Maternal socioeconomic position and inequity in child deaths: An analysis of 2012 South Korean birth cohort of 466,636 children.

Background: Inequalities in child mortality occur via interactions between socio-environmental factors and their constituents. Through childhood developmental stages, we can observe changing patterns of mortality. By investigating these patterns and social inequalities by cause and developmental stage, we aim to gain insights into health policies to reduce and equalize childhood mortality. Methods: Using vital statistics, we examined the Korean birth cohort of 2012, including all children born in 2012 up to five years of age (N = 466,636). The dependent variables were all-cause and cause-specific mortality by developmental stage (i.e., neonatal, post-neonatal, and childhood). A Cox proportional hazard regression model was built to compare child mortality according to maternal education. The distribution of inequalities in cause-specific mortality by child age was calculated using the slope index of inequality (SII). Results: Inequalities in child mortality due to maternal education occur during the neonatal period and increase over time. After adjusting for covariates, the Cox proportional hazard models showed that "injury and external causes" (HR = 2.178; 95% CI = [1.283-3.697]) and "unknown causes" (HR = 2.299; 95% CI = [1.572-3.363]) in the post-neonatal period, and "injury and external causes" (HR = 2.153; 95% CI = [1.347-3.440]) in the childhood period significantly contributed to socioeconomic inequalities in child mortality. For each period, the leading causes of inequality were identified as follows: "congenital" (96.7%) for the neonatal period, "unknown causes" (58.2%) and "injury and external causes" (28.4%) for the post-neonatal period, and "injury and external causes" (56.5%) for the childhood period. Conclusion: We confirmed that the main causes of death in mortality inequality vary according to child age, in accordance with the distinctive context of child development. Strengthening the health system and multisectoral efforts that consider families' and children's needs according to spatial contexts (e.g., home, community) may be necessary to address the social inequalities in child health.

Distribution of Neuroglobin in Pericytes is Associated with Blood-Brain Barrier Leakage against Cerebral Ischemia in Mice.

With emerging data on the various functions of neuroglobin (Ngb), such as neuroprotection and neurogenesis, we investigated the role of Ngb in the neurovascular unit (NVU) of the brain. To study the distribution and function of Ngb after cerebral ischemia, transient middle cerebral artery occlusion (tMCAO) was performed in mice. Brain immunostaining and fluorescence-activated cell sorting were used to analyze the role of Ngb according to the location and cell type. In normal brain tissue, it was observed that Ngb was distributed not only in neurons but also around the brain's blood vessels. Interestingly, Ngb was largely expressed in platelet-derived growth factor receptor ß (PDGFRß)-positive pericytes in the NVU. After tMCAO, Ngb levels were significantly decreased in the core of the infarct, and Ngb and PDGFRß-positive pericytes were detached from the vasculature. In contrast, in the penumbra of the infarct, PDGFRß-positive pericytes expressing Ngb were increased compared with that in the core of the infarct. Moreover, the cerebral blood vessels, which have Ngb-positive PDGFRß pericytes, showed reduced blood-brain barrier (BBB) leakage after tMCAO. It showed that Ngb-positive PDGFRß pericytes stayed around the endothelial cells and reduced the BBB leakage in the NVU. Our results indicate that Ngb may play a role in attenuating BBB leakage in part by its association with PDGFRß. In this study, the distribution and function of Ngb in the pericytes of the cerebrovascular system have been elucidated, which contributes to the treatment of stroke through a new function of Ngb.

Injectable Self-Crosslinkable Thiolated Hyaluronic Acid for Stem Cell Therapy of Atopic Dermatitis.

Stem cell therapies offer great promise in regenerative medicine to reinstate the normal function of diseased tissue, thereby avoiding the need for replacement. In stem cell therapies, damaged cells are replaced or restored by regulating inflammation and the immune system. However, the low survival rate and local retention of transplanted cells pose a significant challenge. In this study, injectable self-crosslinkable hydrogels using thiol-functionalized hyaluronic acid (HA-SH) were developed to improve the efficacy of mesenchymal stem cells (MSCs) for treating atopic dermatitis (AD)-related inflammatory lesions. The gelation kinetics and mechanical properties of HA-SH hydrogels were easily tuned by varying the concentration of the polymer in the precursor solution before injection. The MSC-laden HA-SH hydrogels exhibited high cell viability (>80%) for 1 week and good in vivo biocompatibility after implantation beneath the mouse skin. Moreover, the MSC-laden HA-SH hydrogel showed increased expression of anti-inflammatory cytokines, which can alleviate the immune response. In an AD animal model, a reduction in epidermal thickness and mast cell infiltration was achieved by applying a self-crosslinkable HA-SH solution including MSCs. This HA-based injectable hydrogel represents a potential carrier of stem cells, and its strong immunomodulation capabilities can be utilized for treating inflammation-related diseases.

Investigation of long-term metabolic alteration after stroke in tMCAO (transient middle cerebral artery occlusion) mouse model using metabolomics approach.

Stroke causes serious long-term disability and numerous molecular changes, including inflammation, depression, and immunosuppression. Despite this, the underlying metabolic mechanisms of poststroke complications remain unclear, and assessing metabolic changes may be beneficial. In this study, we investigated the changes in brain damage and long-term metabolic changes caused by stroke in a transient middle cerebral artery occlusion (tMCAO) mouse model. Metabolic profiling was conducted using UPLC-Orbitrap-MS/MS to compare the metabolites that changed 1 day, 1 week, 1 month, and 6 months after stroke. tMCAO caused an infarction that peaked at 1 week, following which atrophy was observed up to 6 months along with metabolomic changes. From the metabolomics analysis, 72 important metabolites associated with poststroke were identified, and the changes in their levels were most at 1 day and less significant at 1 week followed by a significant change 6 months after stroke. Fatty acids, corticosterone, tyrosine, and tryptophan metabolites are involved in immunosuppression and inflammation. These results indicated that the change in metabolic level after stroke was persistent and could be associated with poststroke complications, such as brain atrophy. Therefore, it was concluded that long-term metabolic changes could involve the chronic after-effects of ischemic stroke.

A Metabolomics Investigation of the Metabolic Changes of Raji B Lymphoma Cells Undergoing Apoptosis Induced by Zinc Ions.

Zinc plays a pivotal role in the function of cells and can induce apoptosis in various cancer cells, including Raji B lymphoma. However, the metabolic mechanism of Zn-induced apoptosis in Raji cells has not been explored. In this study, we performed global metabolic profiling using UPLC-Orbitrap-MS to assess the apoptosis of Raji cells induced by Zn ions released from ZnO nanorods. Multivariate analysis and database searches identified altered metabolites. Furthermore, the differences in the phosphorylation of 1380 proteins were also evaluated by Full Moon kinase array to discover the protein associated Zn-induced apoptosis. From the results, a prominent increase in glycerophosphocholine and fatty acids was observed after Zn ion treatment, but only arachidonic acid was shown to induce apoptosis. The kinase array revealed that the phosphorylation of p53, GTPase activation protein, CaMK2a, PPAR-γ, and PLA-2 was changed. From the pathway analysis, metabolic changes showed earlier onset than protein signaling, which were related to choline metabolism. LC-MS analysis was used to quantify the intracellular choline concentration, which decreased after Zn treatment, which may be related to the choline consumption required to produce choline-containing metabolites. Overall, we found that choline metabolism plays an important role in Zn-induced Raji cell apoptosis.

Dynamics of T Lymphocyte between the Periphery and the Brain from the Acute to the Chronic Phase Following Ischemic Stroke in Mice.

Stroke causes systemic immunosuppression. T lymphocytes are involved in infarct size in the early stages of stroke. However, the phenotypes of T lymphocytes and their functions in peripheral immune organs and the brain have not been well analyzed in the acute and chronic phases of stroke. Here, we investigated pathological phenotypic alterations in the systemic immune response, especially changes in T lymphocytes, from one day to six months after ischemic stroke in mice. Impairment in thymocyte numbers, development, proliferation, and apoptosis were observed for up to two weeks. The number of mature T cells in the spleen and blood decreased and showed reduced interferon-γ production. Increased numbers of CD4-CD8-CD3+ double-negative T cells were observed in the mouse brain during the early stages of stroke, whereas interleukin (IL)-10+Foxp3+ regulatory T lymphocytes increased from two weeks during the chronic phase. These phenotypes correlated with body weight and neurological severity scores. The recovery of T lymphocyte numbers and increases in IL-10+Foxp3+ regulatory T lymphocytes may be important for long-term neurological outcomes. Dynamic changes in T lymphocytes between the acute and chronic phases may play different roles in pathogenesis and recovery. This study provides fundamental information regarding the T lymphocyte alterations from the brain to the peripheral immune organs following stroke.

Prunus cerasoides Extract and Its Component Compounds Upregulate Neuronal Neuroglobin Levels, Mediate Antioxidant Effects, and Ameliorate Functional Losses in the Mouse Model of Cerebral Ischemia.

Prunus cerasoides (PC) has been reported to have antimicrobial and anti-inflammatory properties, but its potential as a neuroprotective agent in a mouse model of cerebral ischemia has not been explored. Considering neuroglobin (Ngb), an endogenous neuroprotective factor, as a novel approach to neuroprotection, in this study, Ngb promoter activity, Ngb expression changes, and antioxidant protection by PC extract (PCE) and PC component compounds (PCCs) were analyzed in oxygen-glucose deprivation (OGD)-treated neurons. In vivo analysis involved transient middle cerebral artery occlusion (tMCAO) in mice with pre- and post-treatment exposure to PCE. Following ischemic stroke induction, neurological behavior scores were obtained, and cellular function-related signals were evaluated in the ischemic infarct areas. In addition to PCE, certain component compounds from PCE also significantly increased Ngb levels and attenuated the intracellular ROS production and cytotoxicity seen with OGD in primary neurons. Administration of PCE reduced the infarct volume and improved neurological deficit scores in ischemic stroke mice compared with the vehicle treatment. Increased Ngb levels in infarct penumbra with PCE treatment were also accompanied by decreased markers of apoptosis (activated p38 and cleaved caspase-3). Our findings point to the benefits of Ngb-mediated neuroprotection via PCE and its antioxidant activity in an ischemic stroke model.

Self-Adherent Biodegradable Gelatin-Based Hydrogel Electrodes for Electrocardiography Monitoring.

Patch-type hydrogel electrodes have received increasing attention in biomedical applications due to their high biocompatibility and conformal adherence. However, their poor mechanical properties and non-uniform electrical performance in a large area of the hydrogel electrode should be improved for use in wearable devices for biosignal monitoring. Here, we developed self-adherent, biocompatible hydrogel electrodes composed of biodegradable gelatin and conductive polymers for electrocardiography (ECG) measurement. After incorporating conductive poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS) into gelatin hydrogels crosslinked by natural crosslinkers (genipin), the mechanical properties and electrical conductivity of the hydrogel electrodes were improved and additionally optimized by adjusting the amounts of crosslinker and PEDOT:PSS, respectively. Furthermore, the effect of dimethyl sulfoxide, as a dopant, on the conductivity of hydrogels was investigated. The gelatin-based, conductive hydrogel patch displayed self-adherence to human skin with an adhesive strength of 0.85 N and achieved conformal contact with less skin irritation compared to conventional electrodes with a chemical adhesive layer. Eyelet-type hydrogel electrodes, which were compatible with conventional ECG measurement instruments, exhibited a comparable performance in 12-lead human ECG measurement with commercial ECG clinical electrodes (3M Red Dot). These self-adherent, biocompatible, gelatin-based hydrogel electrodes could be used for monitoring various biosignals, such as in electromyography and electroencephalography.

On-site fabrication of injectable 131I-labeled microgels for local radiotherapy.

Nuclear medicine is a routine but essential clinical option for diagnostic imaging and disease treatment. Encapsulating radioisotopes in injectable biodegradable hydrogels is ideal for localizing radiation sources to target tissues or organs to achieve long-term, low-dose radiotherapy. However, difficulties in the on-site production of radioactive gels upon treatment and the unpredictable radiation level at the target region are major obstacles to their clinical use. In this study, we bypassed these limitations by developing locally injectable hydrogel microparticles based on 131I-labeled photo-crosslinkable hyaluronic acid (HA) and a microfluidic high-throughput droplet generator. This approach enabled rapid on-site production of injectable, radioactive, biodegradable (IRB) HA microgels, thus allowing their immediate therapeutic application with improved local retention and predictable radioactivity. We demonstrated the clinical utility of this comprehensive approach by preparing IRB HA microgels within 15 min and localizing them to the target tissue (rat muscle) with minimal off-target biodistribution and in vivo radioactivity that extended beyond 3 weeks.

Estrogenic properties of Prunus cerasoides extract and its constituents in MCF-7 cell and evaluation in estrogen-deprived rodent models.

Prunus cerasoides (PC) products contain relatively high levels of flavones and isoflavones and may be potential sources of phytoestrogens for postmenopausal symptom relief. We assessed the PC extract (PCE) and its representative constituents in vitro with assays for estrogen receptor alpha binding, estrogen response element transcriptional activity, cell proliferation, and gene expression changes for pS2 in MCF-7 cells. PCE and its compounds showed strong estrogen receptor binding affinities and estrogen response element induction. A previously undescribed compound (designated as compound 18), now identified as being gentisic acid, 5-O-ß-D-(6'-O-trans-4-coumaroyl)-glucopyranoside, also showed potent estrogenic properties and induced proliferation of MCF-7 cells. PCE was evaluated for its in vivo uterotrophic effects in immature female rats as well as for its lipid lowering effects in estrogen-deprived animals. For ovariectomized rats and aged female mice, PCE-treated groups had lower plasma triglyceride levels compared with control and, for the same comparison, had reduced serum levels of liver stress/damage markers. Our results point to strong estrogenic activities and beneficial metabolic effects for PCE, with properties that put PC and its extracts as promising sources of phytoestrogens for symptom relief in menopausal and postmenopausal cases.

Transdermal delivery of Minoxidil using HA-PLGA nanoparticles for the treatment in alopecia.

BACKGROUND: Alopecia has become a very common disease that many people around the world are suffered. Minoxidil (MXD) is the most well-known commercialized drug in its treatment. However, in the case of MXD administration, there are some problems with low efficiency of transdermal delivery and additional side effects. METHOD: MXD and Rhodamine B (Rho B) are encapsulated in poly(Lactide-co-Glycolide) grafted hyaluronate nanoparticles (HA-PLGA/MXD NPs, HA-PLGA/Rho B NPs) which is prepared with W/O/W solvent evaporation method. After then, the investigation is carried out to confirm the feasibility of NPs in alopecia treatment. RESULTS: Both of HA-PLGA/MXD NPs and HA-PLGA/Rho B NPs are successfully prepared. In addition, it is confirmed that HA-PLGA NPs sufficiently delivered to cells without any significant cytotoxicity by cell viability, cellular uptake and skin permeation test. CONCLUSION: Taken together, HA-PLGA NPs as a transdermal delivery carrier to hair follicle cells can be exploited to develop the efficient and effective platform of transdermal drug delivery for the treatment of various diseases.

Acid-Treated Water-Soluble Chitosan Suitable for Microneedle-Assisted Intracutaneous Drug Delivery.

Chitosan has been widely used as a nature-derived polymeric biomaterial due to its high biocompatibility and abundance. However, poor solubility in aqueous solutions of neutral pH and multiple fabrication steps for the molding process limit its application to microneedle technology as a drug delivery carrier. Here, we present a facile method to prepare water-soluble chitosan and its application for sustained transdermal drug delivery. The water-soluble chitosan was prepared by acid hydrolysis using trifluoroacetic acid followed by dialysis in 0.1 M NaCl solutions. We successfully fabricated bullet-shaped microneedle (MN) arrays by the single molding process with neutral aqueous chitosan solutions (pH 6.0). The chitosan MN showed sufficient mechanical properties for skin insertion and, interestingly, exhibited slow dissolving behavior in wet conditions, possibly resulting from a physical crosslinking of chitosan chains. Chitosan MN patches loading rhodamine B, a model hydrophilic drug, showed prolonged release kinetics in the course of the dissolving process for more than 72 h and they were found to be biocompatible to use. Since the water-soluble chitosan can be used for MN fabrication in the mild conditions (neutral pH and 25 °C) required for the loading of bioactive agents such as proteins and achieve a prolonged release, this biocompatible chitosan MN would be suitable for sustained transdermal drug delivery of a diverse range of drugs.

Estrogenic effects of phytoestrogens derived from Flemingia strobilifera in MCF-7 cells and immature rats.

Phytoestrogen (PE) has received considerable attention due to the physiological significance of its estrogenicity. Flemingia strobilifera (FS) has been used as a folk medicine in Asia for the treatment of inflammation, cancer, and infection; however, the estrogenic effects and chemical components of FS have not yet been reported. We aimed to uncover the estrogenic properties and PEs derived from FS using phytochemical and pharmacological evaluation. PEs from FS extract (FSE) were analyzed by NMR, HPLC, and MS. To evaluate estrogenic activity, FSE and its compounds were evaluated by in vitro and in vivo assays, including human estrogen receptor alpha (hERα) binding, estrogen response element (ERE)-luciferase reporter assays, and uterotrophic assays. FSE and its compounds 1-5 showed binding affinities for hERα and activated ERE transcription in MCF-7 cells. Additionally, FSE and compounds 1-5 induced MCF-7 cell proliferation and trefoil factor 1 (pS2) expression. In immature female rats, significant increases in uterine weight and pS2 gene were observed in FSE-treated groups. We identified estrogenic activities of FSE and its bioactive compounds, suggesting their possible roles as PEs via ERs. PEs derived from FSE are promising candidates for ER-targeted therapy for post-menopausal symptoms.

Anti-inflammatory Effect of Glucagon Like Peptide-1 Receptor Agonist, Exendin-4, through Modulation of IB1/JIP1 Expression and JNK Signaling in Stroke.

Glucagon like peptide-1 (GLP-1) stimulates glucose-dependent insulin secretion. Dipeptidyl peptidase-4 (DPP-4) inhibitors, which block inactivation of GLP-1, are currently in clinical use for type 2 diabetes mellitus. Recently, GLP-1 has also been reported to have neuroprotective effects in cases of cerebral ischemia. We therefore investigated the neuroprotective effects of GLP-1 receptor (GLP-1R) agonist, exendin-4 (ex-4), after cerebral ischemia-reperfusion injury. Transient middle cerebral artery occlusion (tMCAO) was induced in rats by intracerebroventricular (i.c.v.) administration of ex-4 or ex9-39. Oxygen-glucose deprivation was also induced in primary neurons, bEnd.3 cells, and BV-2. Ischemia-reperfusion injury reduced expression of GLP-1R. Additionally, higher oxidative stress in SOD2 KO mice decreased expression of GLP-1R. Downregulation of GLP-1R by ischemic injury was 70% restored by GLP-1R agonist, ex-4, which resulted in significant reduction of infarct volume. Levels of intracellular cyclic AMP, a second messenger of GLP-1R, were also increased by 2.7-fold as a result of high GLP-1R expression. Moreover, our results showed that ex-4 attenuated pro-inflammatory cyclooxygenase-2 (COX-2) and prostaglandin E2 after MCAO. C-Jun NH2 terminal kinase (JNK) signaling, which stimulates activation of COX-2, was 36% inhibited by i.c.v. injection of ex-4 at 24 h. Islet-brain 1 (IB1), a scaffold regulator of JNK, was 1.7-fold increased by ex-4. GLP-1R activation by ex-4 resulted in reduction of COX-2 through increasing IB1 expression, resulting in anti-inflammatory neuroprotection during stroke. Our study suggests that the anti-inflammatory action of GLP-1 could be used as a new strategy for the treatment of neuroinflammation after stroke accompanied by hyperglycemia.

TLR5 Activation through NF-κB Is a Neuroprotective Mechanism of Postconditioning after Cerebral Ischemia in Mice.

Postconditioning has been shown to protect the mouse brain from ischemic injury. However, the neuroprotective mechanisms of postconditioning remain elusive. We have found that toll-like receptor 5 (TLR5) plays an integral role in postconditioning-induced neuroprotection through Akt/nuclear factor kappa B (NF-κB) activation in cerebral ischemia. Compared to animals that received 30 min of transient middle cerebral artery occlusion (tMCAO) group, animals that also underwent postconditioning showed a significant reduction of up to 60.51% in infarct volume. Postconditioning increased phospho-Akt (p-Akt) levels and NF-κB translocation to the nucleus as early as 1 h after tMCAO and oxygen-glucose deprivation. Furthermore, inhibition of Akt by Akt inhibitor IV decreased NF-κB promoter activity after postconditioning. Immunoprecipitation showed that interactions between TLR5, MyD88, and p-Akt were increased from postconditioning both in vivo and in vitro. Similar to postconditioning, flagellin, an agonist of TLR5, increased NF-κB nuclear translocation and Akt phosphorylation. Our results suggest that postconditioning has neuroprotective effects by activating NF-κB and Akt survival pathways via TLR5 after cerebral ischemia. Additionally, the TLR5 agonist flagellin can simulate the neuroprotective mechanism of postconditioning in cerebral ischemia.

Neuroprotective effect of mesenchymal stem cell through complement component 3 downregulation after transient focal cerebral ischemia in mice.

Bone marrow-derived mesenchymal stem cells (MSCs) are used in stroke treatment despite the poor understanding of its mode of action. The immune suppressive and anti-inflammatory properties of MSCs possibly play important roles in regulating neuroinflammation after stroke. We investigated whether MSCs reduce the inflammatory complement component 3 (C3) levels, thus, providing neuroprotection during stroke. Mice were subjected to transient focal cerebral ischemia (tFCI), after which MSCs were intravenously injected. The infarct volume of the brain was reduced in MSC-injected tFCI mice, and C3 expression was significantly reduced in both the brain and the blood. Additionally, the profiles of other inflammatory mediators demonstrated neuroprotective changes in the MSCs-treated group. In order to analyze the effect of MSCs on neurons during cerebral ischemia, primary cortical neurons were co-cultured with MSCs under oxygen-glucose deprivation (OGD). Primary neurons co-cultured with MSCs exhibited reduced levels of C3 expression and increased protection against OGD, indicating that treatment with MSCs reduces excessive C3 expression and rescues ischemia-induced neuronal damage. Our finding suggests that reduction of C3 expression by MSCs can help to ameliorate ischemic brain damage, offering a new neuroprotective strategy in stroke therapy.

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88.

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.

Signaling of high mobility group box 1 (HMGB1) through toll-like receptor 4 in macrophages requires CD14.

High mobility group box 1 (HMGB1) is a DNA-binding protein that possesses cytokinelike, proinflammatory properties when released extracellularly in the C23-C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with toll-like receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/myeloid differentiation protein 2 (MD2) complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the coreceptor CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW264.7 macrophages after stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates tumor necrosis factor (TNF)-α release in WT but not in TLR4(-/-), CD14(-/-), TIR domain-containing adapter-inducing interferon-ß (TRIF)(-/-) or myeloid differentiation primary response protein 88 (MyD88)(-/-) macrophages. HMGB1 induces the release of monocyte chemotactic protein 1 (MCP-1), interferon gamma-induced protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in a TLR4- and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.

Mammalian DNA is an endogenous danger signal that stimulates local synthesis and release of complement factor B.

Complement factor B plays a critical role in ischemic tissue injury and autoimmunity. Factor B is dynamically synthesized and released by cells outside of the liver, but the molecules that trigger local factor B synthesis and release during endogenous tissue injury have not been identified. We determined that factor B is upregulated early after cold ischemia-reperfusion in mice, using a heterotopic heart transplant model. These data suggested upregulation of factor B by damage-associated molecular patterns (DAMPs), but multiple common DAMPs did not induce factor B in RAW264.7 mouse macrophages. However, exogenous DNA induced factor B mRNA and protein expression in RAW cells in vitro, as well as in peritoneal and alveolar macrophages in vivo. To determine the cellular mechanisms involved in DNA-induced factor B upregulation we then investigated the role of multiple known DNA receptors or binding partners. We stimulated peritoneal macrophages from wild-type (WT), toll-like receptor 9 (TLR9)-deficient, receptor for advanced glycation end products (RAGE)⁻/⁻ and myeloid differentiation factor 88 (MyD88)⁻/⁻ mice, or mouse macrophages deficient in high-mobility group box proteins (HMGBs), DNA-dependent activator of interferon-regulatory factors (DAI) or absent in melanoma 2 (AIM2), with DNA in the presence or absence of lipofection reagent. Reverse transcription-polymerase chain reaction, Western blotting and immunocytochemical analysis were employed for analysis. Synthesis of factor B was independent of TLR9, RAGE, DAI and AIM2, but was dependent on HMGBs, MyD88, p38 and NF-κB. Our data therefore show that mammalian DNA is an endogenous molecule that stimulates factor B synthesis and release from macrophages via HMGBs, MyD88, p38 and NF-κB signaling. This activation of the immune system likely contributes to damage following sterile injury such as hemorrhagic shock and ischemia-reperfusion.

Mast cells play a critical role in the systemic inflammatory response and end-organ injury resulting from trauma.

BACKGROUND: Much of the morbidity after trauma results from excessive activation of the innate immune system. This is manifested as a systemic inflammatory response and associated end-organ damage. Although mast cells are known to be important in many immune responses, their role in the systemic response to severe trauma is unknown. STUDY DESIGN: C57BL/6J-KitW-sh/BsmJ (mast cell deficient) and wild type mice were subjected to 1.5 hours of hemorrhagic shock plus bilateral femur fracture and soft tissue injury (HS/T), followed by resuscitation at 4.5 hours. Blood withdrawal volumes, mean arterial pressures, circulating cytokine, chemokine, high mobility group box-1 (HMGB-1), double strain DNA (dsDNA), transaminase levels, and histology in liver and lung were compared between groups. RESULTS: Mast cell deficient mice exhibited greater hemodynamic stability than wild type mice. At baseline, the mast cell deficient mice exhibited no difference in any of the organ injury or inflammatory markers measured. As expected, wild type mice subjected to HS/T exhibited end-organ damage manifested by marked increases in circulating alanine transaminase, aspartate aminotransferase, and dsDNA levels, as well as histologic evidence of tissue necrosis. In clear contrast, mast cell deficient mice exhibited almost no tissue damage. Similarly, the magnitude of increased circulating cytokine and chemokine induced by HS/T was much less in the mast cell deficient mice than in the wild type group. CONCLUSIONS: Mast cell deficiency resulted in a damped systemic inflammatory response, greatly attenuated multiple organ injury, and more stable hemodynamics in HS/T. So mast cells appear to be a critical component of the initial host response to severe injury.