Search

Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3 s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3 s at 5 mg/kg prevented weight gain by 13.6 % in high-fat diet-fed mice without any toxicological observation. In addition, compound 3 s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T 1/2 ), and blood exposure ratio of i.p to i.v were significantly improved in compound 3 s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3 s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3 s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation., (Copyright © 2024. Published by Elsevier B.V.)

The purpose of this study was to investigate the role of Lactobacillus rhamnosus GG (LGG) probiotics in radiation enteritis using in vivo mice. A total of 40 mice were randomly assigned to four groups: control, probiotics, radiotherapy (RT), and RT + probiotics. For the group of probiotics, 0.2 mL of solution that contained 1.0 × 10 8 colony-forming units (CFU) of LGG was used and orally administered daily until sacrifice. For RT, a single dose of 14 Gy was administered using a 6 mega-voltage photon beam to the abdominopelvic area. Mice were sacrifice at day 4 (S1) and day 7 (S2) after RT. Their jejunum, colon, and stool were collected. A multiplex cytokine assay and 16 s ribosomal RNA amplicon sequencing were then performed. Regarding cytokine concentrations in tissues, pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein-1, showed significantly decreased protein levels in colon tissues of the RT + probiotics group than in the RT alone group (all p < 0.05). As for comparing microbial abundance through alpha-diversity and beta-diversity, no significant differences were observed between the RT + probiotics and RT alone groups, except for an increase in alpha-diversity in the stool of the RT + probiotics group. Upon analysis of differential microbes based on treatment, the dominance of anti-inflammatory-related microbes, such as Porphyromonadaceae, Bacteroides acidifaciens, and Ruminococcus, was observed in the jejunum, colon, and stool of the RT + probiotics group. With regard to predicted metabolic pathway abundances, the pathways associated with anti-inflammatory processes, such as biosynthesis of pyrimidine nucleotides, peptidoglycans, tryptophan, adenosylcobalamin, and propionate, were differentially identified in the RT + probiotics group compared to the RT alone group. Protective effects of probiotics on radiation enteritis were potentially derived from dominant anti-inflammation-related microbes and metabolites., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Since its introduction as a model organism in the 1980s, the use of zebrafish ( Danio rerio ) in research has expanded worldwide. Despite its now widespread use in research, guidelines to safeguard the ethical treatment of zebrafish, particularly with regard to euthanasia and humane endpoint practices, remain inadequate. One well-recognized example is the use of excess tricaine methanesulfonate (MS-222) as a means to euthanize zebrafish, regardless of life stage. In this study, through nationwide expert elicitation, we provide a detailed account of zebrafish research practices within the Republic of Korea and the challenges of implementing appropriate methods for euthanasia as a humane endpoint, with many opting for hypothermic shock. We report a local expert consensus for establishing national guidelines to improve zebrafish welfare and good research practice. Suggestions and recommendations for national guidelines were offered. Taken together, our findings raise awareness broadly among zebrafish research practitioners in the field, offer an accurate account of the welfare and treatment of zebrafish in research within the Republic of Korea, and advocate for the development and implementation of national guidelines. As such, our study is useful as a model to adopt the expert elicitation approach to investigate, quantify, and address welfare concerns in zebrafish research, and to establish best practice guidelines.

Background: In recent years, there has been considerable interest in the therapeutic targeting of tumor-associated macrophages (TAMs) to modulate the tumor microenvironment (TME), resulting in antitumoral phenotypes. However, key mediators suitable for TAM-mediated remodeling of the TME remain poorly understood. Methods: In this study, we used single-cell RNA sequencing analyses to analyze the landscape of the TME modulated by TAMs in terms of a protumor microenvironment during early tumor development. Results: Our data revealed that the depletion of TAMs leads to a decreased epithelial-to-mesenchymal transition (EMT) signature in cancer cells and a distinct transcriptional state characterized by CD8 + T cell activation. Moreover, notable alterations in gene expression were observed upon the depletion of TAMs, identifying Galectin-1 (Gal-1) as a crucial molecular factor responsible for the observed effect. Gal-1 inhibition reversed immune suppression via the reinvigoration of CD8 + T cells, impairing tumor growth and potentiating immune checkpoint inhibitors in breast tumor models. Conclusion: These results provide comprehensive insights into TAM-mediated early tumor microenvironments and reveal immune evasion mechanisms that can be targeted by Gal-1 to induce antitumor immune responses., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Adipose-derived stem cells (ADSCs) exert immunomodulatory effects in the treatment of transplant rejection. This study aimed to evaluate the effects of ADSCs on the skin graft survival in a human-to-rat xenograft transplantation model and to compare single and multiple injections of ADSCs., Methods: Full-thickness human skin xenografts were transplanted into the backs of Sprague-Dawley rats. The rats were injected subcutaneously on postoperative days 0, 3, and 5. The injections were as follows: triple injections of phosphate-buffered saline (PBS group), a single injection of ADSCs and double injections of PBS (ADSC × 1 group), and triple injections of ADSCs (ADSC × 3 group). The immunomodulatory effects of ADSCs on human skin xenografts were assessed., Results: Triple injections of ADSCs considerably delayed cell-mediated xenograft rejection compared with the PBS and ADSC × 1 groups. The vascularization and collagen type 1-3 ratios in the ADSC × 3 group were significantly higher than those in the other groups. In addition, intragraft infiltration of CD3-, CD4-, CD8-, and CD68-positive cells was reduced in the ADSC × 3 group. Furthermore, in the ADSC × 3 group, the expression levels of proinflammatory cytokine interferon-gamma (IFN-γ) were decreased and immunosuppressive prostaglandin E synthase (PGES) was increased in the xenograft and lymph node samples., Conclusion: This study presented that triple injections of ADSCs appeared to be superior to a single injection in suppressing cell-mediated xenograft rejection. The immunomodulatory effects of ADSCs are associated with the downregulation of IFN-γ and upregulation of PGES in skin xenografts and lymph nodes., (© 2023. Korean Tissue Engineering and Regenerative Medicine Society.)

Increasing evidence indicates a strong interaction between cellular metabolism and innate macrophage immunity. Here, we show that the intracellular replication of Mycobacteroides massiliense in macrophages depends on host pyruvate dehydrogenase kinase (PDK) activity. Infection with M. massiliense induced a metabolic switch in macrophages by increasing glycolysis and decreasing oxidative phosphorylation. Treatment with dichloroacetate (DCA), a PDK inhibitor, converts this switch in M. massiliense-infected macrophages and restricts intracellular bacterial replication. Mechanistically, DCA resulted in AMPKα1 activation via increased AMP/ATP ratio, consequently inducing autophagy to constrain bacterial proliferation in the phagolysosome. This study suggests that the pharmacological inhibition of PDK could be a strategy for host-directed therapy to control virulent M. massiliense infections., Competing Interests: Declaration of competing interest The authors declare that this study was conducted without any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Macrophages are essential innate immune cells found throughout the body that have protective and pathogenic functions in many diseases. When activated, macrophages can mediate the phagocytosis of dangerous cells or materials and participate in effective tissue regeneration by providing growth factors and anti-inflammatory molecules. Ex vivo-generated macrophages have thus been used in clinical trials as cell-based therapies, and based on their intrinsic characteristics, they outperformed stem cells within specific target diseases. In addition to the old methods of generating naïve or M2 primed macrophages, the recently developed chimeric antigen receptor-macrophages revealed the potential of genetically engineered macrophages for cell therapy. Here, we review the current developmental status of macrophage-based cell therapy. The findings of important clinical and preclinical trials are updated, and patent status is investigated. Additionally, we discuss the limitations and future directions of macrophage-based cell therapy, which will help broaden the potential utility and clinical applications of macrophages., (© 2023. The Author(s).)

Immunotoxicity has been an important topic in toxicology since inadvertent exposures to xenobiotics were found to alter immune functions in humans. While rodent toxicity tests can reveal some levels of immunotoxicity, alternative methods must be developed to identify the detailed mechanisms. In this study, a method of in vitro prediction of innate immune suppression by substances was developed using a genomics approach. The primary selection of immune suppressors was based on their ability to downregulate MCP-1, CCL3, TNF, IL-8, and IL-12p40 expression levels in lipopolysaccharide (LPS)-stimulated THP-1 cells. Among 11 substances classified as potent immune suppressors, six including dexamethasone, tacrolimus, tofacitinib, prednisolone, sodium lauryl sulfate, and benzoic acid were used to create a dataset by transcriptomics of chemical-treated THP-1 cells using bulk RNA sequencing. We selected genes that were significantly upregulated by suppressor treatment while filtering out genes also upregulated in LPS-treated THP-1 cells. We identified a 226-gene immunosuppressive gene set (ISG). Innate immune suppressor signature scores were calculated as the median expression of the ISG. In a validation dataset, the signature score predicted acyclovir, cyclosporine, and mercuric chloride as immune suppressors, while selecting genistein as a non-immune suppressor. Although more dataset integration is needed in the future, our results demonstrated the possibility and utility of a novel genomics-based approach, the transcriptome-based determination of innate immune suppressor (TDIS) assay, to evaluate innate immune suppression by different substances. This provides insight into the development of future alternative testing methods because it reflects a comprehensive genetic signature derived from multiple substances rather than one cytokine., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The development of molecular imaging probes to identify key cellular changes within lung metastases may lead to noninvasive detection of metastatic lesions in the lung. In this study, we constructed a macrophage-targeted clickable albumin nanoplatform (CAN) decorated with mannose as the targeting ligand using a click reaction to maintain the intrinsic properties of albumin in vivo . We also modified the number of mannose molecules on the CAN and found that mannosylated serum albumin (MSA) harboring six molecules of mannose displayed favorable pharmacokinetics that allowed high-contrast imaging of the lung, rendering it suitable for in vivo visualization of lung metastases. Due to the optimized control of functionalization and surface modification, MSA enhanced blood circulation time and active/passive targeting abilities and was specifically incorporated by mannose receptor (CD206)-expressing macrophages in the metastatic lung. Moreover, extensive in vivo imaging studies using single-photon emission computed tomography (SPECT)/CT and positron emission tomography (PET) revealed that blood circulation of time-optimized MSA can be used to discern metastatic lesions, with a strong correlation between its signal and metastatic burden in the lung.

Re-emerging viral threats have continued to challenge the medical and public health systems. It has become clear that a significant number of severe viral infection cases are due to an overreaction of the immune system, which leads to hyperinflammation. In this study, we aimed to demonstrate the therapeutic efficacy of the dexamethasone nanomedicine in controlling the symptoms of influenza virus infection. We found that the A/Wisconsin/WSLH34939/2009 (H1N1) infection induced severe pneumonia in mice with a death rate of 80%, accompanied by significant epithelial cell damage, infiltration of immune cells, and accumulation of pro-inflammatory cytokines in the airway space. Moreover, the intranasal delivery of liposomal dexamethasone during disease progression reduced the death rate by 20%. It also significantly reduced the protein level of tumor necrosis factor-alpha (TNFα), interleukin-1β (IL-1β), IL-6, and the C-X-C motif chemokine ligand 2 (CXCL2) as well as the number of infiltrated immune cells in the bronchoalveolar lavage fluids as compared to the control and free dexamethasone. The liposomal dexamethasone was mainly distributed into the monocyte/macrophages as a major cell population for inducing the cytokine storm in the lungs. Taken together, the intranasal delivery of liposomal dexamethasone may serve as a novel promising therapeutic strategy for the treatment of influenza A-induced pneumonia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kwon, Quan, Song, Chung, Jung, Hong, Na and Seok.)

Objective: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive., Design: We investigated the role of the prostaglandin E 2 (PGE 2 ) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4 fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4 + macrophage secreted molecules was investigated on epithelial organoid differentiation., Results: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4 fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE 2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4 + macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing., Conclusion: PTGER4 + intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The dysregulation of fibroblast growth factor (FGF) signaling has been implicated in tumorigenesis, tumor progression, angiogenesis, and chemoresistance. The small-molecule AZD4547 is a potent inhibitor of FGF receptors. This study was performed to investigate the antitumor effects and determine the mechanistic details of AZD4547 in ovarian cancer cells. AZD4547 markedly inhibited the proliferation and increased the apoptosis of ovarian cancer cells. AZD4547 also suppressed the migration and invasion of ovarian cancer cells under nontoxic conditions. Furthermore, it attenuated the formation of spheroids and the self-renewal capacities of ovarian cancer stem cells and exerted an antiangiogenic effect. It also suppressed in vivo tumor growth in mice. Collectively, this study demonstrated the antitumor effect of AZD4547 in ovarian cancer cells and suggests that it is a promising agent for ovarian cancer therapy.

Human cytomegalovirus (HCMV) exploits the interleukin-10 (IL-10) pathway as a part of its infection cycle through the manipulation of the host IL-10 signaling cascade. Based on its immunomodulatory nature, HCMV attenuates the host immune response and facilitates the progression of co-infection with other pathogens in an immune-competent host. To investigate the impact of HCMV infection on the burden of non-tuberculous mycobacteria (NTM), whose prevalence is growing rapidly worldwide, macrophages were infected with HCMV and further challenged with Mycobacterium massiliense in vitro . The results showed that HCMV infection significantly increased host IL-10 synthesis and promoted the proliferation of M. massiliense in an IL-10-dependent manner. Transcriptomic analysis revealed that HCMV infection dampened the regulatory pathways of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1), consequently abrogating the immune responses to M. massiliense coinfection in macrophages. These findings provide a mechanistic basis of how HCMV infection may facilitate the development of pathogenic NTM co-infection by upregulating IL-10 expression., (Copyright © 2020 Quan, Kim, Na, Kim, Kim, Kim, Hong, Hwang and Seok.)

Interleukin-10 (IL-10) is the most potent anti-inflammatory cytokine in the body and plays an essential role in determining outcomes of many inflammatory diseases. Cellular metabolism is a critical determinant of immune cell function; however, it is currently unclear whether metabolic processes are specifically involved in IL-10 production. In this study, we aimed to find the central metabolic molecule regulating IL-10 production of macrophages, which are the main producers of IL-10. Transcriptomic analysis identified that metabolic changes were predominantly enriched in Kupffer cells at the early inflammatory phase of a mouse endotoxemia model. Among them, pyruvate dehydrogenase kinase (PDK)-dependent acute glycolysis was negatively involved in IL-10 production. Inhibition or knockdown of PDK selectively increased macrophage IL-10 expression. Mechanistically, PDK inhibition increased IL-10 production via profound phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha 1 (AMPKα1) by restricting glucose uptake in lipopolysaccharide-stimulated macrophages. AMPKα1 consequently activated p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and cyclic AMP-responsive element-binding protein to regulate IL-10 production. Our study uncovers a previously unknown regulatory mechanism of IL-10 in activated macrophages involving an immunometabolic function of PDK., (© The Author(s) (2020). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

As current therapies benefit only a minority of cancer patients, additional therapeutic targets are needed. Tumor-associated macrophages (TAMs) have attracted attention for improving therapeutic responses, yet regulatory strategies remain elusive. Here, we show that the protein kinase A catalytic subunit (PKA-C) acts as a molecular switch, inducing a pro-tumoral immunosuppressive macrophage phenotype within tumors. In human and murine breast cancer, overactivated PKA in TAMs creates a detrimental microenvironment for cancer progression by inducing vascular endothelial growth factor A (VEGFA), interleukin-10 (IL-10), and macrophage-derived arginase 1 (ARG1) expression. Macrophages with genetic deletion of PKA-C are prone to be pro-inflammatory, suggesting a possible immunotherapeutic target. Delivery of liposomal PKA inhibitor facilitates tumor regression and abrogates pro-tumoral TAM functions in mice. The therapeutic effect of targeting PKA is pronounced when combined with αCTLA-4 antibody, increasing cluster of differentiation 8 (CD8) + GranzymeB + T cells by about 60-fold. Our findings demonstrate critical roles of TAM PKA-C in tumor progression and suggest that targeting PKA-C efficiently augments cancer treatment responses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: The identification of novel targets for developing synergistic drug-radiation combinations would pave the way to overcome tumor radioresistance. We conducted cell-based screening of a human kinome siRNA library to identify a radiation-specific kinase that has a synergistic toxic effect with radiation upon inhibition and is not essential for cell survival in the absence of radiation., Experimental Design: Unbiased RNAi screening was performed by transfecting A549 cells with a human kinome siRNA library followed by irradiation. Radiosensitizing effects of a target gene and involved mechanisms were examined., Results: We identified the nonreceptor protein tyrosine kinase FES (FEline Sarcoma oncogene) as a radiosensitizing target. The expression of FES was increased in response to irradiation. Cell viability and clonogenic survival after irradiation were significantly decreased by FES knockdown in lung and pancreatic cancer cell lines. In contrast, FES depletion alone did not significantly affect cell proliferation without irradiation. An inducible RNAi mouse xenograft model verified in vivo radiosensitizing effects. FES-depleted cells showed increased apoptosis, DNA damage, G 2 -M phase arrest, and mitotic catastrophe after irradiation. FES depletion promoted radiation-induced reactive oxygen species formation, which resulted in phosphorylation of S6K and MDM2. The radiosensitizing effect of FES knockdown was partially reversed by inhibition of S6K activity. Consistent with the increase in phosphorylated MDM2, an increase in nuclear p53 levels was observed, which appears to contribute increased radiosensitivity of FES-depleted cells., Conclusions: We uncovered that inhibition of FES could be a potential strategy for inducing radiosensitization in cancer. Our results provide the basis for developing novel radiosensitizers., (©2019 American Association for Cancer Research.)

Macrophages are the gatekeepers of intestinal immune homeostasis as they discriminate between innocuous antigens and potential pathogens to maintain oral tolerance. However, in individuals with a genetic and environmental predisposition, regulation of intestinal immunity is impaired, leading to chronic relapsing immune activation and pathologies of the gastrointestinal tract, such as IBD. As evidence suggests a causal link between defects in the resolution of intestinal inflammation and altered monocyte-macrophage differentiation in patients with IBD, macrophages have been considered as a novel potential target to develop new treatment approaches. This Review discusses the molecular and cellular mechanisms involved in the differentiation and function of intestinal macrophages in homeostasis and inflammation, and their role in resolving the inflammatory process. Understanding the molecular pathways involved in the specification of intestinal macrophages might lead to a new class of targets that promote remission in patients with IBD.

Fasiglifam (TAK-875) a G-protein coupled receptor 40 (GPR40) agonist, significantly improves hyperglycemia without hypoglycemia and weight gain, the major side effects of conventional anti-diabetics. Unfortunately, during multi-center Phase 3 clinical trials, unexpected liver toxicity resulted in premature termination of its development. Here, we investigated whether TAK-875 directly inflicts toxicity on hepatocytes and explored its underlying mechanism of toxicity. TAK-875 decreased viability of 2D and 3D cultures of HepG2, a human hepatocarcinoma cell line, in concentration- (＞50 µM) and time-dependent manners, both of which corresponded with ROS generation. An antioxidant, N-acetylcysteine, attenuated TAK-875-mediated hepatotoxicity, which confirmed the role of ROS generation. Of note, knockdown of GPR40 using siRNA abolished the hepatotoxicity of TAK-875 and attenuated ROS generation. In contrast, TAK-875 induced no cytotoxicity in fibroblasts up to 500 µM. Supporting the hepatotoxic potential of TAK-875, exposure to TAK-875 resulted in increased mortality of zebrafish larvae at 25 µM. Histopathological examination of zebrafish exposed to TAK-875 revealed severe hepatotoxicity as manifested by degenerated hypertrophic hepatocytes with cytoplasmic vacuolation and acentric nuclei, confirming that TAK-875 may induce direct hepatotoxicity and that ROS generation may be involved in a GPR40-dependent manner.

Purpose: Lactic acid, the end product of glycolysis, has emerged as an immune-modulating metabolite in various diseases. In this study, we aimed to examine whether lactic acid contributes to the disease pathogenesis of choroidal neovascularization (CNV) and to investigate the role of macrophages in CNV pathogenesis., Methods: CNV was induced by laser photocoagulation in C57BL/6J mice. Lactic acid concentration was measured in the RPE-choroid region. Macrophage infiltration and VEGF were quantified by flow cytometry. VEGF-positive areas and CNV lesions were measured by flat-mount immunofluorescence staining. To inhibit lactic acid uptake in vivo, alpha-cyano-4-hydroxycinnamic acid (α-CHC), a monocarboxylate transporter (MCT) blocker, was injected intravitreally 1 day after laser. VEGF productions were measured in ARPE-19, THP-1 cells, and human umbilical vein endothelial cells (HUVECs) by quantitative PCR and ELISA. Angiogenic activity of lactic acid-treated macrophages was assessed by HUVEC tube formation assay., Results: Lactic acid was significantly increased in the RPE-choroid region of CNV-induced mice. Lactic acid upregulated VEGFA mRNA and VEGF protein expressions in THP-1 macrophages, but did not in ARPE-19 or HUVECs. THP-1 macrophages treated with lactic acid increased the angiogenesis of endothelial cells independent of MCT activity. Intravitreal injection of α-CHC substantially reduced the VEGF-positive area that colocalized with F4/80-positive macrophages. CNV lesions were also significantly reduced following α-CHC injection compared with vehicle-injected controls., Conclusions: To our knowledge, these results show for the first time the role of lactic acid in facilitating neovascularization through macrophage-induced angiogenesis. We suggest that targeting macrophage metabolism can be a promising strategy for CNV treatment.

Macrophages are now considered to be important players in various inflammatory diseases as well as tumor progression. Emerging evidence reveals that macrophage metabolic features are deeply associated with their immune functions. Understanding the interaction between cellular metabolism and immune signaling pathways in macrophages can help us to develop appropriate therapeutic approaches for inflammatory diseases. In this review, we briefly summarize key metabolic features of M1 and M2 macrophages as well as signaling interactions between major metabolic molecules with TLRs and NLRs. Current knowledges of cellular metabolism are focused on macrophages in various disease situations including sepsis, atherosclerosis, obesity, tuberculosis and cancer. Novel insights and present targets for regulating macrophage metabolism are also discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective To investigate the incidence of postoperative sore throat (POST) in Korean patients undergoing general anaesthesia with endotracheal intubation and to assess potential risk factors. Methods This prospective study enrolled patients who underwent all types of elective surgical procedures with endotracheal intubation and general anaesthesia. The patients were categorized into group S (those with a POST) or group N (those without a POST). The demographic, clinical and anaesthetic characteristics of each group were compared. Results This study enrolled 207 patients and the overall incidence of POST was 57.5% ( n = 119). Univariate analysis revealed that significantly more patients in group S had a cough at emergence and hoarseness in the postanaesthetic care unit compared with group N. Receiver operating characteristic curve analysis showed that an intracuff pressure ≥17 cmH 2 O was associated with POST. Multivariate analysis identified an intracuff pressure ≥17 cmH 2 O and cough at emergence as risk factors for POST. At emergence, as the intracuff pressure over ≥17 cmH 2 O increased, the incidence of hoarseness increased. Conclusions An intracuff pressure ≥17 cmH 2 O and a cough at emergence were risk factors for POST in Korean patients. Intracuff monitoring during anaesthesia and a smooth emergence are needed to prevent POST.

Background: There is a need for investigating the analgesic method as part of early recovery after surgery tailored for laparoscopic colorectal cancer (LCRC) surgery. In this randomized trial, we aimed to investigate the analgesic efficacy of an inverse 'v' shaped bilateral, subfascial ropivacaine continuous infusion in LCRC surgery., Methods: Forty two patients undergoing elective LCRC surgery were randomly allocated to one of two groups to receive either 0.5% ropivacaine continuous infusion at the subfascial plane (n = 20, R group) or fentanyl intravenous patient controlled analgesia (IV PCA) (n = 22, F group) for postoperative 72 hours. The primary endpoint was the visual analogue scores (VAS) when coughing at postoperative 24 hours. Secondary end points were the VAS at 1, 6, 48, and 72 hours, time to first flatus, time to first rescue meperidine requirement, rescue meperidine consumption, length of hospital stay, postoperative nausea and vomiting, sedation, hypotension, dizziness, headache, and wound complications., Results: The VAS at rest and when coughing were similar between the groups throughout the study. The time to first gas passage and time to first rescue meperidine at ward were significantly shorter in the R group compared to the F group (P = 0.010). Rescue meperidine was administered less in the R group; however, without statistical significance. Other study parameters were not different between the groups., Conclusions: Ropivacaine continuous infusion with an inverse 'v ' shaped bilateral, subfascial catheter placement showed significantly enhanced bowel recovery and analgesic efficacy was not different from IV PCA in LCRC surgery.

GM-CSF induces proinflammatory macrophages, but the underlying mechanisms have not been studied thus far. In this study, we investigated the mechanisms of how GM-CSF induces inflammatory macrophages. First, we observed that GM-CSF increased the extent of LPS-induced acute glycolysis in murine bone marrow-derived macrophages. This directly correlates with an inflammatory phenotype because glycolysis inhibition by 2-deoxyglucose abolished GM-CSF-mediated increase of TNF-α, IL-1β, IL-6, and IL-12p70 synthesis upon LPS stimulation. Increased glycolytic capacity is due to de novo synthesis of glucose transporter (GLUT)-1, -3, and -4, as well as c-myc. Meanwhile, GM-CSF increased 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting enzyme of the mevalonate pathway. Inhibition of acute glycolysis or 3-hydroxy-3-methyl-glutaryl-CoA reductase abrogated the inflammatory effects of GM-CSF priming in macrophages. Finally, mice with inflamed colons exposed to dextran sodium sulfate containing GLUT-1 high macrophages led to massive uptake of [ 18 F]-fluorodeoxyglucose, but GM-CSF neutralization reduced the positron-emission tomography signal in the intestine and also decreased GLUT-1 expression in colonic macrophages. Collectively, our results reveal glycolysis and lipid metabolism created by GM-CSF as the underlying metabolic constructs for the function of inflammatory macrophages., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Granulocyte-macrophage colony stimulating factor (GM-CSF) has a role in inducing emergency hematopoiesis upon exposure to inflammatory stimuli. Although GM-CSF generated murine bone marrow derived cells have been widely used as macrophages or dendritic cells in research, the exact characteristics of each cell population have not yet been defined. Here we discriminated GM-CSF grown bone marrow derived macrophages (GM-BMMs) from dendritic cells (GM-BMDCs) in several criteria. After C57BL/6J mice bone marrow cell culture for 7 days with GM-CSF supplementation, two main populations were observed in the attached cells based on MHCII and F4/80 marker expressions. GM-BMMs had MHCII low F4/80 high as well as CD11c + CD11b high CD80 - CD64 + MerTK + phenotypes. In contrast, GM-BMDCs had MHCII high F4/80 low and CD11c high CD8α - CD11b + CD80 + CD64 - MerTK low phenotypes. Interestingly, the GM-BMM population increased but GM-BMDCs decreased in a GM-CSF dose-dependent manner. Functionally, GM-BMMs showed extremely high phagocytic abilities and produced higher IL-10 upon LPS stimulation. GM-BMDCs, however, could not phagocytose as well, but were efficient at producing TNFα, IL-1β, IL-12p70 and IL-6 as well as inducing T cell proliferation. Finally, whole transcriptome analysis revealed that GM-BMMs and GM-BMDCs are overlap with in vivo resident macrophages and dendritic cells, respectively. Taken together, our study shows the heterogeneicity of GM-CSF derived cell populations, and specifically characterizes GM-CSF derived macrophages compared to dendritic cells.

Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 μM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hypoxia-inducible factor-1α (HIF-1α) is one of the key transcription factors that mediate adaptation to hypoxia. Despite increasing evidence implicating the PKC family as potential modulators of HIF-1α, the molecular mechanisms of PKC isoform-dependent HIF-1α activity under hypoxic conditions have not been systematically elucidated in cancer cell lines. Here, we collectively investigated how each isoform of the PKC family contributes to HIF-1α accumulation in the human cervical cancer cell line HeLa. Among the abundant PKC isoforms, blockade of either PKCα or PKCδ was found to substantially reduce HIF-1α accumulation and transcriptional activity in hypoxic cells. Knockdown of PKCδ resulted in a reduction of HIF-1α mRNA levels, whereas the HIF-1α mRNA level was unchanged regardless of PKCα knockdown. Upon searching for the downstream effectors of these kinases, we found that PKCα controls HIF-1α translation via AKT-mTOR under hypoxic conditions. On the other hand, one of the well-known transcriptional regulation pathways of HIF-1α, nuclear factor-κB (NF-κB) is identified as a downstream effector of PKCδ. Taken together, our findings provide insights into the roles of PKC isoforms as additional, discrete modulators of hypoxia-stimulated HIF-1α accumulation through different signaling pathways., (© 2015 Wiley Periodicals, Inc.)

Interleukin-10 (IL-10) is an important anti-inflammatory cytokine that is produced primarily by macrophages. We investigated mechanisms by which the timing of IL-10 production was controlled in macrophages and found that cyclin-dependent kinase 5 (CDK5) activity was markedly increased in lipopolysaccharide (LPS)-stimulated macrophages through the synthesis of the CDK5-binding partner and activator p35. Degradation of p35 released the inhibition on anti-inflammatory signaling mediated by CDK5-p35 complexes. The transiently active CDK5-p35 complexes limited the LPS-stimulated phosphorylation and activation of various mitogen-activated protein kinases (MAPKs), thereby preventing the premature production of SOCS3 (suppressor of cytokine signaling 3), an inhibitor of inflammatory responses in macrophages, and IL-10. Furthermore, we showed that dextran sodium sulfate failed to induce colitis in p35-deficient mice, which was associated with the enhanced production of IL-10 by macrophages. Together, our results suggest that CDK5 enhances the inflammatory function of macrophages by inhibiting the MAPK-dependent production of IL-10., (Copyright © 2015, American Association for the Advancement of Science.)

Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Macrophages have important functions in tissue homeostasis, but the exact mechanisms regarding wide spectrum of macrophage phenotype remain unresolved. In this study, we report that mouse bone marrow derived naïve macrophages produce prostaglandin E2 (PGE2 ) endogenously, resulting in anti-inflammatory gene expression upon differentiation induced by macrophage colony stimulating factor (M-CSF). Cyclooxygenase (COX) inhibition by indomethacin reduced endogenous PGE2 production of macrophages and subsequently reduced arg1, IL10 and Mrc1, YmI and FizzI gene expressions. Of note, PGE2 phosphorylates CREB via EP2 and EP4 receptor ligation, thereby transcriptionally increasing C/EBP-β expression in BALB/c bone marrow derived macrophages. Activated CREB directly binds to the CREB-responsive element of the C/EBP-β promoter, such that PGE2 ultimately reinforces arg1, IL10 and Mrc1 gene expression. Cyclic AMP activator forskolin also phosphorylated CREB and induced the C/EBP-β cascade, but this was completely blocked by the PKA inhibitor, H89. Consequently, M-CSF grown macrophages inhibited T-cell proliferation but the inhibition ability was reduced when the COX is inhibited by indomethacin or macrophage C/EBP-β expression was decreased by siRNA transduction. Our results collectively describe the molecular basis for homeostatic macrophage differentiation by endogenous PGE2 ., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report herein that Zn(II) selectively inhibits the hypoxia-inducible factor prolyl hydroxylase PHD3 over PHD2, and does not compete with Fe(II). Independent of the oligomer formation induced by Zn(II), inhibition of the activity of PHD3 by Zn(II) involves Cys42 and Cys52 residues distantly located from the active site.

Macrophages play important roles in defense against infection, as well as in homeostasis maintenance. Thus alterations of macrophage function can have unexpected pathological results. Cyclooxygenase (COX) inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated. Using bone marrow-derived macrophage culture and long-term COX inhibitor treatments in BALB/c mice and zebrafish, we showed that chronic COX inhibition drives macrophages into an inflammatory state. Macrophages differentiated in the presence of SC-560 (COX-1 inhibitor), NS-398 (COX-2 inhibitor) or indomethacin (COX-1/2 inhibitor) for 7 days produced more TNFα or IL-12p70 with enhanced p65/IκB phosphoylation. YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype. We further observed that indomethacin or NS-398 delivery accelerated zebrafish death rates during LPS induced sepsis. When COX inhibitors were released over 30 days from an osmotic pump implant in mice, macrophages from peritoneal cavities and adipose tissue produced more TNFα in both the basal state and under LPS stimulation. Consequently, indomethacin-exposed mice showed accelerated systemic inflammation after LPS injection. Our findings suggest that macrophages exhibit a more inflammatory phenotype when COX activities are chronically inhibited.

Background: Cell Saver (CS; Haemonemic Corp, Braintree, Mass, United States) is frequently used to decrease transfusion requirements of homologous blood during liver transplantation (OLT). However, the use of CS in hepatitis C virus (HCV)-infected recipients is still debated owing to the potential elevation of HCV RNA level. In this study, we compared HCV RNA levels of CS blood with a series of blood samples obtained from HCV-infected OLT recipients., Methods: Twelve HCV-infected patients with >50,000 copies/mL of HCV RNA were enrolled. HCV RNA was measured immediately after induction (I), at the end of anhepatic period (II), at the end of operation (III), and from the first returned blood in CS (CSb). HCV RNA level at each time period was compared., Results: HCV RNA levels ranged from 77,931 to 9,072,000 copies/mL at I. When compared to I, HCV RNA levels were reduced to 11.1% ± 13.0% and 0.7% ± 1.0% at II and III, respectively. Also, the RNA level reduced to 3.0% ± 2.0% of I after CS processing. The HCV RNA level at I was significantly higher than the levels at II, III, and CSb (P = .012, each), and the level at II was significantly higher than the level at III (P = .012). The HCV RNA level at CSb showed no statistical difference with the levels at II, but it was significantly higher than the level at III (P = .042)., Conclusions: The use of CS in HCV-infected OLT recipients seems to carry no additional risk with respect to intraoperative HCV RNA kinetics., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Graft-recipient weight ratio (GRWR) is the only documented predictor that influences the lactate elimination after reperfusion in living-donor liver transplantation (LDLT). This study was performed to investigate the predictors of lactate elimination after reperfusion in recipients of adult LDLT., Methods: The medical records of 159 patients who underwent LDLT were analyzed. Lactate level (mmol/L) was measured from just before the initiation of surgery (P0) and 5, 60, and 120 minutes after reperfusion of graft (R0, R1, and R2, respectively). The change of lactate level after reperfusion was defined as difference between lactate level measured at R0 and R2. Patients were divided into accumulation and elimination groups. Donor and recipient factors were compared between the 2 groups., Results: Lactate accumulation occurred in 80 of 159 recipients (50.3%), and elimination occurred in 79 (49.7%). GRWR and Model for End-Stage Liver Disease (MELD) score were higher in the elimination group. Lactate at R0 was lower in the elimination group., Conclusions: Higher GRWR and MELD score and lower lactate level immediate after reperfusion of graft were predictors of lactate elimination after reperfusion during adult LDLT., (Copyright © 2014 Elsevier Inc. All rights reserved.)

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

Hypoxia-inducible factor (HIF)-1α mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy termini of HIF-1α and p300. The nature of the constructed reporter protein was verified by observing localized distribution, degradation, and stabilization kinetics in cells transfected with the HIF-1α containing plasmid. A mutant HIF-1α incapable of binding to p300 was then utilized to demonstrate insignificant FRET efficiency, thereby confirming that our constructs could effectively probe the direct interaction between HIF-1α and p300. We further examined the effects of small molecules known to modulate the HIF-1α-p300 interaction and transcriptional activity on FRET. Finally, by inhibiting activities of two HIF-specific hydroxylases, HIF-specific prolyl hydroxylase (PHD) 2 and factor inhibiting HIF-1 (FIH-1) with their specific siRNAs, we explored how these HIF-specific hydroxylases contribute to the HIF-1α-p300 interaction by FRET measurements along with HIF-1 mediated transcriptional activation. Therefore, this technique would provide a way to study selective inhibition of either PHD2 or FIH-1 within living cells, and to screen specific inhibitors of HIF-mediated transcription activity for therapeutic applications., (© 2013 Wiley Periodicals, Inc.)

Melanoma patients frequently have elevated serum levels of interleukin-6 (IL-6), which is correlated with a poor prognosis. IL-6 activates STAT3 phosphorylation, inducing the transcription of genes that regulate tumor cell proliferation and antiapoptosis. In addition, recent evidence suggests that IL-6 induces the epithelial-to-mesenchymal transition and enhances the invasiveness of tumor cells of epithelial origin. However, it is unknown whether IL-6 affects mesenchymal tumor cells. In this study, we examined the effects of IL-6 on melanoma cells and found that IL-6 can enhance their metastatic potential by regulating the expression of Twist and N-cadherin. First, we confirmed that human melanoma tissues express IL-6 (especially at the lesion site), the IL-6 receptor, N-cadherin, and nuclear Twist. Next, we found that IL-6 induces STAT3 phosphorylation in WM-266-4 human melanoma cells, resulting in transient upregulation of Twist, which is a key regulator of metastasis. Importantly, the expression of N-cadherin, a protein downstream of Twist, was also increased on the cell surface after treatment with IL-6. These cells showed enhanced invasiveness, assessed using an invasion assay, and formed more metastatic nodules in the lungs of NOD-SCID mice after an intravenous injection. Importantly, melanoma cells with knocked-down N-cadherin formed less lung nodules compared with control in the NOD-SCID mouse model. Our data suggest that increased serum IL-6 in cancer patients could increase the invasiveness of melanoma cells and accelerate metastasis. Blocking IL-6 in the melanoma microenvironment may therefore inhibit disease progression.

The angiopoietin/Tie2 system is an important regulator of angiogenesis and inflammation. In addition to its functions in endothelial cells, Tie2 expression on non-endothelial cells allows for angiopoietin ligands to stimulate the cells. Although Ang1 is a strong Tie2 receptor agonist, little is known regarding the effect of Ang1 on non-endothelial cells, such as monocytes and macrophages. In this study, we found that Ang1 functionally binds to and stimulates monocytes via p38 and Erk1/2 phosphorylation. Ang1-mediated monocyte stimulation is associated with proinflammatory cytokine TNF-α expression. We also determined that Ang1 switched macrophage differentiation toward a pro-inflammatory phenotype, even in the presence of an anti-inflammatory mediator. These findings suggest that Ang1 plays a role in stimulating pro-inflammatory responses and could provide a new strategy by which to manage inflammatory responses.

Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, we identified inhibitors against the interaction between HIF-1α and p300 by a fluorescence polarization-based assay employing a fluorescently-labeled peptide containing the C-terminal activation domain of HIF-1α. Two small molecule inhibitors, menadione (MD) and ethacrynic acid (EA), were found to decrease expression of luciferase under the control of hypoxia-responsive elements in hypoxic cells as well as to efficiently block the interaction between the full-length HIF-1α and p300. While these compounds did not alter the expression level of HIF-1α, they down-regulated expression of a HIF-1α target vascular endothelial growth factor (VEGF) gene. Considering hypoxia-induced VEGF expression leading to highly aggressive tumor growth, MD and EA may provide new scaffolds for development of tumor therapeutic reagents as well as tools for a better understanding of HIF-1α-mediated hypoxic regulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tumor cells are often associated with abundant macrophages that resemble the alternatively activated M2 subset. Tumor-associated macrophages (TAMs) inhibit anti-tumor immune responses and promote metastasis. Cyclooxygenase-2 (COX-2) inhibition is known to prevent breast cancer metastasis. This study hypothesized that COX-2 inhibition affects TAM characteristics potentially relevant to tumor cell metastasis. We found that the specific COX-2 inhibitor, etodolac, inhibited human M2 macrophage differentiation, as determined by decreased CD14 and CD163 expressions and increased TNFα production. Several key metastasis-related mediators, such as vascular endothelial growth factor-A, vascular endothelial growth factor-C, and matrix metalloproteinase-9, were inhibited in the presence of etodolac as compared to untreated M2 macrophages. Murine bone marrow derived M2 macrophages also showed enhanced surface MHCII IA/IE and CD80, CD86 expressions together with enhanced TNFα expressions with etodolac treatment during differentiation. Using a BALB/c breast cancer model, we found that etodolac significantly reduced lung metastasis, possibly due to macrophages expressing increased IA/IE and TNFα, but decreased M2 macrophage-related genes expressions (Ym1, TGFβ). In conclusion, COX-2 inhibition caused loss of the M2 macrophage characteristics of TAMs and may assist prevention of breast cancer metastasis.

Increased fat intake is known to be a major cause of prostate cancer. In this study, we investigated the effect of dietary high fat on prostate intraepithelial neoplasia using transgenic adenocarcinoma mouse prostate (TRAMP) mice. Six-week-old male TRAMP mice were fed AIN93G (control group, 4.0 kcal/kg, n=6) and AIN93G-HFD (experimental group, 4.8 kcal/kg, n=7) for 10 weeks. Prostate histopathology, urogenital tract (UGT) weight, epididymal white adipose tissue weight, argyrophilic nucleolar organizer regions (AgNORs) counts, and serum leptin levels were examined. AIN93G-HFD fed group showed progressed neoplastic lesions in the prostate (P<0.05) compared to AIN93G fed group. AIN93G-HFD intake resulted in a increase in the weight of UGT (P<0.05) and epididymal white adipose tissue. The number of Ag-NOR positive dots significantly increased in each prostate lobe and final serum leptin levels in AIN93G-HFD fed group were about twice those of AIN93G fed group (P<0.05). Dietary high fat was related to the prostate cancer progression in the early stage of TRAMP mice and increased serum leptin levels, suggesting that the regulation of dietary components could delay the progression of prostate cancer.

Developing a detailed understanding of enzyme function in the context of an intracellular signal transduction pathway requires minimally invasive methods for probing enzyme activity in situ. Here, we describe a new method for monitoring enzyme activity in living cells by sandwiching live cells between two vertical silicon nanowire (NW) arrays. Specifically, we use the first NW array to immobilize the cells and then present enzymatic substrates intracellularly via the second NW array by utilizing the NWs' ability to penetrate cellular membranes without affecting cells' viability or function. This strategy, when coupled with fluorescence microscopy and mass spectrometry, enables intracellular examination of protease, phosphatase, and protein kinase activities, demonstrating the assay's potential in uncovering the physiological roles of various enzymes.

We previously found that clioquinol (CQ) increases functional hypoxia-inducible factor-1α (HIF-1α) with enhanced transcription of its target genes. Here we report that compounds derived from 8-hydroxyquinoline including CQ, broxyquinoline (BQ), iodoquinol (IQ) and chloroacetoxyquinoline (CAQ) promote neovascularization effectively based on chick chorioallantoic membrane assays. The CQ analogues induce stabilization of HIF-1α as well as enhance HIF-1-mediated vascular endothelial growth factor transcription. These analogues also exert inhibitory effects on the activity of prolyl and asparaginyl hydroxylations of HIF-1α in vitro. Despite metal ion-dependent restoration of the inhibited HIF-1α hydroxylase activity, the cellular HIF-1α-inducing effects of the CQ analogues are reversed to varying degrees by Zn(2+) and Fe(2+). While CQ and BQ are completely reversed by Zn(2+), co-administration of Zn(2+) and IQ has only a partial reversing effect. On the other hand, CAQ-mediated stabilization of HIF-1α is reversed by Fe(2+) but not by Zn(2+). These phenomena are found to coincide with elevation of the intracellular Zn(2+) and Fe(2+) levels by the CQ analogues, suggesting that metal ion effects on HIF-1α in cells likely reflect the differential transporting capability of the analogues.

Currently, murine noroviruses (MNV) are the most prevalent viral pathogens identified in laboratory animal facilities. While several reports exist concerning the prevalence of MNV in North American research facilities, very few reports are available for other parts of the world, including Korea. This study evaluated the prevalence of MNV infection in 745 murine sera collected from 15 animal facilities in Korea by enzyme linked immunosorbent assay (ELISA). Positive cases were subcategorized by murine strain/genetics, housing environments and animal sources. In summary, 6.6% of inbred/outbred mice purchased from commercial vendors were seropositive, 9.6% of in-house colonies were seropositive and 27.0% of genetically modified mice (GMM) were seropositive. Partial gene amplification of fecal isolates from infected animals showed that they were homologous (100%) with MNV-4.

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.

Chlorella vulgaris is a green microalga that contains various antioxidants, such as carotenoids and chlorophylls. In this study, antioxidants from C. vulgaris were extracted using pressurized liquid extraction (PLE), which has been recently used for bioactive compound extraction. The antioxidant capacity of individual compounds in chlorella was determined by online HPLC ABTS(*+) analysis. According to the antioxidant analysis of total extracts, the extraction yield, radical scavenging activity, and phenolic compounds using PLE were relatively high compared to those obtained using maceration or ultrasound-assisted extraction. On the basis of online HPLC ABTS(*+) analysis, the 15 major antioxidants from chlorella extracts were identified as hydrophilic compounds, lutein and its isomers, chlorophylls, and chlorophyll derivatives. Using PLE at high temperature (85-160 degrees C) significantly increased antioxidant extraction from chlorella, improving the formation of hydrophilic compounds and yielding more antioxidative chlorophyll derivatives. Online HPLC ABTS(*+) analysis was a useful tool for the separation of main antioxidants from PLE extracts and allowed the simultaneous measurement of their antioxidant capacity, which clearly showed that PLE is an excellent method for extracting antioxidants from C. vulgaris.

Ras proteins regulate signaling pathways that control many cellular responses, such as proliferation, survival, and differentiation. However, there are intriguing questions about the relationship between the developmental timing of specific mutations and the resultant phenotypes in individual cells. In this study, we used the Cre/loxP system for maintaining transgenic zebrafish lines harboring oncogenic Kras(V12) under the nestin promoter, and investigated the developmental effects of Ras activation in neural progenitor cells. Activated human Kras(V12) was induced within pDSNesLCherryLEGFPKRas(V12) transgenic fish by Cre mRNA injection. Cre-mediated gene excision was confirmed by polymerase chain reaction, and the injected embryos were investigated for Kras(V12) effects using the hemotoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and in situ hybridization. pDSNesLCherryLEGFPKRas(V12) transgenic embryos normally expressed mCherry in their central nervous system throughout the developmental stage. However, Cre mRNA injection efficiently excised the flanking stop sequence, and the injected embryos expressed enhanced green fluorescent protein in their brain with severe edema. Brain histology showed that neuronal cell differentiation could occur in spite of oncogenic Kras(V12) overexpression, but massive apoptosis and brain edema caused early embryonal death. In summary, the overexpression of Kras(V12) induces extensive apoptosis of neural progenitor cells followed by severe edema of the brain. However, some neural progenitor cells are resistant to Kras(V12) and can retain their ability to differentiate into neurons. Finally, our transgenic model demonstrates the inability of Kras(V12) alone to induce brain tumors at the early stage of development.

Spontaneous basal cell carcinoma (BCC) is very rare in rats, with an incidence rate of only 0.14% reported in aged animals. A spontaneous BCC occurred in a 7-week-old Sprague-Dawley rat housed in a specific-pathogen-free animal facility. The tumor was a single, well-delineated reddish-brown subcutaneous mass measuring 2 x 2 cm and located in the left inguinal region. Microscopically, the tumor consisted of basaloid cells in lobular and cribriform growth patterns and with a high mitotic rate. Immunohistochemically, cytokeratin 14 (an indicator for basal keratinocytes of the epidermis) showed strong reactions throughout the whole tumor, and cytokeratin 18 showed weak but positive reaction in the majority of nested tumor cells. To the authors' knowledge, this is the first report of spontaneous BCC occurrence in young Sprague-Dawley rats.

Regular monitoring of commercial laboratory rodents and institutional research animal residents is essential for microbiological quality control programs. The objective of our study was to investigate the recent prevalence of infectious pathogens in laboratory mice from eight experimental animal vendors and 56 institutional animal facilities in Korea. Our investigation was conducted in 2006-2007. Specific Pathogen Free (SPF) mice from four commercial breeders were clean according to serological, bacteriological, parasitological, and histopathological examination results. However, mice from one intermediate vendor that distributed SPF animals from main commercial vendors to local districts had Syphacia obvelata and Mycoptes musculinus infections. Additionally, mice from conventional animal breeders were highly contaminated. Among the 56 institutional animal facilities, mouse hepatitis virus (MHV), Sendai virus and Mycoplasma pulmonis positive results were obtained in 23.2, 8.9, and 1.8% of animals tested, respectively. These results indicate that quarantine and eradication efforts of infectious pathogens in these facilities are sub-optimal and need to be improved. The use of commercial conventional mice for research should be eliminated and appropriate vendor selection as well as thorough quarantine before releasing animals into a facility are needed. Finally we suggest qualified veterinary experts are needed at each animal facility to ensure an adequate health surveillance program.

Bone morphogenetic protein (BMP) 7 counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity in developmental stages. Because epithelial-to-mesenchymal transition and its reversed process mesenchymal-to-epithelial transition (MET) are also involved in cancer progression, we investigated whether BMP7 plays a role in WM-266-4 melanoma cell growth and metastasis. An MTT assay was conducted in WM-266-4 and HEK293T cell lines to show the cell growth inhibition ability of BMP7 and cisplatin. Semiquantitative RT-PCR was used to determine MET in morphologically changed BMP7-treated melanoma cells. MET-induced cells expressed less a basic helix-loop-helix transcription factor (TWIST) in western blot analysis, and we confirm that BMP receptor (Alk2) siRNA transduction could restore TWIST protein expression via blocking of Smad 1, 5 and 8 signaling. Matrigel invasion and cell migration assays were done to investigate the BMP7-induced metastasis inhibition ability. BMP7 treatment only slightly reduced cell growth rate, but induced apparent MET. BMP7 also reduced the invasion and migration ability. Furthermore, BMP7 reduced the resistance of WM-266-4 cells to cisplatin. Collectively, our findings indicate that the metastatis inhibition ability of BMP7 is involved in MET, and that BMP7 could be used as a potential metastasis inhibitor in human melanoma cells.