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.

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.

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.

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.

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.

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.

Biofilm Degradation by Seashell-Derived Calcium Hydroxide and Hydrogen Peroxide.

Microbial cells and self-produced extracellular polymeric substances assembled to form biofilms that are difficult to remove from surfaces, causing problems in various fields. Seashell-derived calcium hydroxide, a sustainable inorganic material, has shown high bactericidal activity even for biofilms due to its alkalinity. However, its biofilm removal efficacy is relatively low. Herein, we report a biofilm degradation strategy that includes two environmentally friendly reagents: seashell-derived calcium hydroxide and hydrogen peroxide. A biofilm model of Escherichia coli was prepared in vitro, treated with calcium hydroxide-hydrogen peroxide solutions, and semi-quantified by the crystal violet stain method. The treatment significantly improved biofilm removal efficacy compared with treatments by calcium hydroxide alone and hydrogen peroxide alone. The mechanism was elucidated from calcium hydroxide-hydrogen peroxide solutions, which suggested that perhydroxyl anion and hydroxyl radical generated from hydrogen peroxide, as well as the alkalinity of calcium hydroxide, enhanced biofilm degradation. This study showed that concurrent use of other reagents, such as hydrogen peroxide, is a promising strategy for improving the biofilm degradation activity of seashell-derived calcium hydroxide and will contribute to developing efficient biofilm removal methods.

Viability Improvement of Three-Dimensional Human Skin Substitutes by Photobiomodulation during Cultivation.

Three-dimensional (3D) cultured skin containing vascular networks is a useful skin substitute that enables rapid reperfusion after transplantation. During its cultivation, however, insufficient nutrient delivery to the thick cultured tissue from the surrounding culture medium decreases the tissue viability. To solve this problem, in this study, we applied photobiomodulation (PBM), which can optically activate the electron transport chain of mitochondria, to human 3D skin cultures constructed using the layer-by-layer cell coating technique. PBM was applied once 5 days after the start of epidermal differentiation using a light-emitting diode array with a center wavelength of 440, 523, 658 or 823 nm at a constant light intensity of 15 mW cm-2 for 50 or 600 s. Two days after PBM, we assessed the viability of the tissues by a water-soluble tetrazolium-8 assay, adenosine triphosphate measurements and live/dead cell imaging, and the results showed that the PBM at 823 nm for 50 s (0.75 J cm-2 ) significantly improved the viability of the 3D-cultured skin.

Recent Progress on Heparin-Protamine Particles for Biomedical Application.

Biomolecules are attractive building blocks with self-assembly ability, structural diversity, and excellent functionality for creating artificial materials. Heparin and protamine, a clinically relevant pair of biomolecules used in cardiac and vascular surgery, have been shown to coassemble into particulate polyelectrolyte complexes in vitro. The resulting heparin-protamine particles exhibit adhesive properties that enable advantageous interactions with proteins, cells, and various other substances and have been employed as functional materials for biomedical applications. In this review article, we summarize recent progress in research on the use of heparin-protamine particles as drug carriers, cell adhesives, and cell labels. Studies have demonstrated that heparin-protamine particles are potentially versatile in biomedical fields from drug delivery and regenerative medicine to plastic surgery.

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.

Effects of Selective Serotonin Reuptake Inhibitors on Depression-Like Behavior in a Laser-Induced Shock Wave Model.

Primary blast injury can result in depression-like behavior in the long-term. However, the effects of the selective serotonin reuptake inhibitor (SSRI) on the depression induced by mild blast traumatic brain injury (bTBI) in the long-term remain unclear. We generated a mouse model of mild bTBI using laser-induced shock wave (LISW) and administered an SSRI to mice by oral gavage for 14 days after LISW exposure. This study aimed to investigate the mechanisms of SSRI-mediated alleviation of depression-like behavior induced by mild bTBI. Animals were divided into three groups: sham, LISW-Vehicle, and LISW-SSRI. LISW was applied to the head of anesthetized mice at 0.5 J/cm2. Twenty-eight days after the LISW, mice in the LISW-SSRI group exhibited reduced depression-like behavior, a significant increase in the number of cells co-stained for 5-bromo-2'-deoxyuridine (Brd-U) and doublecortin (DCX) in the dentate gyrus (DG) as well as increased brain-derived neurotrophic factor (BDNF) and serotonin levels in the hippocampus compared to the sham and LISW-Vehicle groups. Additionally, levels of phosphorylated cAMP response element binding protein (pCREB) in the DG were significantly decreased in the LISW-Vehicle group compared to that in the sham group. Importantly, pCREB levels were not significantly different between LISW-SSRI and sham groups suggesting that SSRI treatment may limit the downregulation of pCREB induced by mild bTBI. In conclusion, recovery from depression-like behavior after mild bTBI may be mediated by hippocampal neurogenesis induced by increased BDNF and serotonin levels as well as the inhibition of pCREB downregulation in the hippocampus.

A Potential Role of Adhesion Molecules on Lung Metastasis Enhanced by Local Inflammation.

BACKGROUND/AIM: Local and systemic inflammations are associated with negative long-term outcomes; however, their precise mechanism of action remains unclear. We previously demonstrated that hepatocyte growth factor (HGF)/c-Met signaling contributed to the enhancement of liver metastasis associated with peritonitis model. The aim of this study is to investigate the effect of local inflammation on the development of lung metastasis. MATERIALS AND METHODS: NL-17 cells were injected into BALB/c mice via the tail vein to produce a high potential model for lung metastasis. After injection of NL-17 cells, lipopolysaccharide (LPS) and live Pseudomonas aeruginosa, and phosphate-buffered saline were administered intratracheally to induce acute lung injury (ALI) and pneumonia, respectively. RESULTS: In both ALI and pneumonia mice, lung metastasis was significantly promoted compared to control mice. Concentrations of Interleukin-6, tumor necrosis factor-α, and HGF in the bronchoalveolar lavage fluid were significantly higher in ALI and pneumonia mice than in control mice. Neither administration of recombinant mouse HGF nor c-Met knockdown in NL-17 cells influenced the magnitude of lung metastasis. Yet stimulation with LPS increased the expression of α2 integrin, vascular cell-adhesion protein-1, and intercellular adhesion molecule-1 (ICAM-1) in the lung. Invasive activity of NL-17 cells was significantly up-regulated by LPS, but was suppressed by anti-ICAM-1 antibody. While LPS-stimulated NL-17 cells showed significantly promoted lung metastasis, E-selectin expression in the lungs of mice with ALI or pneumonia was significantly enhanced compared with control mice. CONCLUSION: Up-regulation of adhesion molecules, but not HGF/c-Met signaling, may contribute to the lung metastasis enhanced by local infection/inflammation.

A Potential Mechanism of Tumor Progression during Systemic Infections Via the Hepatocyte Growth Factor (HGF)/c-Met Signaling Pathway.

BACKGROUND: Increasing evidence has demonstrated that postoperative infectious complications (PICs) after digestive surgery are significantly associated with negative long-term outcomes; however, precise mechanisms of how PICs affect the poor long-term survival remain unclear. Here, we focused on the hepatocyte growth factor (HGF)/c-Met signaling pathway as one of those mechanisms. Methods: In the clinical setting, serum HGF levels were measured in the patients with sepsis and those with PICs after undergoing esophagectomy. Using a liver metastasis mouse model with cecal ligation and puncture (CLP), expressions of HGF and the roles of the HGF/c-Met pathway in the progression of tumor cells were examined. Results: Serum HGF levels were very high in the patients with intra-abdominal infection on postoperative days (PODs) 1, 3, and 5; similarly, compared to the patients without PICs, those with PICs had significantly higher serum HGF levels on 1, 3, and 5 days after esophagectomy. The patients with PICs showed poorer overall survival than those without PICs, and the patients with high serum HGF levels on POD 3 showed poorer prognosis than those with low HGF levels. Similarly, at 24 and 72 h after operation, serum levels of HGF in CLP mice were significantly higher than those in sham-operated mice. Intraperitoneal injection of mouse recombinant HGF significantly promoted liver metastases in sham-operated mice on 14 days after surgery. Knocking down c-Met expression on NL17 tumor cells by RNAi technology significantly inhibited the promotion of CLP-induced liver metastases. Conclusions: Infections after surgery increased serum HGF levels in the clinical as well as experimental settings. Induction of high serum HGF levels by CLP promoted liver metastases in a murine liver metastasis model, suggesting the involvement of the HGF/c-Met signaling pathway in tumor promotion mechanisms. Thus, targeting the HGF/c-Met signaling pathway may be a promising approach for malignant tumors, particularly in the patients with PICs.

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.

A novel mouse model of mild traumatic brain injury using laser-induced shock waves.

Blast-induced mild traumatic brain injury (mild bTBI) has been a frequent battlefield injury in soldiers during the conflicts in Iraq and Afghanistan. Understanding the pathophysiology and determining effective treatments for mild bTBI has become an international problem in the field of neurotrauma research. Contributing to this problem is a lack of an experimental model that accurately mimics the characteristics of mild bTBI. To date, the "mild'' versions of common experimental models of TBI have simply been less severe degrees of traumatic injury; these animals do not necessarily exhibit the clinical characteristics of mild bTBI seen in humans. Therefore, our first objective was to develop a highly controlled mouse model of bTBI using laser-induced shockwaves (LISWs). We established the parameters necessary to cause a reproducible injury of very mild severity, the most important feature seen in clinical practice. We defined very mild bTBI as having no traumatic change on the head visible to the naked eye after the insult was applied using very mild shockwaves to the heads of mice. Our very mild bTBI mouse model exhibited neurobehavioral changes in the chronic phase, such as cognitive impairment and depression-like behavior. We also observed an increase in 5-bromo-2'-deoxyuridine-positive, proliferating cells in the dentate gyrus during the acute phase and a subsequent decrease during the chronic phase. This model appears to be an accurate representation of the damage occurring in actual mild bTBI patients. We also found that an increase in cell proliferation in the dentate gyrus during the acute phase is the most prominent feature after a TBI.

A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment.

Autologous split-thickness skin grafts are the preferred treatment for excised burn wounds, but donor sites for autografting are often limited in patients with extensive burns. A number of alternative treatments are already in use to treat large burns and ulcers. Despite intense efforts to develop tissue-engineered skin, delayed or absent vascularization is one of the major reasons for tissue-engineered skin engraftment failure. To overcome these problems, we developed a scaffold-free 3-dimensional (3D) skin substitute containing vascular networks that combine dermal fibroblasts, endothelial cells, and epidermal keratinocytes based on our layer-by-layer cell coating technique. We transplanted the pre-vascularized 3D skin substitutes onto full-thickness skin defects on severe combined immunodeficiency mice to assess their integration with the host tissue and effects on wound healing. We used non-vascularized 3D skin substitutes as a control. Vessels containing red blood cells were evident in the non-vascularized control by day 14. However, blood perfusion of the human-derived vasculature could be detected within 7 days of grafting. Moreover, the pre-vascularized 3D skin substitutes had high graft survival and their epidermal layers were progressively replaced by mouse epidermis. We propose that a novel dermo-epidermal 3D skin substitute containing blood vessels can promote efficient reconstruction of full-thickness skin defects.

The reduced bactericidal activity of neutrophils as an incisive indicator of water-immersion restraint stress and impaired exercise performance in mice.

The incisive evaluation of psychological stress may be required to determine the exercise performance of stressed hosts. We investigated objective markers of psychological stress that reflect exercise performance, focusing on the neutrophil function. We used murine water-immersion restraint (WIR) stress for our assessments. After receiving WIR for 1 or 2 h, mice were exercised on an airtight treadmill that monitors their respiratory exchange ratio. The neutrophil function was analyzed after WIR stress. Although the control mice (without WIR) showed good combustion of both carbohydrates and lipids as energy sources during treadmill exercise, mice that underwent 2-h WIR did not combust carbohydrates or lipids effectively, drastically reducing their performance. In contrast, the 1-h WIR mice showed carbohydrate combustion (albeit a slow response) but did not use lipids for energy, thereby running longer than the 2-h WIR mice but shorter than the control mice. The bactericidal activity of neutrophils, but not their superoxide production or microsphere-phagocytic activity, was significantly reduced by 1-h WIR and further reduced by 2-h WIR, indicating a significant association between WIR stress and exercise performance. The neutrophil bactericidal activity may be a good indicator of psychological stress and a useful tool for precisely assessing exercise performance.

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.