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Glioblastoma (GBM) is the central nervous system tumor with the most aggressive behavior, and no definitive therapy has yet been found. The tumor microenvironment of GBM is immunosuppressive and is considered a ‘cold tumor’ with low lymphocytic infiltration, but is characterized by a high proportion of glioma-associated macrophages/microglia (GAMs). GAMs promote tumor growth and also affect treatment resistance in GBM. In this review, we describe the origin and classification of GAMs in humans and describe the mechanisms of their activation and the cell-cell interactions between tumor cells and GAMs. We also describe the history of GAM detection methods, especially immunohistochemistry, and discusses the merits and limitations of these techniques. In addition, we summarized chemotactic factors for GAMs and the therapies targeting these factors. Recent single-cell RNA analysis and spatial analysis add new insights to our previous knowledge of GAMs. Based on these studies, GBM therapies targeting GAMs are expected to be further developed.

Abstract Background Photodynamic therapy (PDT) is an antitumor therapy and has traditionally been regarded as a localized therapy in itself. However, recent reports have shown that it not only exerts a direct cytotoxic effect on cancer cells but also enhances body's tumor immunity. We hypothesized that the immunological response induced by PDT could potentially enhance the efficacy of programmed death‐1 (PD‐1) / programmed death‐ligand 1 (PD‐L1) blockade. Methods The cytotoxic effects of PDT on colon 26 cells were investigated in vitro using the WST assay. We investigated whether the antitumor effect of anti‐PD‐1 antibodies could be amplified by the addition of PDT. We performed combination therapy by randomly allocating tumor‐bearing mice to four treatment groups: control, anti‐PD‐1 antibodies, PDT, and a combination of anti‐PD‐1 antibodies and PDT. To analyze the tumor microenvironment after treatment, the tumors were resected and pathologically evaluated. Results The viability rate of colon 26 cells decreased proportionally with the laser dose. In vivo experiments for combined PDT and anti‐PD‐1 antibody treatment, combination therapy showed an enhanced antitumor effect compared with the control. Immunohistochemical findings of the tumor microenvironment 10 days after PDT indicated that the number of CD8+ cells, the area of Iba‐1+ cells and the area expressing PD‐L1 were significantly higher in tumors treated with combination therapy than in tumors treated with anti‐PD‐1 antibody alone, PDT alone, or the control. Conclusions PDT increased immune cell infiltration into the tumor microenvironment. The immunological response induced by PDT may enhance the efficacy of PD‐1/PD‐L1 blockade.

After endoscopic sinus surgery (ESS), nasal packing is often used to stop bleeding and promote wound healing. Because maintaining a moist environment is important to enhance wound healing, hydrogel-based wound dressings are effective to promote wound healing. Chitosan is used in the medical field because of its high hemostatic and wound healing properties. We developed a pH-neutral and non-toxic chitosan hydrogel, which was difficult to achieve using conventional methods. In this study, we show in animal experiments that the chitosan hydrogel (hydrogel particles) had higher wound healing properties than a commercially available solid wound dressing (dry state) composed of the same polymer. Additionally, we applied the injectable chitosan hydrogel particles as nasal packing materials to patients with bilateral chronic sinusitis undergoing ESS in a pilot clinical study. Concerning symptom scores, though the results narrowly missed statistical differences (p < 0.05), the average scores of our chitosan hydrogel were superior to those of a commercially available wound dressing (especially p = 0.09 for nasal bleeding). These findings suggest that the injectable chitosan hydrogel could be a viable option as a packing material following ESS.

Micro/nanoplastics (MPs/NPs) in the environment exhibit various effects on ecosystems as well as living organisms, such as immune system toxicity. To understand the effects of MPs/NPs experimentally, preparing synthetic MPs/NPs with their geometrical morphology closely similar to the MPs/NPs present in natural environments and with specific material composition is necessary. A pin-on-disc method with plastic pins and micro-textured glass discs has been employed to generate MPs/NPs with defined composition for biological tests. However, these previous studies did not clarify the repeated elastic deformation and stress distribution inside the pin causing the fatigue of degraded areas. Accordingly, in this study, MPs/NPs were generated using a pin-on-disc method, and the interfacial pressure of the pin and disc and the micro-texture pattern were assessed as factors that could change the elastic deformation and the stress distribution inside the pin; additionally, how these factors affect the MP/NP geometrical morphology were investigated. Polyethylene, polypropylene, polyvinyl chloride, polystyrene, and polyethylene terephthalate were used as test materials. Almost all the MPs/NPs of these materials had a fragmented morphology. Further, these MPs/NPs were compared to those identified and they showed almost the same geometrical morphology as the fragmented MPs/NPs in the environment. The equivalent circle diameters of the generated MPs/NPs were suggested to be affected by micro-textures on the discs, which promoted fatigue failure. Additionally, by increasing the interfacial pressure, the stress was distributed deeper inside the pin depending on the plastic materials, which accelerated the crack propagation and generated a large amount of MPs/NPs. From these results, the fragmented morphology of MP/NP which is similar to those present in environments is expected to be generated with defined morphology and material composition for application to biological tests.

Introduction: As inhibitors of advanced glycation end products (AGEs), such as pyridoxamine, significantly inhibit the development of retinopathy and neuropathy in rats with streptozotocin-induced diabetes, treatment with AGE inhibitors is believed to be a potential strategy for the prevention of aging, age-related diseases, and lifestyle-related diseases, including diabetic complications. In the present study, the MeOH extract of Epimedii Herba (EH; aerial parts of Epimedium spp.) was found to inhibit the formation of Nε-(carboxymethyl)lysine (CML) and Nω-(carboxymethyl) arginine (CMA) during the incubation of collagen-derived gelatin with ribose.Materials and methods: EH was purchased from Uchida Wakan-yaku Co., and a MeOH extract was prepared. Several steps of column chromatography purified the extract. Each fraction was tested for inhibitory activity by ELISA using monoclonal antibodies for CML and CMA.Results: After activity-guided fractionation and purification by column chromatography, three new prenylflavonoids [named Koreanoside L (1), Koreanoside E1 (2), and Koreanoside E2 (3)] and 40 known compounds (4–43) were isolated from EH, and their inhibitory effects against CML and CMA formation were tested. Among these, epimedokoreanin B (8), epimedonin E (21), epicornunin B (22), and epicornunin F (24) inhibited the formation of both CML and CMA, with epimedokoreanin B (8) having the most potent inhibitory effect among the isolated compounds. To obtain the structure–activity relationships of 8, the phenolic hydroxy groups of 8 were methylated by trimethylsilyl-diazomethane to afford the partially and completely methylated compounds of 8. Prenyl derivatives of propolis (artepillin C, baccharin, and drupanin) were used in the assay.Discussion: As only 8 showed significant activity among these compounds, the catechol group of the B ring and the two prenyl groups attached to the flavanone skeleton were essential for activity. These data suggest that 8 could prevent the clinical complications of diabetes and age-related diseases by inhibiting AGEs.

Abstract Objectives C‐reactive protein (CRP) is a well‐known acute‐phase protein that increases remarkably under various inflammatory conditions and is elevated in patients with malignant tumors. In this study, we investigated the influence of CRP on the tumor microenvironment in clear cell renal cell carcinoma (ccRCC). Methods This study explored CRP's role in ccRCC by co‐culturing human macrophages with ccRCC cells and employing antibody blocking, RNA sequencing and in vitro experiments for functional insights. We also analysed The Cancer Genome Atlas Program (TCGA) data to link CD64 expression with ccRCC prognosis and used immunohistochemistry to associate CD64+ macrophages with tumor severity and systemic CRP levels. Results A co‐culture study using human macrophages and RCC cell lines showed that CRP‐stimulated macrophages secrete IL‐6, which induces RCC proliferation via STAT3 activation. CRP‐induced protumor activation of macrophages was suppressed by CD64 blocking antibodies. Furthermore, CRP elevates PD‐L1 expression in macrophages via the CD64‐STAT1 signalling pathway. Statistical analysis of TCGA data indicated that increased CD64 expression was associated with a worse clinical course in ccRCC. Immunohistochemical analysis of pathological specimens revealed that high CD64 expression in tumor‐associated macrophages (TAMs), and a high density of CD64+ TAMs, was linked to high nuclear grade and stage. High CD64 expression was also correlated with increased serum CRP levels. Conclusions The CRP‐CD64 signal was linked to the protumor activation of TAMs and could be a promising target for anticancer immunotherapy in ccRCC.

Abstract Aims Mismatch‐repair deficiency and microsatellite instability‐high (dMMR/MSI‐H) colorectal cancer (CRC) is treated with programmed death (PD)‐1 antibody regardless of PD‐ligand (L)1 expression in tumor cells. We previously found that abundant CD169+ macrophages in regional lymph node (RLN) sinuses and CD8+ tumor‐infiltrating lymphocytes (TILs) positively correlated in CRC and were associated with a favorable prognosis. However, associations between dMMR/MSI‐H CRC and CD8+ TILs or prognoses vary among studies. In this study, we attempted to compare the association between MMR status, CD169+ macrophages in RLNs, CD8+ TILs, PD‐L1 scores, and prognoses in CRC. Methods and Results We immunostained 83 surgically resected CRC tumors that we previously analyzed for MMR proteins, and identified 9 that were dMMR. The number of CD169+ macrophages in RLNs and CD8+ TILs significantly correlated with overall survival, whereas MMR status did not. The number of cells positive for the TIL markers CD3, CD4, CD8, and TIA‐1, and macrophage markers CD68 and CD169 in RLNs did not significantly differ between groups according to MMR status. Furthermore, combined positive scores (CPS) for PD‐L1 expression in five of nine dMMR CRCs were all

Abstract Ever since immune checkpoint inhibitors have been approved for anti-cancer therapy in several cancers, including triple-negative breast cancer, the significance of programmed death-1 ligand 1 (PD-L1) expression in the tumor immune microenvironment has been a topic of interest. In the present study, we investigated the detailed mechanisms of PD-L1 overexpression on tumor-associated macrophages (TAMs) in breast cancer. In in vitro culture studies using human monocyte-derived macrophages, lymphocytes, and breast cancer cell lines, PD-L1 overexpression on macrophages was induced by the conditioned medium (CM) of activated lymphocytes, but not that of cancer cells. Granulocyte–macrophage colony-stimulating factor (GM-CSF) derived from activated lymphocytes was found to be involved in PD-L1 overexpression, in addition to interferon (IFN)-γ, via STAT3 pathway activation. Macrophages suppressed lymphocyte activation, and this inhibition was impaired by PD-1 blocking. The CM of activated lymphocytes also induced the overexpression of PD-L2, but GM-CSF did not affect PD-L2 expression. In the murine E0771 breast cancer model, anti-GM-CSF therapy did not affect PD-L1 expression on TAMs, and the mechanisms of PD-L1 expression on TAMs might differ between humans and mice. However, not only PD-L1, but also PD-L2 was overexpressed on TAMs in the E0771 tumor model, and their expression levels were significantly lower in the tumors in nude mice than in wild-type mice. Anti-PD-L1 antibody and anti-PD-L2 antibody synergistically inhibited E0771 tumor development. In conclusion, PD-L1 and PD-L2 were overexpressed on TAMs, and they potentially contributed to immunosuppression. The GM-CSF-STAT3 pathway is thought to represent a new mechanism of PD-L1 overexpression on TAMs in human breast cancer microenvironment.

Chronic obstructive pulmonary disease (COPD) is a complex pulmonary condition characterized by bronchitis, emphysema, and mucus stasis. Due to the variability in symptoms among patients, traditional approaches to treating COPD as a singular disease are limited. This led us to focus on phenotype/endotype classifications. In this study, we explore the potential therapeutic role of thyroid hormone (T3) by using mouse models: emphysema-dominant elastase-induced COPD and airway-dominant C57BL/6-βENaC-Tg to represent different types of the disease. Here, we showed that intratracheal T3 treatment (40, 80 μg/kg, i.t., every other day) resulted in significant improvements regarding emphysema and the enhancement of respiratory function in the elastase-induced COPD model. T3-dependent improvement is likely linked to the up-regulation of Ppargc1a, a master regulator of mitochondrial biogenesis, and Gclm, a factor associated with oxidative stress. Conversely, neither short- nor long-term T3 treatments improved COPD pathology in the C57BL/6-βENaC-Tg mice. Because the up-regulation of extrathyroidal T3-producing enzyme Dio2, which is also considered a marker of T3 requirement, was specifically observed in elastase-induced COPD lungs, these results demonstrate that exogenous T3 supplementation may have therapeutic potential for acute but not chronic COPD exacerbation. Moreover, this study highlights the relevance of considering not only COPD phenotypes but also COPD endotypes (expression levels of Ppargc1a and/or Dio2) in the research and development of better treatment approaches for COPD.

Abstract Hepatoblastoma is the most common pediatric liver tumor, but little research has been done on the role of macrophages in hepatoblastoma. The purpose of this study was to gain insight into potential roles for macrophages in hepatoblastoma. Paraffin‐embedded specimens from 56 patients who underwent surgical resection were examined with immunohistochemical staining for the macrophage‐specific markers, Iba1 and CD163. Significant differences were seen among histological subtypes. Significantly increased numbers of macrophages were detected in embryonal components compared to fetal components in the mixed epithelial type. In vitro studies using human monocyte‐derived macrophages and two hepatoblastoma cell lines (HepG2 and Huh6) were performed. Conditioned medium from these cell lines induced increased CD163 expression in macrophages. Direct co‐culture with macrophages induced tumor cell proliferation via induction of protumor cytokine secretion from macrophages. Direct co‐culture with macrophages also induced interleukin (IL)‐34 overexpression by Huh6 cells via Brd4 signaling. IL‐34 overexpression promoted tumor cell proliferation and chemoresistance. High IL‐34 and Brd4 expression was detected in embryonal components, which have potentially higher proliferation activity than fetal components. In conclusion, IL‐34 expression in embryonal components may induce macrophage chemotaxis in a paracrine manner, and tumor cell proliferation and chemoresistance in an autocrine manner. IL‐34 is a potential therapeutic target for hepatoblastoma.

Lymph nodes are secondary lymphoid organs that appear as bean-like nodules usually

Abstract Once disseminated tumor cells (DTCs) arrive at a metastatic organ, they remain there, latent, and become seeds of metastasis. However, the clonal composition of DTCs in a latent state remains unclear. Here, we applied high-resolution DNA barcode tracking to a mouse model that recapitulated the metastatic dormancy of head and neck squamous cell carcinoma (HNSCC). We found that clones abundantly circulated peripheral blood dominated DTCs. Through analyses of multiple barcoded clonal lines, we identified specific subclonal population that preferentially generated homotypic circulating tumor cell (CTC) clusters and dominated DTCs. Despite no notable features under static conditions, this population significantly generated stable cell aggregates that were resistant to anoikis under fluid shear stress (FSS) conditions in an E-cadherin-dependent manner. Our data from various cancer cell lines indicated that the ability of aggregate-constituting cells to regulate cortical actin-myosin dynamics governed the aggregates’ stability in FSS. The CTC cluster-originating cells were characterized by the expression of a subset of E-cadherin binding factors enriched with actin cytoskeleton regulators. Furthermore, this expression signature was associated with locoregional and metastatic recurrence in HNSCC patients. These results reveal a biological selection of tumor cells capable of generating FSS-adaptive CTC clusters, which leads to distant colonization.

Summary: Subtilase cytotoxin (SubAB) is an AB5 toxin mainly produced by the locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli (STEC) strain such as O113:H21, yet the contribution of SubAB to STEC infectious disease is unclear. We found that SubAB reduced activation of the STEC O113:H21 infection-induced non-canonical NLRP3 inflammasome and interleukin (IL)-1β and IL-18 production in murine macrophages. Downstream of lipopolysaccharide signaling, SubAB suppressed caspase-11 expression by inhibiting interferon-β/STAT1 signaling, followed by disrupting formation of the NLRP3/caspase-1 assembly. These inhibitions were regulated by PERK/IRE1α-dependent endoplasmic reticulum (ER) stress signaling initiated by cleavage of the host ER chaperone BiP by SubAB. Our murine model of SubAB-producing Citrobacter rodentium demonstrated that SubAB promoted C. rodentium proliferation and worsened symptoms such as intestinal hyperplasia and diarrhea. These findings highlight the inhibitory effect of SubAB on the NLRP3 inflammasome via ER stress, which may be associated with STEC survival and infectious disease pathogenicity in hosts.

Moist wound healing is known to heal wounds faster than dry wound healing. Hydrogel wound dressings are suitable for moist wound healing because of their hyperhydrous structure. Chitosan, a natural polymer, promotes wound healing by stimulating inflammatory cells and releasing bioactive compounds. Therefore, chitosan hydrogel has great potential as a wound dressing. In our previous study, physically crosslinked chitosan hydrogels were successfully prepared solely by freeze-thawing of chitosan-gluconic acid conjugate (CG) aqueous solution without using any toxic additives. Furthermore, the CG hydrogels could be sterilized by autoclaving (steam sterilization). In this study, we showed that autoclaving (121 °C, 20 min) of a CG aqueous solution simultaneously achieved gelation of the solution and sterilization of the hydrogel. Hydrogelation of CG aqueous solution by autoclaving is also physically crosslinking without any toxic additives. Further, we showed that the CG hydrogels retained favorable biological properties of the CG hydrogels prepared by freeze-thawing and subsequent autoclaving. These results indicated that CG hydrogels prepared by autoclaving were promising as wound dressings.

Many patients with impaired renal function undergoing dialysis are subject to severe dietary restrictions. Especially overdose of salt is related to crisis of their life, so their meals are basically salt-free or low salt. Therefore, their quality of life is declined due to their yearning for salty taste. In the present study, we searched new salt-adsorbing food materials in dietary fibers to develop food ingredients preventing salt-sensitive hypertension and kidney dysfunction. As a result, calcium alginate and ammonium alginate possessed sodium-binding capacity without releasing potassium which causes a problem in chronic kidney injury. Furthermore, the administration of those fibers inhibited blood NaCl concentration and induced NaCl excretion in mice model.Therefore, calcium alginate and ammonium alginate are new candidate materials as salt-adsorbing materials, thus indicating that the health foods and/or health supplements containing those fibers may be a potentially new tool for prevention of salt-sensitive hypertension and kidney dysfunction.

To investigate the effects of microparticles on the immune system, a device was designed for an optimal culture environment. Macrophages phagocytose microparticles and produce inflammatory cytokines. In a culture environment in which macrophages phagocytose microparticles mixed in a culture medium, it remains unclear whether the macrophages can physically access all the microparticles present in the culture medium. In the culturing of fine particles, such as microparticles, it is necessary to devise methods that can realize a close biological contact between the macrophages and the microparticles. To enable macrophages to appropriately phagocytose microparticles, a microchamber with a glenoid hole for cell culturing was designed and manufactured. To clarify the effects of the size, administration amount, and administration time of the microparticles on the production of inflammatory cytokines, a system that can continuously deliver and collect the culture medium was introduced. The results obtained using these systems helped clarify the aforementioned effects. Our study confirms the possibility of employing a system that can optimally adjust the biological contact between macrophages and microparticles in a culture environment.

M2-like tumor-associated macrophages (TAMs) in the tumor tissues promote tumor progression by various mechanisms and represent possible targets of antitumor therapy. In the present study, we tested whether compounds from Epimedii Herba inhibit macrophage polarization to the M2/protumorigenic phenotype and prevent tumor progression, using human monocyte-derived macrophages (HMDMs) and an animal sarcoma model. Four Epimedii Herba-derived flavonoid compounds, namely, limonianin, epimedokoreanin B, icaritin, and desmethylicaritin, inhibited CD163 expression and interleukin (IL)-10 production, which are known M2 markers, suggesting that these compounds inhibit M2 polarization. Among these compounds, epimedokoreanin B and limonianin suppressed STAT3 activation in HMDMs. Notably, epimedokoreanin B also suppressed cell proliferation by blocking STAT3 activation in Saos-2 human sarcoma and LM8 mouse sarcoma cell lines. Furthermore, oral administration of epimedokoreanin B inhibited tumor growth in an LM8 tumor-bearing murine model. These results indicate that Epimedii Herba and Epimedii Herba-derived compounds, such as epimedokoreanin B, may be potentially new agents that can be used for the treatment and prevention of various malignant tumors. They may also be promising compounds for targeting the tumor microenvironment by inhibiting M2 polarization of the TAMs.

Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.

Abstract Objectives Septic (or endotoxin) shock is a severe systemic inflammatory disease caused by bacteraemia or endotoxaemia. Although it is known that increased serum levels of CD163 are observed in septic/endotoxin shock patients, the exact function and significance of CD163 in macrophage activation remain unclear. Therefore, in the current study, we tested whether CD163 contributes to the pathogenesis of endotoxin shock in mice. Methods and results In samples obtained from autopsy, the number of CD163‐positive macrophages was increased in the kidney, liver, heart, bone marrow and spleen of patients who had died from septic/endotoxin shock when compared to patients who had died from other causes. The animal study revealed a significantly lower survival rate in CD163‐deficient mice after lipopolysaccharide (LPS) injection. Several cytokines and oxidative stress‐related molecules were significantly elevated in the sera of LPS‐induced endotoxin shock mice models. Higher concentrations of IL‐6, TNF‐α, IL‐1β, nitrite (NO2‐) and nitrate (NO3‐) and a lower concentration of IL‐10 were observed in CD163‐deficient mice treated with LPS. Similar results were observed in CD163‐deficient LPS‐stimulated macrophages. Furthermore, in an antitype II collagen antibody‐induced arthritis (CAIA), rheumatoid arthritis model, inflammation and bone erosion scores as well as the expression of IL‐6 and IL‐1β were significantly increased in CD163‐deficient mice. Conclusions CD163 was suggested to be involved in the regulation of inflammatory cytokine expression in septic/endotoxin shock and CAIA.

The relationship between the wear behaviour of an ultra-high-molecular-weight polyethylene (GUR1050) pin and surface profile of a Co-28Cr-6Mo alloy (ASTM F-75) disc was investigated. Tribological tests have been performed by pin-on-disc wear testing machine with multi-directional pathways, obtaining the influence of micro slurry-jet erosion (MSE) processed Co–Cr–Mo alloy. The specific wear and the wear particles have been investigated. It was elucidated that the Co–Cr–Mo alloy surface processed by MSE influenced the specific wear rate of polyethylene; however, the morphological aspect of polyethylene wear particles was not drastically changed. The wear particles isolated from the lubricating liquid were added to a culture medium, and human monocyte-derived macrophages were incubated by using an upright/inverted method. The production of TNF-α and IL-6 seemed to have a correlation with the amount of wear particles added, however, the influence of particle size on the production of TNF-α and IL-6 was not obvious. This means that the improvements for the incubation method, i.e. upright/inverted method, need further investigation for the accurate analysis.

Tumour-associated macrophages (TAMs) polarized to the protumoural M2 phenotype promote tumour proliferation and are considered to be a therapeutic target in patients with malignant tumours. In this study, we tried to identify natural compounds that inhibit TAM polarization into the M2 phenotype. We found that an extract of soybeans suppressed M2 polarization, and that soyasapogenols, major compounds contained in soybeans, significantly inhibited the M2 polarization and increased the secretion of IL-12 produced by M1 macrophages. Soyasapogenols suppressed the activation of STAT3 in both macrophages and tumour cells, and inhibited the proliferation of U373, SaOS2 and LM8 tumour cells. Furthermore, oral administration of soyasapogenol B significantly suppressed both subcutaneous tumour development and lung metastasis in a LM8 tumour-bearing mice. An anti-tumour immune response was also induced by the administration of soyasapogenol B. Therefore, soyasapogenols may represent new agents that can be used for the prevention and/or treatment of various malignant tumours.

ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. Here, we isolated ER membranes and ER-derived vesicles to examine their ACAT enzyme activity in vitro. The results showed that when macrophages are grown under normal conditions, ACAT1 is located in high density ER membrane; its enzymatic activity is relatively low. Loading macrophages with cholesterol did not increase the total cellular ACAT1 protein content significantly but caused more ACAT1 to appear in ER-derived vesicles. These vesicles exhibit lower density and are associated with markers of both ER and the trans-Golgi network. When normalized with equal ACAT1 protein mass, the enzymatic activities of ACAT1 in ER-derived vesicles were 3-fold higher than those present in ER membrane. Results using reconstituted ACAT enzyme assay showed that the increase in enzyme activity in ER-derived vesicles is not due to an increase in the cholesterol content associated with these vesicles. Overall, our results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles with elevated ACAT1 enzyme activity without having to produce more ACAT1 protein.

Reactive oxygen species (ROS) and nitric oxide (NO) are major pathogenic molecules produced during viral lung infections, including influenza. While fluoroquinolones are widely used as antimicrobial agents for treating a variety of bacterial infections, including secondary infections associated with the influenza virus, it has been reported that they also function as anti-oxidants against ROS and as a NO regulator. Therefore, we hypothesized that levofloxacin (LVFX), one of the most frequently used fluoroquinolone derivatives, may attenuate pulmonary injuries associated with influenza virus infections by inhibiting the production of ROS species such as hydroxyl radicals and neutrophil-derived NO that is produced during an influenza viral infection. The therapeutic impact of LVFX was examined in a PR8 (H1N1) influenza virus-induced lung injury mouse model. ESR spin-trapping experiments indicated that LVFX showed scavenging activity against neutrophil-derived hydroxyl radicals. LVFX markedly improved the survival rate of mice that were infected with the influenza virus in a dose-dependent manner. In addition, the LVFX treatment resulted in a dose-dependent decrease in the level of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress) and nitrotyrosine (a nitrative marker) in the lungs of virus-infected mice, and the nitrite/nitrate ratio (NO metabolites) and IFN-γ in BALF. These results indicate that LVFX may be of substantial benefit in the treatment of various acute inflammatory disorders such as influenza virus-induced pneumonia, by inhibiting inflammatory cell responses and suppressing the overproduction of NO in the lungs.

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

Background/Aim: a1-Acid glycoprotein (AGP), also known as orosomucoid, is an acute-phase protein that has been found increased in plasma of cancer patients. This study investigates the role of AGP expression in clear cell renal cell carcinoma (ccRCC) and its association with clinical outcomes. Materials and Methods: We investigated the correlation between AGP levels and the prognosis of ccRCC through an analysis of The Cancer Genome Atlas (TCGA) database. To examine AGP expression and its clinicopathological associations, immunostaining was performed on paraffin-embedded tissue samples of 92 ccRCC cases. Results: AGP expression was found to be higher in RCC cell lines compared to normal renal epithelial cells. Analysis of the TCGA dataset showed that patients with AGP gene expression had significantly worse overall survival. However, AGP expression was not correlated with age, sex, or cancer stage. A mouse monoclonal antibody against AGP was generated. This antibody reacted with human and mouse hepatocytes, but not in AGP-deficient mice. From 92 examined ccRCC cases, AGP protein expression was detected in 89 cases, with only 3 being negative. AGP expression levels did not correlate with clinicopathological factors, such as age, tumor size, or nuclear grade. CD14, a receptor of AGP, was found to be expressed in Iba1-positive monocytes and tumor-associated macrophages (TAMs) but not in other cell types like lymphocytes or cancer cells. No significant correlation was found between AGP expression and the number of Iba1-positive cells in ccRCC tissues. Iba1-positive cells were correlated with Fuhrman grade, and patients with =30% Iba1-positive cells were, on average, significantly younger and had more aggressive tumor. Conclusion: AGP expression is linked to poorer survival in ccRCC, but its association with immune cell infiltration (via Iba1-positive cells) is unclear. [ABSTRACT FROM AUTHOR]

Background: Immunohistochemistry for p53 was a well-established method for cancer diagnosis in pathology. Aberrant cytoplasmic p53 positivity reflects the accumulation of p53 aggregates, which has been shown to be associated with chemoresistance and to be a predictive marker of a worse clinical course in ovarian cancer. Case Report: A 65-year-old Japanese man was diagnosed with lung cancer, and surgical resection was performed. Multiple metastasis were found 21 months post-surgery. The lesions were resistant to chemotherapy, and he succumbed to the disease 29 months post-surgery. The resected primary lesion was pathologically diagnosed as squamous cell carcinoma, with notable cytoplasmic p53 positivity indicated by immunohistochemistry. Conclusion: Notable aberrant cytoplasmic accumulation of p53 aggregate was observed in the cancer cells of this case. Chemotherapy was ineffective for the recurrent lesions, suggesting a role of p53 aggregates in chemoresistance. Pathological analysis of p53 via immunohistochemistry may be useful in predicting chemoresistance of lung squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Background/Aim: CXCL10, a member of the CXC chemokine family, plays a crucial role in immune response by facilitating the chemotaxis of CXCR3-positive immune cells. We examined the expression of CXCL10 to unravel its functional significance in colorectal cancer. Materials and Methods: Bioinformatics analysis was performed to investigate CXCL10 expression and its clinicopathological relevance. Subsequently, we examined the correlation between the serum levels of CXCL10 and its expression within cancer tissues. Results: Analysis of the TCGA database revealed that elevated CXCL10 expression in CRC tissues correlates with improved long-term survival and is inversely associated with lymph node infiltration and metastasis. Insights from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes further established a connection between increased CXCL10 and co-regulated gene expression with enhanced immune activation and regulation, mediated by the inhibition of the NOD-like receptor signaling pathway. Single-cell analysis pinpointed myeloid cells and macrophages as the primary sources of CXCL10. Immunohistochemical assessments revealed that a subset of cancer cells and macrophages are positive for CXCL10 expression. CXCL10-positive cells are predominantly located at the invasive front of the tumor. Intriguingly, our findings reveal an inverse correlation between serum CXCL10 levels and its expression in cancer tissues. Conclusion: The expression of CXCL10 may play a role in mediating the inflammatory responses at the invasive front in colorectal cancer and is observed to be inversely correlated with serum CXCL10 levels. It is pivotal to elucidate the distinct roles of CXCL10 in colorectal cancer, particularly different functions of cancer-tissue CXCL10 from serum CXCL10. [ABSTRACT FROM AUTHOR]