Novel phenotypical and functional sub-classification of liver macrophages highlights changes in population dynamics in experimental mouse models.

Liver macrophages are critical components of systemic immune system defense mechanisms. F4/80high Kupffer cells (KCs) are the predominant liver-resident macrophages and the first immune cells to contact pathogens entering the liver. F4/80low monocyte-derived macrophages (MoMφs) are essential macrophages that modulate liver immune functions. Here we report a novel method of identifying subpopulations of these two populations using traditional flow cytometry and examine each subpopulation for its putative roles in the pathogenesis of an experimental non-alcoholic steatohepatitis model. Using male C57BL/6 mice, we isolated and analyzed liver non-parenchymal cells by flow cytometry. We identified F4/80high and F4/80low macrophage populations and characterized subpopulations using uniform manifold approximation and projection. We identified three subpopulations in F4/80high macrophages: CD163(+) KCs, CD163(-) KCs, and liver capsular macrophages. CD163(+) KCs had higher phagocytic and bactericidal activities and more complex cellular structures than CD163(-) KCs. We also identified four subpopulations of F4/80low MoMφs based on Ly6C and MHC class II expression: infiltrating monocytes, pro-inflammatory MoMφs, Ly6C(-) monocytes, and conventional dendritic cells. CCR2 knock-out mice expressed lower levels of these monocyte-derived cells, and the count varied by subpopulation. In high-fat- and cholesterol-diet-fed mice, only one subpopulation, pro-inflammatory MoMφs, significantly increased in count. This indicates that changes to this subpopulation is the first step in the progression to non-alcoholic steatohepatitis. The community can use our novel subpopulation and gating strategy to better understand complex immunological mechanisms in various liver disorders through detailed analysis of these subpopulations.

Validation study for in vitro skin irritation test using reconstructed human skin equivalents constructed by layer-by-layer cell coating technology.

The aim of this study is to validate an in vitro skin irritation test (SIT) using three-dimensional reconstructed human epidermal (RhE) skin equivalents prepared by layer-by-layer (LbL) method (LbL-3D Skin) in a series of interlaboratory studies. The goal of these validation studies is to evaluate the ability of this in vitro test to reliably discriminate skin irritant from nonirritant chemicals, as defined by OECD and UN GHS. This me-too validation study is to assess the within- and between-laboratory reproducibility, as well as the predictive capacity, of the LbL-3D Skin SIT in accordance with performance standards for OECD TG 439. The developed skin model, LbL-3D Skin had a highly differentiated epidermis and dermis, similar to the validated reference methods (VRM) and native human skin. The quality parameters (cell survival in controls, tissue integrity, and barrier function) were similar to VRM and in accordance with OECD TG 439. The LbL-3D Skin SIT validation study was performed by three participating laboratories and consisted of three independent tests using 20 reference chemicals. The results obtained with the LbL-3D Skin demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from nonirritants. The predictive potency of LbL-3D Skin SIT using total 54 test chemicals were comparable to those in other RhE models in OECD TG 439. The validation study demonstrated that LbL-3D Skin has proven to be a robust and reliable method for predicting skin irritation.

Performance evaluation of the new chemiluminescent intact FGF23 assay relative to the existing assay system.

INTRODUCTION: This study assessed the performance of a new fully automated immunoassay for fibroblast growth factor (FGF) 23 (Determinar CL FGF23 CL) among healthy individuals and those with chronic hypophosphatemia compared with the previous assay (Kainos FGF23 KI). MATERIALS AND METHODS: A total of 380 serum samples from healthy participants were collected to determine the reference range of FGF23 levels with CL. A total of 200 serum samples from 22 hypophosphatemic patients were collected simultaneously to compare the difference in FGF23 levels between CL and KI. The Mann-Whitney U test and linear regression analysis were adopted to assess the differences and linearity between the two assays. RESULTS: The median FGF23 levels among healthy individuals was 31.7 (interquartile: 26.4-37.5) pg/mL. When the reference range was calculated as the mean ± 2 standard deviation (2SD), it was 16.1-49.3 pg/mL. A total of 363 individuals (96%) among normal cases fell in this range. Among 200 samples from patients with chronic hypophosphatemic disorder, the median FGF23 levels analyzed by CL and KI were 123.0 (90.2-237.7) and 172.5 (115.8-290.7) pg/mL. KI yielded significantly higher FGF23 values than CL (p < 0.001). A linear regression model revealed the correlation between KI (x) and CL (y), which had a slope of 0.76 with a y-intercept of -0.32 and high linearity (R2 = 0.99). CONCLUSION: The new measurement kit yielded lower FGF23 values when compared with the previous assay. Clinicians should consider this discrepancy when they assay intact FGF23 values with CL.

Pioglitazone Modifies Kupffer Cell Function and Protects against Escherichia coli-Induced Bacteremia in Burned Mice.

Infectious complications and subsequent sepsis in severely burned patients lead to high morbidity and mortality in response to uncontrolled innate immune responses mediated by macrophages. Peroxisome proliferator-activated receptor gamma (PPARγ) has anti-inflammatory activity and acts as a master regulator of macrophage polarization. In this study, we investigated whether the administration of a PPARγ agonist could modulate the Kupffer cell phenotype and thereby ameliorate the dysregulated innate response during post-burn bacterial infection. C57BL/6 mice were subjected to severe burns and randomized to receive either the PPARγ agonist, pioglitazone, or the vehicle control five days after injury, followed by the subsequent analysis of hepatic macrophages. Survival from the bacterial infection was monitored for seven days. Pioglitazone protected burned mice against bacterial infection. A single treatment with pioglitazone significantly enhanced phagocytosis, phagosome acidification, bacterial clearance, and reduction in inflammatory mediators in Kupffer cells. In conclusion, PPARγ activation by pioglitazone prevents clinical deterioration due to post-burn bacterial infection and improves survival. Our findings suggest that pioglitazone may be an effective therapeutic candidate for post-burn infectious complications.

Clinical Significance of Programmed Death Ligand-1 Expression in Esophageal Squamous Cell Carcinoma.

BACKGROUND: The aim of this study was to evaluate the association between the expression of programmed death ligand-1 (PD-L1) and clinical outcomes in patients with surgically resected esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: We included 76 patients with primary ESCC who underwent surgical resection between January 2009 and December 2014 at National Defense Medical College Hospital. Using the tumor tissues, we evaluated PD-L1 expression in tumor cells and stromal reactive lymphocytes via immunohistochemistry. Furthermore, the relationship between PD-L1 expression and the clinicopathological status of patients with ESCC was investigated. RESULTS: PD-L1 expression in tumor cells was detected in 39.5% of the patients. In addition, 51.3% of the patients had PD-L1-positive stromal reactive lymphocytes and exhibited significantly longer overall survival than those with lack of PD-L1 expression in stromal reactive lymphocytes (median survival time, 56.0 versus 27.3 mo; log-rank test, P = 0.04). Patients with lack of PD-L1 expression in both tumor cells and stromal reactive lymphocytes showed worse overall survival than those with the PD-L1-positive expression in tumor cells and/or stromal reactive lymphocytes (P = 0.02). CONCLUSIONS: PD-L1-positive expression in stromal reactive lymphocytes, rather than in tumor cells, is associated with a longer survival in patients with ESCC.

Effects of semielemental diet containing whey peptides on Peyer's patch lymphocyte number, immunoglobulin A levels, and intestinal morphology in mice.

BACKGROUND: Enteral nutrition (EN) is the gold standard of nutritional therapy for critically ill or severely injured patients, because EN promotes gut and hepatic immunity, thereby preventing infectious complications as compared with parenteral nutrition. However, there are many EN formulas with different protein and fat contents. Their effects on gut-associated lymphoid tissue remain unclear. Recently, semielemental diets (SEDs) containing whey peptides as a nitrogen source have been found to be beneficial in patients with malabsorption or pancreatitis. Herein, we examined the influences of various dietary formulations on gut immunity to clarify the advantages of SEDs over elemental diets. METHODS: Forty-four male Institute of Cancer Research mice were randomized to four groups: chow (CH: n = 5), intragastric total parenteral nutrition (IG-TPN: n = 13), elemental diet (ED: n = 13), and SED (n = 13). The CH group received CH diet ad libitum, whereas the IG-TPN, ED (Elental, Ajinomoto, Japan), and SED (Peptino, Terumo, Japan) groups were given their respective diets for 5 day via gastrostomy. After 5 days, the mice were killed to obtain whole small intestines. Peyer's patch (PP) lymphocytes were harvested and counted. Their subpopulations were evaluated by flow cytometry. Immunoglobulin A (IgA) levels in intestinal and respiratory tract washings were measured with enzyme-linked immunosorbent assay. Villous height (VH) and crypt depth in the distal intestine were measured by light microscopy. RESULTS: SED increased the PP cell number and intestinal or respiratory IgA levels to those of CH mice, while ED partially restored these parameters. The IG-TPN group showed the lowest PP cell number and IgA levels among the four groups. VH was significantly greater in the CH than in the other groups. VH in the ED and SED groups also exceeded in the IG-TPN group, while being similar in these two groups. No significant crypt depth differences were observed among the four groups. CONCLUSIONS: SED administration can be recommended for patients unable tolerate complex enteral diets or a normal diet in terms of not only absorption and tolerability but also maintenance of gut immunity.

The efficacy of basic fibroblast growth factor-loaded poly(lactic-co-glycolic acid) nanosheet for mouse wound healing.

Although human recombinant basic fibroblast growth factor (bFGF) is widely used for wound healing, daily treatment with bFGF is required because of its short half-life. An effective controlled-release system of bFGF is, therefore, desired in clinical settings. To investigate the efficacy of a bFGF-loaded nanosheet for wound healing, focusing on the controlled-release of bFGF, bFGF-loaded poly(lactic-co-glycolic acid) (PGLA) nanosheets were developed, and their in vitro release profile of bFGF and their in vivo efficacy for wound healing were examined. A polyion complex of positively charged human recombinant bFGF and negatively charged alginate was sandwiched between PLGA nanosheets (70 nm thick for each layer). The resulting bFGF-loaded nanosheet robustly adhered to silicon skin by observation using a microscratch test. bFGF was gradually and continuously released over three days in an in vitro incubation study. Treatment with the bFGF-loaded nanosheets (every 3 day for 15 days) as well as with a conventional bFGF spray effectively promoted wound healing of mouse dorsal skin defects with accelerated tissue granulation and angiogenesis, although the dose of bFGF used in the treatment with the bFGF nanosheets was approximately 1/20 of the sprayed bFGF. In conclusion, we developed a bFGF-loaded nanosheet that sustained a continuous release of bFGF over three days and effectively promoted wound healing in mice.

Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion.

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open.

Fibrinogen γ-Chain Peptide-Coated Adenosine 5' Diphosphate-Encapsulated Liposomes Rescue Mice From Lethal Blast Lung Injury via Adenosine Signaling.

OBJECTIVES: Fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes can accumulate via dodecapeptide HHLGGAKQAGDV interactions at bleeding sites where they release adenosine 5'-diphosphate that is rapidly metabolized to adenosine, which has tissue-protective effects. We investigated the efficacy of fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes to treat blast lung injury, with a focus on adenosine signaling. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: Mice were pretreated with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes, dodecapeptide HHLGGAKQAGDV-(phosphate-buffered saline)-liposomes, adenosine 5' diphosphateliposomes, or phosphate-buffered saline-liposomes. Five minutes after treatment the mice received a single laser-induced shock wave (1.8 J/cm) that caused lethal blast lung injury, and their survival times and lung injuries were then assessed. We also evaluated the therapeutic effect of posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes or H12-(phosphate-buffered saline)-liposomes 1 minute after laser-induced shock wave exposure. To examine the effect of adenosine signaling, adenosine A2A receptor (ZM241385) or adenosine A2B receptor (PSB 1115) antagonists were administered to the mice 1 hour before the pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes that was followed by laser-induced shock wave exposure. MEASUREMENTS AND MAIN RESULTS: Pre- and posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes significantly increased mouse survival [fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes: 58% survival vs H12-(phosphate-buffered saline)-liposomes: 8%; p < 0.05 (posttreatment)] and mitigated pulmonary tissue damage/hemorrhage and neutrophil accumulation after laser-induced shock wave exposure. fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes accumulated at pulmonary vessel injury sites after laser-induced shock wave exposure with both pre- and posttreatment. Furthermore, pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes reduced albumin and macrophage inflammatory protein-2 levels in bronchoalveolar lavage fluid. Although fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes pretreatment did not affect blood coagulation activity in the injured mice, its beneficial effect on blast lung injury was significantly abrogated by A2A or A2B adenosine receptor antagonists (A2A antagonist: 17% survival; A2B antagonist: 33% vs dimethyl sulfoxide control: 80%; p < 0.05, respectively). CONCLUSIONS: Fibrinogen γ-chain (dodecapeptide HHLGGAKQA GDV)-coated adenosine 5'-diphosphate-encapsulated liposomes may be effective against blast lung injury by promoting tissue-protective adenosine signaling and could represent a novel controlled-release drug delivery system.

Abdominal Infection Suppresses the Number and Activity of Intrahepatic Natural Killer Cells and Promotes Tumor Growth in a Murine Liver Metastasis Model.

BACKGROUND: Increasing evidence suggests that postoperative infection is associated with poorer long-term outcome in various malignancies. However, the mechanism of poor prognosis induced by postoperative infection has not been clearly explained. We sought to determine whether abdominal infection promotes cancer metastases in a murine liver metastasis model, and to investigate the role of liver natural killer (NK) cells on antitumor immunity during abdominal infection. METHODS: Female BALB/c (8-10 weeks old) mice were inoculated with NL-17 colon cancer cells into the spleen and then subjected to abdominal infection induced by cecal ligation and puncture (CLP) or sham treatment. The extent of liver metastases and cytokine production in the serum and liver were investigated. Cell fraction and cytotoxic activities of liver mononuclear cells (MNCs) were elucidated. RESULTS: CLP mice had poorer survival and their serum levels of IL-6, -10, and -12p70 were significantly elevated on day 1 compared with sham-treated and control mice. No obvious differences in cytokine levels of the liver homogenates were identified among the three groups, except IL-12p70 levels in CLP mice on day 7 significantly decreased. The cytotoxic activities of liver MNCs were significantly suppressed in CLP mice soon after tumor inoculation. Flow cytometry revealed a decrease in NK cells in the liver and perforin and granzyme B expression levels. CONCLUSIONS: Abdominal infection promoted liver metastases in a murine liver metastasis model, which may be partially caused by a decrease in the number and activity of NK cells during abdominal infection.

Theranostic Photosensitive Nanoparticles for Lymph Node Metastasis of Gastric Cancer.

BACKGROUND: Preoperative and intraoperative diagnoses of lymph node (LN) metastasis in patients with gastric cancer is essential to determine the extent of LN dissection in order to establish individualized treatment strategies. We investigated the theranostic value of a newly developed drug delivery system employing nanoparticles loaded with the indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm) using a murine draining LN metastasis model of gastric cancer. METHODS: In the experimental draining LN metastasis model of human gastric cancer, the right hind footpads of nude mice were injected with cancer cells. Three weeks later, either ICGm or ICG solution was injected through the tail vein. Forty-eight hours after the administration of a photosensitizer, in vivo and ex vivo imaging and photodynamic therapy (PDT) were performed, and size of the LNs was measured. RESULTS: In vivo imaging revealed metastatic LNs in the ICGm-treated mice but not in the ICG-treated mice. PDT using ICGm induced apoptosis and significantly inhibited the growth of metastatic LNs. CONCLUSIONS: ICGm presents a novel theranostic nanodevice for LN metastasis of gastric cancer.

Thoracic shock wave injury causes behavioral abnormalities in mice.

BACKGROUND: Mild traumatic brain injury (mTBI) is caused by complex mechanisms of systemic, local and cerebral responses to blast exposure. However, the molecular mechanisms of cognitive impairment after exposure to blast waves are not clearly known. We tested the hypothesis that thoracic injury induced functional and morphological impairment in the brain, leading to behavioral abnormalities. METHODS: Mice were exposed to laser-induced shock waves (LISWs) impacting the thorax and assessed for behavioral outcome at 7 and 28 days post injury. Hippocampus and lung were collected for histopathological analysis and gene expression profiling after injury. RESULTS: Thoracic injury transiently decreased the heart rate, blood pressure, peripheral oxyhemoglobin saturation and cerebral blood flow immediately after LISW exposure. Although LISWs exposure caused pulmonary contusions, hemorrhage was not apparent in the brain. At 7 and 28 days after, the injured mice exhibited impaired short-term memory and depression-like behavior compared with controls. Histological assessments showed an increase in neuronal cell death after shock wave exposure, especially in the CA3 region of the hippocampus. Moreover, shock wave exposure altered the expression of functionally relevant genes in the hippocampus at 1 h and 1 day post injury. CONCLUSIONS: Our findings indicate that the LISW-induced thoracic injury with no direct impact on the brain affected the hippocampal gene expression and led to morphological alterations, resulting in behavioral abnormalities. Therefore, body protection may be extremely important in the effective prevention against blast-induced alterations in brain function.

Photodynamic therapy using nanoparticle loaded with indocyanine green for experimental peritoneal dissemination of gastric cancer.

Although there have been multiple advances in the development of novel anticancer agents and operative procedures, prognosis of patients with advanced gastric cancer remains poor, especially in patients with peritoneal metastasis. In this study, we established nanoparticles loaded with indocyanine green (ICG) derivatives: ICG loaded lactosomes (ICGm) and investigated the diagnostic and therapeutic value of photodynamic therapy (PDT) using ICGm for experimental peritoneal dissemination of gastric cancer. Experimental peritoneal disseminated xenografts of human gastric cancer were established in nude mice. Three weeks after intraperitoneal injection of the cancer cells, either ICGm (ICGm-treated mice) or ICG solution (ICG-treated mice) was injected through the tail vein. Forty-eight hours after injection of the photosensitizer, in vivo and ex vivo imaging was carried out. For PDT, 48 h after injection of the photosensitizer, other mice were irradiated through the abdominal wall, and the body weight and survival rate were monitored. In vivo imaging revealed that peritoneal tumors were visualized through the abdominal wall in ICGm-treated mice, whereas only non-specific fluorescence was observed in ICG-treated mice. The PDT reduced the total weight of the disseminated nodules and significantly improved weight loss and survival rate in ICGm-treated mice. In conclusion, ICGm can be used as a novel diagnostic and therapeutic nanodevice in peritoneal dissemination of gastric cancer.

Sequential Approach for Water Purification Using Seashell-Derived Calcium Oxide through Disinfection and Flocculation with Polyphosphate for Chemical Pollutant Removal.

Safe water supply is usually inadequate in areas without water treatment plants and even in a city under emergency conditions due to a disaster, even though safe water is essential for drinking and other various purposes. The purification of surface water from a river, lake, or pond requires disinfection and removal of chemical pollutants. In this study, we report a water purification strategy using seashell-derived calcium oxide (CaO) via disinfection and subsequent flocculation with polyphosphate for chemical pollutant removal. Seashell-derived CaO at a concentration (2 g L-1) higher than its saturation concentration caused the >99.999% inactivation of bacteria, mainly due to the alkalinity of calcium hydroxide (Ca(OH)2) produced by hydration. After the disinfection, the addition of sodium polyphosphate at 2 g L-1 allowed for the flocculation of CaO/Ca(OH)2 particles with adsorbing chemical pollutants, such as Congo red, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate, and polychlorinated biphenyls, for removing these pollutants; purified water was obtained through filtration. Although this purified water was initially highly alkaline (pH ∼ 12.5), its pH decreased into a weak alkaline region (pH ∼ 9) during exposure to ambient air by absorbing carbon dioxide from the air with the precipitating calcium carbonate. The advantages of this water purification strategy include the fact that the saturation of CaO/Ca(OH)2 potentially serves as a visual indicator of disinfection, that the flocculation by polyphosphate removes excessive CaO/Ca(OH)2 as well as chemical pollutants, and that the high pH and Ca2+ concentrations in the resulting purified water are readily decreased. Our findings suggest the usability of seashell-derived material-polymer assemblies for water purification, especially under emergency conditions due to disasters.

Pivotal role of myeloid-derived suppressor cells in infection-related tumor growth.

BACKGROUND: In this study, we investigated infection-related tumor growth, focusing on myeloid-derived suppressor cells (MDSCs) in clinical and experimental settings. PATIENTS AND METHODS: In the clinical study, a total 109 patients who underwent gastrectomy or esophagectomy were included. Blood samples were collected from a preoperative time point through 3 months after surgery, and MDSCs were analyzed using flow cytometry. In animal experiments, peritonitis model mice were created by CLP method. We investigated the number of splenic MDSCs in these mice using flow cytometry. Malignant melanoma cells (B16F10) were inoculated on the back of the mice, and tumor growth was monitored. We compared the level of MDSC infiltration around the tumor and the migration ability between CLP and sham-operated mice-derived MDSCs. Finally, we focused on PD-L1+ MDSCs to examine the effectiveness of anti-PD-L1 antibodies on tumor growth in CLP mice. RESULTS: In patients with postoperative infectious complication, MDSC number was found to remain elevated 3 months after surgery, when the inflammatory responses were normalized. CLP mice showed increased numbers of MDSCs, and following inoculation with B16F10 cells, this higher number of MDSCs was associated with significant tumor growth. CLP-mice-derived MDSCs had higher levels of accumulation around the tumor and had more enhanced migration ability. Finally, CLP mice had increased numbers of PD-L1+ MDSCs and showed more effective inhibition of tumor growth by anti-PD-L1 antibodies compared to sham-operated mice. CONCLUSION: Long-lasting enhanced MDSCs associated with infection may contribute to infection-related tumor progression.

Glucocorticoid receptor-dependent immunomodulatory effect of ursodeoxycholic acid on liver lymphocytes in mice.

Although ursodeoxycholic acid (UDCA) has long been used for patients with chronic cholestatic liver diseases, particularly primary biliary cirrhosis, it may modulate the host immune response. This study investigated the effect of UDCA feeding on experimental hepatitis, endotoxin shock, and bacterial infection in mice. C57BL/6 mice were fed a diet supplemented with or without 0.3% (wt/vol) UDCA for 4 wk. UDCA improved hepatocyte injury and survival in concanavalin-A (Con-A)-induced hepatitis by suppressing IFN-γ production by liver mononuclear cells (MNC), especially NK and NKT cells. UDCA also increased survival after lipopolysaccharide (LPS)-challenge; however, it increased mortality of mice following Escherichia coli infection due to the worsening of infection. UDCA-fed mice showed suppressed serum IL-18 levels and production of IL-18 from liver Kupffer cells, which together with IL-12 potently induce IFN-γ production. However, unlike normal mice, exogenous IL-18 pretreatment did not increase the serum IFN-γ levels after E. coli, LPS, or Con-A challenge in the UDCA-fed mice. Interestingly, however, glucocorticoid receptor (GR) expression was significantly upregulated in the liver MNC of the UDCA-fed mice but not in their whole liver tissue homogenates. Silencing GR in the liver MNC abrogated the suppressive effect of UDCA on LPS- or Con-A-induced IFN-γ production. Furthermore, RU486, a GR antagonist, restored the serum IFN-γ level in UDCA-fed mice after E. coli, LPS, or Con-A challenge. Taken together, these results suggest that IFN-γ-reducing immunomodulatory property of UDCA is mediated by elevated GR in the liver lymphocytes in an IL-12/18-independent manner.

Nanospiked paper: Microfibrous cellulose materials nanostructured via partial hydrolysis and self-assembly.

Nanocelluloses, such as cellulose nanofibers and nanocrystals, are sustainable nanomaterials that are generally extracted from natural raw materials in a top-down manner. These nanomaterials and their assemblies are facilitating new applications of biopolymers. However, creating nanostructures from conventional cellulosic materials including paper and cloth remains challenging. Herein, we report an approach for bottom-up nanostructuring of conventional microfibrous cellulose materials via a molecular self-assembly strategy. As a precursor cellulose material, paper was allowed to swell with aqueous phosphoric acid for the partial dissolution and hydrolysis of cellulose while maintaining its microfibrous structure. The generated cello-oligosaccharides in a dissolved state started to self-assemble upon adding water as a coagulant, resulting in nanospike-like assemblies on the microfiber surfaces. The resultant nanospiked papers were found to serve as a precursor for synthesizing silver nanoparticle-cellulose composites with bactericidal activities. Our findings promote the development of cellulose-based functional materials with nanostructures designed via molecular self-assembly.

Cultivation and Transplantation of Three-Dimensional Skins with Laser-Processed Biodegradable Membranes.

For the treatment of irreversible, extensive skin damage, artificial skins or cultured skins are useful when allogeneic skins are unavailable. However, most of them lack vasculature, causing delayed perfusion and hence delay or failure in engraftment of the tissues. We previously developed a prevascularized three-dimensional (3D) cultured skin based on the layer-by-layer cell coating technique (LbL-3D skin), in which cells are seeded and laminated on a porous polymer membrane for medium supply to the thick cultured tissue. Recent animal studies have demonstrated that LbL-3D skin can achieve rapid perfusion and high graft survival after transplantation. However, there were practical issues with separating LbL-3D skins from the membranes before transplantation and the handling separated LbL-3D skins for transplantation. To address these problems, in this study, we examined the use of biodegradable porous polymer membranes that enabled the transplantation of LbL-3D skins together with the membranes, which could be decomposed after transplantation. Thin films made from poly (lactic-co-glycolic acid) (PLGA) were irradiated with femtosecond laser pulses to create micro through-holes, producing porous membranes. We designed and fabricated culture inserts with the PLGA membranes and cultivated LbL-3D skins with 2 × 106 neonatal normal human dermal fibroblasts and 1 × 104 human umbilical vein endothelial cells in the dermis of 20 cell layers and 1 × 105 neonatal human epidermal keratinocytes in the epidermis. Histological analyses revealed that the skins cultured on the PLGA membranes had thickness of about 400 µm and that there were no defects in the quality of the skins cultured on the PLGA membranes when compared with those cultured on the conventional (nonbiodegradable) commercial membranes. The cultured LbL-3D skins were then transplanted together with the PLGA membranes onto full-thickness excisional wounds in mice. At 7 days posttransplantation onto a mouse, the tissues above and below the membrane were connected through the holes with collagen-positive fibers that appeared to migrate from both the host and donor sides, and favorable reepithelization was observed throughout the transplanted skin region. However, insufficient engraftment was observed in some cases. Thus, further optimization of the membrane conditions would be needed to improve the transplantation outcome.

Clinical performance of a new intact FGF23 immunoassay in healthy individuals and patients with chronic hypophosphatemia.

While the positive association between automated intact fibroblast growth factor (FGF) 23 measurement kit (Determinar CL FGF23 [CL]) and the former assay (Kainos [KI]), and clinical utility of CL was well established, the clinical performance of Medfrontier FGF23 (MED), which was the manual intact FGF23 measurement kit with same antibody set as CL, has not yet been validated. Therefore, this study aims to compare MED FGF23 levels to KI FGF23 levels. A total of 380 samples were collected from healthy individuals, and 200 samples were collected from 20 patients with chronic hypophosphatemia. The intact FGF23 level of each sample was measured by KI and MED. Among the healthy individuals, the reference range of MED FGF23 levels was 18.6-59.8 pg/mL when calculated as the average ± 2 standard deviations. When compared with KI FGF23 levels, MED FGF23 levels were lower than KI levels both among samples from healthy individuals (KI FGF23, 40.9 [interquartile (IQR), 31.1-50.6]; MED FGF23, 38.0 [IQR, 31.5-45.7]; p value = 0.02) and among samples from patients with chronic hypophosphatemia (KI FGF23, 172.5 [IQR, 115.8-290.7]; MED FGF23, 130.2 [IQR, 93.6-247.0]; p value = 0.003). The linear regression analysis showed that the correlation between KI FGF23 and MED FGF23 was interpreted as a slope of 0.83 with a y-intercept of 0.53, revealing good linearity (R2 = 0.99). This study showed that the discrepancy between KI and MED was very similar to the previously reported data between KI and CL.

Insulin treatment directly restores neutrophil phagocytosis and bactericidal activity in diabetic mice and thereby improves surgical site Staphylococcus aureus infection.

Bacterial infections, including surgical site infections (SSI), are a common and serious complication of diabetes. Staphylococcus aureus, which is eliminated mainly by neutrophils, is a major cause of SSI in diabetic patients. However, the precise mechanisms by which diabetes predisposes to staphylococcal infection are not fully elucidated. The effect of insulin on this infection is also not well understood. We therefore investigated the effect of insulin treatment on SSI and neutrophil function in diabetic mice. S. aureus was inoculated into the abdominal muscle in diabetic db/db and high-fat-diet (HFD)-fed mice with or without insulin treatment. Although the diabetic db/db mice developed SSI, insulin treatment ameliorated the infection. db/db mice had neutrophil dysfunction, such as decreased phagocytosis, superoxide production, and killing activity of S. aureus; however, insulin treatment restored these functions. Ex vivo treatment (coincubation) of neutrophils with insulin and euglycemic control by phlorizin suggest that insulin may directly activate neutrophil phagocytic and bactericidal activity independently of its euglycemic effect. However, insulin may indirectly restore superoxide production by neutrophils through its euglycemic effect. HFD-fed mice with mild hyperglycemia also developed more severe SSI by S. aureus than control mice and had impaired neutrophil phagocytic and bactericidal activity, which was improved by insulin treatment. Unlike db/db mice, in HFD mice, superoxide production was increased in neutrophils and subsequently suppressed by insulin treatment. Glycemic control by insulin also normalized the neutrophil superoxide-producing capability in HFD mice. Thus, insulin may restore neutrophil phagocytosis and bactericidal activity, thereby ameliorating SSI.