Macrophage expressed tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder involving scarring of pulmonary tissue and a subsequent decrease in respiratory capacity, ultimately resulting in death. Tartrate resistant acid phosphatase 5 (ACP5) plays a role in IPF but the exact mechanisms are yet to be elucidated. In this study, we have utilized various perturbations of the bleomycin mouse model of IPF including genetic knockout, RANKL inhibition, and macrophage adoptive transfer to further understand ACP5's role in pulmonary fibrosis. Genetic ablation of Acp5 decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix-production) as well as histological damage. Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il-1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions. Furthermore, antibody-based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development. Adoptive transfer of Acp5-/- bone marrow-derived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages. Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.

Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro.

Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

Impact of spontaneous liposome modification with phospholipid polymer-lipid conjugates on protein interactions.

Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in humans. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10 to 100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only the outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.

A Robust Method to Store Complement C3 With Superior Ability to Maintain the Native Structure and Function of the Protein.

Complement components have a reputation to be very labile. One of the reasons for this is the spontaneous hydrolysis of the internal thioester that is found in both C3 and C4 (but not in C5). Despite the fact that ≈20,000 papers have been published on human C3 there is still no reliable method to store the protein without generating C3(H2O), a fact that may have affected studies of the conformation and function of C3, including recent studies on intracellular C3(H2O). The aim of this work was to define the conditions for storage of native C3 and to introduce a robust method that makes C3 almost resistant to the generation of C3(H2O). Here, we precipitated native C3 at the isoelectric point in low ionic strength buffer before freezing the protein at -80°C. The formation of C3(H2O) was determined using cation exchange chromatography and the hemolytic activity of the different C3 preparations was determined using a hemolytic assay for the classical pathway. We show that freezing native C3 in the precipitated form is the best method to avoid loss of function and generation of C3(H2O). By contrast, the most efficient way to consistently generate C3(H2O) was to incubate native C3 in a buffer at pH 11.0. We conclude that we have defined the optimal storage conditions for storing and maintaining the function of native C3 without generating C3(H2O) and also the conditions for consistently generating C3(H2O).

Effect of liposome surface modification with water-soluble phospholipid polymer chain-conjugated lipids on interaction with human plasma proteins.

Alternative liposome surface coatings for PEGylation to evade the immune system, particularly the complement system, have garnered significant interest. We previously reported poly(2-methacryloyloxyethyl phosphorylcholine) (MPC)-based lipids (PMPC-lipids) and investigated the surface modification of liposomes. In this study, we synthesize PMPC-lipids with polymerization degrees of 10 (MPC10-lipid), 20 (MPC20-lipid), 50 (MPC50-lipid), and 100 (MPC100-lipid), and coated liposomes with 1, 5, or 10 mol% PMPC-lipids (PMPC-liposomes). Non-modified and PEGylated liposomes are used as controls. We investigate the liposome size, surface charge, polydispersity index, and adsorption of plasma proteins to the liposomes post incubation in human plasma containing N,N,N',N'-ethylenediamine tetraacetic acid (EDTA) or lepirudin by some methods such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and automated capillary western blot, with emphasis on the binding of complement protein C3. It is shown that the coating of liposome PMPC-lipids can suppress protein adsorption more effectively with an increase in the molecular weight and molar ratio (1-10 mol%). Apolipoprotein A-I is detected on PMPC-liposomes with a higher molecular weight and higher molar ratio of PMPC-lipids, whereas α2-macroglobulin is detected on non-modified, PEGylated, and PMPC-liposomes with a shorter polymer chain. In addition, a correlation is shown among the PMPC molecular weight, molar ratio, and C3 binding. The MPC10-lipid cannot inhibit C3 binding efficiently, whereas surface modifications with 10 mol% MPC20-lipid and 5 mol% and 10 mol% MPC50-lipid suppress both total protein and C3 binding. Hence, liposome modification with PMPC-lipids can be a possible strategy for avoiding complement activation.

Synthesis of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated lipids and their characterization and surface properties of modified liposomes for protein interactions.

Poly(ethylene glycol) (PEG) is frequently used for liposomal surface modification. However, as PEGylated liposomes are cleared rapidly from circulation upon repeated injections, substitutes of PEG are being sought. We focused on a water-soluble polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units, and synthesized poly(MPC) (PMPC)-conjugated lipid (PMPC-lipid) with degrees of MPC polymerization ranging from 10 to 100 (calculated molecular weight: 3 to 30 kDa). In addition, lipids with three different alkyl chains, myristoyl, palmitoyl, and stearoyl, were applied for liposomal surface coating. We studied the interactions of PMPC-lipids with plasma albumin, human complement protein C3 and fibrinogen using a quartz crystal microbalance with energy dissipation, and found that adsorption of albumin, C3 and fibrinogen could be suppressed by coating with PMPC-lipids. In particular, the effect was more pronounced for PMPC chains with higher molecular weight. We evaluated the size, polydispersity index, surface charge, and membrane fluidity of the PMPC-lipid-modified liposomes. We found that the effect of the coating on the dispersion stability was maintained over a long period (98 days). Furthermore, we also demonstrated that the anti-PEG antibody did not interact with PMPC-lipids. Thus, our findings suggest that PMPC-lipids can be used for liposomal coating.

Animal-Free Human Whole Blood Sepsis Model to Study Changes in Innate Immunity.

Studying innate immunity in humans is crucial for understanding its role in the pathophysiology of systemic inflammation, particularly in the complex setting of sepsis. Therefore, we standardized a step-by-step process from the venipuncture to the transfer in a human model system, while closely monitoring the inflammatory response for up to three hours. We designed an animal-free, human whole blood sepsis model using a commercially available, simple to use, tubing system. First, we analyzed routine clinical parameters, including cell count and blood gas analysis. Second, we demonstrated that extracellular activation markers (e.g., CD11b and CD62l) as well as intracellular metabolic (intracellular pH) and functional (generation of radical oxygen species) features remained stable after incubation in the whole blood model. Third, we mimicked systemic inflammation during early sepsis by exposure of whole blood to pathogen-associated molecular patterns. Stimulation with lipopolysaccharide revealed the capability of the model system to evoke a sepsis-like inflammatory phenotype of innate immunity. In summary, the presented model serves as a convenient, economic, and reliable platform to study innate immunity in human whole blood, which may yield clinically important insights.

Assessment of the Role of C3(H2O) in the Alternative Pathway.

In this study we investigate the hydrolysis of C3 to C3(H2O) and its ability to initiate activation via the alternative pathway (AP) of the complement system. The internal thioester bond within C3 is hydrolyzed by water in plasma because of its inherent lability. This results in the formation of non-proteolytically activated C3(H2O) which is believed have C3b-like properties and be able to form an active initial fluid phase C3 convertase together with Factor B (FB). The generation of C3(H2O) occurs at a low but constant rate in blood, but the formation can be greatly accelerated by the interaction with various surfaces or nucleophilic and chaotropic agents. In order to more specifically elucidate the relevance of the C3(H2O) for AP activation, formation was induced in solution by repeated freeze/thawing, methylamine or KCSN treatment and named C3(x) where the x can be any of the reactive nucleophilic or chaotropic agents. Isolation and characterization of C3(x) showed that it exists in several forms with varying attributes, where some have more C3b-like properties and can be cleaved by Factor I in the presence of Factor H. However, in common for all these variants is that they are less active partners in initial formation of the AP convertase compared with the corresponding activity of C3b. These observations support the idea that formation of C3(x) in the fluid phase is not a strong initiator of the AP. It is rather likely that the AP mainly acts as an amplification mechanism of complement activation that is triggered by deposition of target-bound C3b molecules generated by other means.

Is generation of C3(H2O) necessary for activation of the alternative pathway in real life?

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(H2O) 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(H2O) 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.

Self-referral psychological treatment centre for young adults: a 2-year observational evaluation of routine practice before and after treatment.

OBJECTIVES: To examine a self-referral psychological service provided to young adults with regard to effects on anxiety, depression and psychological distress and to explore client factors predicting non-adherence and non-response. DESIGN: Observational study over a 2-year period. SETTING: Young Adults Centre providing psychological services by self-referral (preprimary care) to Linköping, Åtvidaberg, and Kinda municipalities (combined population 145,000) in Östergötland county, Sweden. PARTICIPANTS: 607 young adults (16-25 years of age); 71% females (n=429). INTERVENTION: Individually scheduled cognitive behavioural therapy delivered in up to six 45 min sessions structured according to an assessment of the client's mental health problems: anxiety, depression, anxiety and depression combined, or decreased distress without specific anxiety or depression. PRIMARY OUTCOME MEASURES: Pre-post intervention changes in psychological distress (General Health Questionnaire-12, GHQ-12), Hospital Anxiety and Depression Scale Anxiety/Depression (HADS-A/D). RESULTS: 192 clients (32.5%) discontinued the intervention on their own initiative and 39 clients (6.6%) were referred to a psychiatric clinic during the course of the intervention. Intention-to-treat analyses including all clients showed a medium treatment effect size (d=0.64) with regard to psychological distress, and small effect sizes were observed with regard to anxiety (d=0.58) and depression (d=0.57). Restricting the analyses to clients who adhered to the agreed programme, a large effect size (d=1.26) was observed with regard to psychological distress, and medium effect sizes were observed with regard to anxiety (d=1.18) and depression (d=1.19). Lower age and a high initial HADS-A score were the strongest risk factors for non-adherence, and inability to concentrate and thinking of oneself as a worthless person increased the risk for discontinuation. CONCLUSIONS: We conclude that provision of psychological services to young people through a self-referral centre has potential to improve long-term mental health in communities, but management of non-adherence remains a central challenge.

Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction.

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis.