypotension and long-term angiogenesis in vivo, with implications for cancer, arteriosclerosis and pulmonary disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The complement system (CS) plays a role in the pathogenesis of a number of ocular diseases, including diabetic retinopathy (DR), glaucoma, uveitis, and age-related macular degeneration (AMD). Given that many of the complex eye-related degenerative diseases have limited treatment opportunities, we aimed to mimic the in vivo retinal degenerative process by developing a relevant co-culture system., Method and Materials: The adult porcine retina was co-cultured with the spontaneously arising human retinal pigment epithelial cells-19 (ARPE-19)., Results: Inflammatory activity was found after culture and included migrating microglial cells, gliosis, cell death, and CS activation (demonstrated by a minor increase in the secreted anaphylotoxin C3a in co-culture). CS components, including C1q, C3, C4, soluble C5b-9, and the C5a receptor, were expressed in the retina and/or ARPE cells after culture. C1q, C3, and CS regulators such as C4 binding protein (C4BP), factor H (CFH), and factor I (CFI) were secreted after culture., Discussion: Thus, our research indicates that this co-culturing system may be useful for investigations of the CS and its involvement in experimental neurodegenerative diseases.

The complement system is a vital component of the immune-priveliged human eye that is always active at a low-grade level, preventing harmful intraocular injuries caused by accumulation of turnover products and controlling pathogens to preserve eye homeostasis and vision. The complement system is a double-edged sword that is essential for protection but may also become harmful and contribute to eye pathology. Here, we review the evidence for the involvement of complement system dysregulation in age-related macular degeneration, glaucoma, uveitis, and neuromyelitis optica, highlighting the relationship between morphogical changes and complement system protein expression and regulation in these diseases. The potential benefits of complement inhibition in age-related macular degeneration, glaucoma, uveitis, and neuromyelitis optica are abundant, as are those of further research to improve our understanding of complement-mediated injury in these diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Retinal neurodegenerative disorders like retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and retinal detachment decrease retinal functionality leading to visual impairment. The pathological events are characterized by photoreceptor degeneration, synaptic disassembly, remodeling of postsynaptic neurons and activation of glial cells. Despite intense research, no effective treatment has been found for these disorders. The current study explores the potential of human neural progenitor cell (hNPC) derived factors to slow the degenerative processes in adult porcine retinal explants. Retinas were cultured for 3 days with or without hNPCs as a feeder layer and investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemical, western blot and quantitative real time-polymerase chain reaction (qRT-PCR) techniques. TUNEL showed that hNPCs had the capacity to limit photoreceptor cell death. Among cone photoreceptors, hNPC coculture resulted in better maintenance of cone outer segments and reduced opsin mislocalization. Additionally, maintained synaptic structural integrity and preservation of second order calbindin positive horizontal cells was also observed. However, Müller cell gliosis only seemed to be alleviated in terms of reduced Müller cell density. Our observations indicate that at 3 days of coculture, hNPC derived factors had the capacity to protect photoreceptors, maintain synaptic integrity and support horizontal cell survival. Human neural progenitor cell applied treatment modalities may be an effective strategy to help maintain retinal functionality in neurodegenerative pathologies. Whether hNPCs can independently hinder Müller cell gliosis by utilizing higher concentrations or by combination with other pharmacological agents still needs to be determined., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate rod and cone photoreceptor degeneration in organotypic cultures of adult porcine retina. Our hypothesis was that the photoreceptors accumulate opsins, which, together with exposure to cyclic dim light illumination, induce autophagy and endoplasmic reticulum stress (ER-stress) to overcome damaging protein overload. For this purpose, retinas were cultured for 48 h and 72 h during which they were illuminated with dim light for 8h/day; specimens were analyzed by means of immunohistochemistry, Western blot, real-time polymerase chain reaction (PCR) and transmission electron microscopy. ER-stress and photoreceptor degeneration was observed in conventionally cultured retinas. The additional stress in the form of dim light illumination for 8h/day resulted in increased levels of the ER-stress markers GRP78/BiP and CHOP, as well as increased level of active caspase-12. Increased autophagic processes in cone and rod photoreceptors were detected by LC3B-II increases and occurrence of autophagosomes at the ultrastructural level. Illumination also resulted in altered protein expression for autophagy inducers such as p62 and Beclin-1. Moreover, there was a decrease in phosphorylated mammalian target of rapamycin (mTOR), which further indicate an increase of autophagy. Rod and cone photoreceptors in retinas from a diurnal animal that were exposed to dim light illumination in vitro displayed autophagy and ER-stress processes. As no alteration of rhodopsin mRNA was observed, autophagy and ER-stress are suggested to decrease rhodopsin protein at the posttranscriptional level., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background/aims: Increased nitric oxide (NO) production or inducible form of NO synthase activity have been documented in patients suffering from urinary tract infection (UTI), but the role of NO in this infection is unclear. We investigated whether NO can affect the host response in human renal epithelial cells by modulating IL-6 production and mRNA expression., Methods: The human renal epithelial cell line A498 was infected with a uropathogenic Escherichia coli (UPEC) strain and/or the NO donor DETA/NO. The IL-6 production and mRNA expression were evaluated by ELISA and real-time RT-PCR. IL-6 mRNA stability was evaluated by analyzing mRNA degradation by real-time RT-PCR., Results: DETA/NO caused a significant (p < 0.05) increase in IL-6 production. Inhibitors of p38 MAPK and ERK1/2 signaling, but not JNK, were shown to significantly suppress DETA/NO-induced IL-6 production. UPEC-induced IL-6 production was further increased (by 73 ± 23%, p < 0.05) in the presence of DETA/NO. The IL-6 mRNA expression increased 2.1 ± 0.17-fold in response to DETA/NO, while the UPEC-evoked increase was pronounced (20 ± 4.5-fold). A synergistic effect of DETA/NO on UPEC-induced IL-6 expression was found (33 ± 7.2-fold increase). The IL-6 mRNA stability studies showed that DETA/NO partially attenuated UPEC-induced degradation of IL-6 mRNA., Conclusions: NO was found to stimulate IL-6 in renal epithelial cells through p38 MAPK and ERK1/2 signaling pathways and also to increase IL-6 mRNA stability in UPEC-infected cells. This study proposes a new role for NO in the host response during UTI by modulating the transcription and production of the cytokine IL-6., (Copyright © 2012 S. Karger AG, Basel.)

Explanted rat retinas show progressive photoreceptor degeneration that appears to be caspase-12-dependent. Decrease in photoreceptor density eventually affects the inner retina, particularly in the bipolar cell population. Explantation and the induced photoreceptor degeneration are accompanied by activation of Müller and microglia cells. The goal of this study was to determine whether the presence of a feeder layer of human neural progenitor cells (hNPCs) could suppress the degenerative and reactive changes in the explants. Immunohistochemical analyses showed considerable sprouting of rod photoreceptor axon terminals into the inner retina and reduced densities of cone and rod bipolar cells. Both sprouting and bipolar cell degenerations were significantly lower in retinas cultured with feeder layer cells compared to cultured controls. A tendency toward reduced microglia activation in the retinal layers was also noted in the presence of feeder layer cells. These results indicate that hNPCs or factors produced by them can limit the loss of photoreceptors and secondary injuries in the inner retina. The latter may be a consequence of disrupted synaptic arrangement.

Adenosine has been identified as a significant inhibitor of inflammation by acting on adenosine A(2A) receptors. In this study, we examined the role of adenosine and A(2A) receptors in the transmigration of human neutrophils across an in vitro model of the transitional bladder urothelium. Human uroepithelial cells (UROtsa) were grown on transwell inserts; uropathogenic Escherichia coli (UPEC) and neutrophils were added to the transwell system; and the number of migrating neutrophils was evaluated. Reverse transcription-PCR (RT-PCR), immunohistochemistry, and flow cytometry were used to investigate the expression of adenosine receptors, the epithelial adhesion molecule ICAM-1, and the neutrophil integrin CD11b. Levels of proinflammatory interleukin-8 (IL-8) and phosphorylated IκBα were measured by enzyme-linked immunosorbent assays (ELISA) and Luminex assays, respectively. The neutrophils expressed all four adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3) receptors), but A(3) receptors were not expressed by UROtsa cells. UPEC stimulated neutrophil transuroepithelial migration, which was significantly decreased in response to the specific A(2A) receptor agonist CGS 21680. The inhibitory effect of CGS 21680 on neutrophil migration was reversed by the A(2A) receptor antagonist SCH 58261. The production of chemotactic IL-8 and the expression of the adhesion molecule ICAM-1 or CD11b were not significantly affected by CGS 21680. However, a significant decrease in the level of phosporylated IκBα was revealed in response to CGS 21680. In conclusion, UPEC infection in vitro evoked neutrophil migration through a multilayered human uroepithelium. The UPEC-evoked neutrophil transmigration decreased in response to A(2A) receptor activation, possibly through inhibition of NF-κB signaling pathways.

Here we suggest that endoplasmic reticulum (ER)-stress may be induced following aberrant rhodopsin accumulation in photoreceptors in explanted rat retinas. Rhodopsin accumulation was accompanied by increased phosphorylation of pancreatic ER-kinase and eukaryotic initiator factor 2α as well as increased levels of C/EBP homologous protein, glucose-regulated protein 78 and eventually increased cleaved caspase-12 and cleaved caspase-3. Glucose-regulated protein 78, pancreatic ER-kinase, caspase-12 and cleaved caspase-3 were present in photoreceptors, indicating that ER-stress and apoptosis are induced in this cell population. These results suggest that ER-stress and subsequent apoptosis is induced in healthy photoreceptors, presumably by aberrant accumulation of rhodopsin and the phosphorylation of eukaryotic initiator factor 2α. The explant culture system may allow investigations of neuroprotective strategies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Objective: To assess the expression and regulation of adenosine receptors in unstimulated, uropathogenic Escherichia coli (UPEC)-infected and cytokine-stimulated human urinary tract epithelial cells, and to examine the regulation of interleukin (IL)-6 secretion in response to A(2A) receptor activation., Materials and Methods: Human urinary tract epithelial cells (A498, T24 and RT4) were grown in cell culture and stimulated with a mixture of pro-inflammatory cytokines (CM) or UPEC. The expression of adenosine receptors was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry. IL-6 secretion was measured with an enzyme-linked immunosorbent assay., Results: RT-PCR analysis showed the presence of transcripts for the A(1), A(2A) and A(2B) receptor subtypes but not for the A(3) receptor in A498 kidney epithelial cells. The expression of A(2A) receptor mRNA increased in A498 epithelial cells exposed to CM and UPEC, while A(1) and A(2B) receptor transcripts decreased or remained unchanged. Up-regulation of A(2A) receptors was confirmed at the protein level using Western blot analysis and immunocytochemistry. There was also an increase in A(2A) receptor mRNA in human bladder epithelial cells (T24 and RT4) and in mouse bladder uroepithelium in response to cytokines and UPEC. IL-6 secretion in UPEC-infected A498 cells was decreased by 38% when exposed to the A(2A) receptor agonist CGS 21680., Conclusion: Our data showed a subtype-selective plasticity among adenosine receptors in urinary tract epithelial cells in response to UPEC-infection and cytokines. There was a consistent up-regulation of A(2A) receptors in kidney and bladder epithelial cells. Functionally, A(2A) receptor activation reduced UPEC-induced IL-6 secretion. These findings suggest that adenosine might be a previously unrecognized regulator of the mucosal response in urinary tract infection.

Aims: Extracellular ATP may be metabolized to AMP and adenosine by the ectonucleotidases CD39 and CD73 and, in this study, we characterized the pathways for adenosine formation in human urinary tract epithelial cells., Methods: Bladder (RT4) and kidney (A498) epithelial cells were grown in cell culture and the expression of CD39 and CD73 was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. High-performance liquid chromatography was used to determine adenosine formation in cell medium., Results: RT-PCR and immunohistochemistry revealed a high CD73 and a low CD39 expression in human urinary tract epithelial cells, whereas neutrophils had a higher CD39 than CD73 expression. Adenosine was produced when the cells were exposed to 5'-AMP (substrate for CD73), but not when exposed to 5'-ATP (substrate for CD39). A pronounced inhibition of 5'-AMP-induced adenosine formation by the CD73 inhibitor AMP-CP confirmed the involvement of CD73. Adenosine production from 5'-ATP was slightly increased (p < 0.05) when epithelial cells were cocultured with neutrophils., Conclusions: The data demonstrate that adenosine formation from extracellular ATP is negligible in urinary tract epithelial cells due to low CD39 expression in this cell type. However, the epithelial cells express CD73 and are able to convert extracellular AMP to adenosine.