Exploring the roles of SPARC as a proinflammatory factor and its potential as a novel therapeutic target against cardiovascular disease.

Cardiovascular disease (CVD) is a leading cause of death worldwide, and the number of patients with CVD continues to increase despite extensive research and developments in this field. Chronic inflammation is a pivotal pathological component of CVD, and unveiling new proinflammatory factors will help devise novel preventive and therapeutic strategies. The extracellular matrix (ECM) not only provides structural support between cells but also contributes to cellular functions. Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein that is particularly induced during development and tissue remodeling. A proinflammatory role for SPARC has been demonstrated in various animal models, such as in the lipopolysaccharide-induced footpad model and dextran sodium sulfate-induced colitis model. Recent clinical studies reported a positive correlation between elevated plasma SPARC levels and hypertension, obesity, and the inflammatory marker high-sensitive C-reactive protein. In addition, SPARC gene deletion attenuates the cardiac injury induced by aging, myocardial infarction, and pressure load, suggesting that SPARC has deleterious effects on CVD. This review summarizes the regulatory and proinflammatory mechanisms of SPARC on CVD, chronic kidney disease (CKD), and cerebrovascular disease and discusses the rationale behind measuring SPARC as a biomarker of CVD and the effects of inhibition of SPARC in the prevention and treatment of CVD.

IL-18 primes T cells with an antigen-inexperienced memory phenotype for proliferation and differentiation into effector cells through Notch signaling.

Recent studies have revealed that a subset of CD8+ T cells exhibit innate features and can be activated by cytokines. However, the precise mechanisms underlying the proliferation and differentiation of these cells remain unclear. Here, we demonstrated that CD44highCD8+ T cells in the mouse spleen express functional interleukin-18 (IL-18) receptors, whereas CD44lowCD8+ T cells do not. In response to IL-18 stimulation, these cells activated various metabolic pathways, upregulated the expression of surface molecules, such as c-Kit (CD117), CD25, and PD-1, and induced progression through the G1/S phase in the cell cycle. IL-18-primed cells, expressing a high-affinity receptor for IL-2, exhibited robust proliferation in response to IL-2 and underwent differentiation into effector cells. The splenic CD44highCD8+ T cells exhibited high expression levels of CD122, CD62L, CCR7, and CXCR3, along with CD5, indicating their potential for migration to the lymph nodes, where they could undergo expansion and terminal differentiation into effector cells. Additionally, in a tumor model, administration of IL-18 increased the accumulation of CD8+ T cells in both the lymph nodes and tumors. It is noteworthy that stimulation of CD44highCD8+ T cells with IL-18 upregulated the Notch-1 receptor and c-Myc. Moreover, inclusion of γ-secretase inhibitors attenuated the effect of IL-18 on both proliferation and interferon-γ production in the cells. These results demonstrate that IL-18 primes CD44highCD122highCXCR3highCD62LhighCD8+ T cells for expansion and differentiation into effector cells in a Notch signaling-dependent manner.

Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Experimental infection of goats with pVAPN-harboring Rhodococcus equi causes latent infection in the lymph nodes.

Rhodococcus equi has recently been identified in various animals, including ruminants. Several studies have highlighted the emergence of pVAPN-harboring strains, isolated from multiple abscesses, in the liver and lungs of ruminants. Epidemiological evidence strongly suggests that pVAPN-harboring strains are pathogenic in ruminants. This study aims to replicate the disease in goats through experimental infection. Intravenous administration of the pVAPN-harboring strain (Yokkaichi), pVAPA-harboring strain (ATCC33701), and pVAPN-cured strain (Yokkaichi_P-), each at 1.0 × 107 CFU/head, was conducted in 24-month-old goats (n = 1 per group). During the observation period, goats treated with Yokkaichi or ATCC33701 exhibited transient increases in body temperature and white blood cell count, alongside a decrease in body weight from the administration day. Conversely, goats treated with Yokkaichi_P- displayed no significant changes in these values. The Yokkaichi-treated goat demonstrated a >10-fold increase in anti-VapN antibody titers from 11 to 14 days postadministration, whereas the other two goats exhibited no variation in anti-VapA and VapN antibody titers. Pathological autopsy analysis of organs harvested 28 days postadministration revealed no characteristic lesions on gross examination. However, the inoculated strain (vapN-positive R. equi) was exclusively recovered from the tracheobronchial lymph node in the Yokkaichi-treated goat. Immunohistochemistry detected a VapN-positive reaction in the tracheobronchial lymph node, confirming latent infection despite the absence of dramatic suppurative lesions seen in ruminants. Overall, this study highlights the latent infection in lymph nodes induced by the pVAPN-harboring strain, despite the absence of overt pathological manifestations.

Isolation of vapB-positive Rhodococcus equi from submaxillary lymph nodes with or without granulomatous lesions in growing-finishing pigs in Japan.

To investigate the etiological role of vapB-positive Rhodococcus equi in pigs, R. equi was isolated from the submaxillary lymph nodes with or without macroscopically detectable lesions of apparently healthy growing-finishing pigs at a slaughterhouse in Toyama Prefecture, Japan. R. equi was isolated from 57 (24.6%) of 232 pigs with macroscopically detectable lymph node lesions, and 56 (98.2%) of the 57 isolates were vapB-positive. R. equi was isolated from 10 (2.4%) of 420 pigs without lymph node lesions, and six (60%) of the 10 isolates were vapB-positive. Plasmid DNA was isolated from the 62 vapB-positive isolates and digested with EcoRI and NsiI to obtain the plasmid profile. Fifty-two (83.9%), three (4.8%), and four (6.5%) isolates contained pVAPB subtypes 1, 2, and 3, respectively, while the remaining three isolates were of pVAPB subtypes 9, 13, and 14, respectively. Twelve specimens from lymph nodes with macroscopically detectable lesions were randomly selected for histopathological staining. Granulomatous lesions resembling tuberculosis were found in 11 of the 12 specimens, and the remaining specimen showed typical foci of malakoplakia in the lymph node. The isolation rates of R. equi and vapB-positive R. equi from lymph nodes with macroscopically detectable lesions were significantly higher (P<0.05) than those of lymph nodes without lesions, suggesting an etiologic association between vapB-positive R. equi and macroscopically detectable granulomatous lesions in porcine submaxillary lymph nodes. Previous reports on the prevalence of vapB-positive R. equi in pigs are reviewed and discussed.

Evaluation of Alacepril Administration in Canine Patent Ductus Arteriosus According to Plasma Chymase Activity.

Chymase in the renin-angiotensin system (RAS) actively contributes to cardiac disease progression. Chymase is activated to produce angiotensin II during tissue injury and is involved in hemodynamics. A recent study demonstrated that plasma chymase activity reflects hemodynamic changes and aids in understanding patent ductus arteriosus (PDA) pathophysiology. The present study examined the relationship between plasma chymase activity and the administration of angiotensin-converting enzyme (ACE) inhibitor. Alacepril was administered to 13 puppies with PDA. Conventional echocardiographic parameters and non-invasive blood pressure were measured before and after medication. Plasma chymase activity was calculated using the colorimetric absorbance method. Plasma chymase activity significantly increased, but blood pressure significantly decreased. We detected an increase in plasma chymase activity due to ACE inhibition in PDA cases treated with alacepril. Plasma chymase activity was affected and altered by alacepril. In veterinary medicine, plasma chymase activity may be a novel method for assessing the pathology of and therapy for cardiac diseases.

[Rhodococcus equi infections in humans: an emerging zoonotic pathogen].

Rhodococcus equi is a facultative intracellular gram-positive coccobacillus which is a well-known cause of foal pneumonia and/or enteritis in equine veterinary medicine. More than 300 cases of R. equi infection have been reported since the first description of human disease in 1968. Most patients who become infected with R equi are immunocompromised, such as those infected with human immunodeficiency virus (HIV), recipients of organ transplantation, and patients receiving cancer treatment. However, there are increasing reports of the immunocompetent hosts. The pathogenicity of R. equi has been attributed to the presence of plasmid-encoded virulence-associated proteins (Vap). To date, three host-associated virulence plasmid types of R. equi have been identified as follows: the circular pVAPA and pVAPB, related, respectively, to equine and porcine isolates in 1991 and 1995, and a recently described linear pVAPN plasmid associated with bovine and caprine strains in 2015. More recently, these three plasmid types have been re-found in the human isolates which were isolated during 1980s to 1990s. Not only horses, but also pigs, goats, cattle and their environment should be considered as a potential source of R. equi for humans. In this review, we shed light on the current understanding of R. equi as an emerging zoonotic pathogen.

Isolation and genetic characterization of Staphylococcus aureus from wild animal feces and game meats.

The populations of Japanese deer and boar have increased dramatically and have a serious impact on farming and mountain villages. Although the Japanese government promotes the use of captured wild animals, game meat is not subject to sanitary control considering that it is not subject to meat inspection or quality control. Here, we have attempted to isolate Staphylococcus aureus, a typical foodborne pathogen, as a part of an investigation of contamination in the meats of wild animals and their processing stages. We examined 390 samples of deer feces, 117 samples of wild boar feces, and 75 samples of disemboweled deer meat for isolation of S. aureus; ultimately, 30 (positive rate: 7.7%), 2 (1.7%), and 21 (28.0%) strains were isolated, respectively, from the samples. The genome sequences of these isolates were analyzed and were subjected to multilocus sequence typing. We identified 12 new sequence types (STs) and a dominant population of S. aureus with a characteristic genetic background in wild animals, namely, the ST groups derived from CC121 (number of strains = 39). These strains did not harbor the enterotoxin gene or only harbored egc-related enterotoxin, which is of low involvement in Staphylococcal food poisoning. However, one ST2449 strain, which produces causative enterotoxins, was isolated from a deer's feces. Since there are several common STs isolated from feces and dismembered meat and because fecal contamination during dismemberment is suspected, continuous monitoring and guidance for improving sanitary management conditions during processing and handling of the meat are highly warranted with immediate effect.

Short review: Geographical distribution of equine-associated pVAPA plasmids in Rhodococcus equi in the world.

Virulent Rhodococcus equi strains expressing virulence-associated 15-17 kDa protein (VapA) and having a large virulence plasmid (pVAPA) of 85-90 kb containing vapA gene are pathogenic for horses. In the last two decades, following pVAPA, two host-associated virulence plasmid types of R. equi have been discovered: a circular plasmid, pVAPB, associated with porcine isolates in 1995, and a recently detected linear plasmid, pVAPN, related to bovine and caprine isolates. Molecular epidemiological studies of R. equi infection in foals on horse-breeding farms in Japan and many countries around the world have been conducted in the last three decades, and the epidemiological studies using restriction enzyme digestion patterns of plasmid DNAs from virulent isolates have shown 14 distinct pVAPA subtypes and their geographical preference. This short review summarizes previous reports regarding equine-associated pVAPA subtypes in the world and discusses their geographic distribution from the standpoint of horse movements.

Zinc Acetate Hydrate Supplementation versus Polaprezinc Supplementation for Improving Hypozincemia in Hemodialysis Patients: A Randomized Clinical Trial.

Zinc supplementation may ameliorate zinc deficiency in maintenance hemodialysis patients; however, no standard protocol has been established. This study aimed to investigate the effects of zinc acetate hydrate (ZAH) and polaprezinc (PPZ) as zinc supplements in hemodialysis patients. We enrolled 75 hemodialysis patients with serum zinc levels <60 µg/dL for this study and randomly assigned Zinc supplementation to these 75 patients: 37 received ZAH (50 mg/day), and 38 received PPZ (34 mg/day). Serum zinc levels of both groups were compared every 4 weeks for 1 year. In both groups, serum zinc levels significantly increased at 4-52 weeks. Serum zinc levels were significantly higher in the ZAH group at 4-12 weeks; however, no significant differences were observed between the groups at 16-52 weeks. Adverse events requiring a reduction in the zinc dose, including copper deficiency, occurred significantly more frequently in the ZAH group. In conclusion, PPZ can safely maintain serum zinc levels for 1 year. ZAH provides rapid zinc supplementation but can cause adverse events.

Effects of Angiotensin Receptor Blockers on Streptozotocin-Induced Diabetic Cataracts.

This study aimed to determine the role of oxidative stress produced by the renin-angiotensin system (RAS) in cataract formation in streptozotocin-induced diabetic rats (STZ) using angiotensin II receptor blockers (ARBs). Rats were treated with streptozotocin and orally administered candesartan (2.5 mg/kg/day) or a normal diet for 10 weeks until sacrifice. Cataract progression was assessed through a slit-lamp examination. Animals were euthanized at 18 weeks, and the degree of cataract progression was evaluated. Oxidative stress was also assessed. In STZ-treated rats, lens opacity occurred at 12 weeks. Cataract progression was inhibited in the ARB-treated group compared with the placebo group (p < 0.05). STZ-treated rats exhibited upregulated angiotensin-converting enzyme (ACE) gene expression than control rats. Oxidative stress-related factors were upregulated in the placebo-treated group but suppressed in the ARB-treated group. A correlation coefficient test revealed a positive correlation between ACE gene expression and oxidative stress-related factors and a negative correlation between ACE and superoxide dismutase. Immunostaining revealed oxidative stress-related factors and advanced glycation end products in the lens cortex of the placebo-treated group. The mechanism of diabetic cataracts may be related to RAS, and the increase in focal ACE and angiotensin II in the lens promotes oxidative stress-related factor production.

Development of monoclonal antibodies against Rhodococcus equi virulence-associated protein N and their application to pathological diagnosis.

IMPORTANCE: Rhodococcus equi can cause infection in ruminants, and its pathogenicity is suggested to be associated with VapN. Despite its wide distribution, no immunological diagnostic method has been developed for VapN-producing R. equi. Against this background, we attempted to develop monoclonal antibodies targeting VapN and assess their application in immunostaining. In the study, mice were immunized with recombinant VapN, and cell fusion and cloning by limiting dilution permitted the generation of three antibody-producing hybridomas. The utility of the antibodies produced from the hybridomas in immunostaining was demonstrated using an infected mouse model, and the antibodies were further applied to previously reported cases of R. equi infection in goats and cattle. Although the 4H4 antibody induced the strongest reactions, the reactivity of two other antibodies was improved by antigen retrieval. Our monoclonal antibodies will be utilized to support the definitive diagnosis of suspected R. equi infection, including cases that were previously missed.

Tryptase-Positive Mast Cells Promote Adipose Fibrosis in Secondary Lymphedema through PDGF.

Lymphedema is a chronic and progressive condition that causes physical disfigurement and psychological trauma due to the accumulation of lymphatic fluid in the interstitial space. Once it develops, lymphedema is difficult to treat because it leads to the fibrosis of adipose tissue. However, the mechanism behind this remains unclear. The purpose of this study was to investigate the involvement of mast cells (MCs) in the adipose tissues of patients with lymphedema. We found that fibrosis spread through blood vessels in the adipose tissues of lymphedema patients, and the expression of the collagen I and III genes was significantly increased compared to that of those in normal adipose tissue. Immunostaining of vimentin and α-smooth muscle actin showed that fibroblasts were the main cellular components in severely fibrotic regions. Toluidine blue staining confirmed a significant increase in the number of MCs in the adipose tissues of lymphedema patients, and immunostaining of serial sections of adipose tissue showed a significant increase in the number of tryptase-positive cells in lymphedema tissues compared with those in normal adipose tissues. Linear regression analyses revealed significant positive correlations between tryptase and the expressions of the TNF-α, platelet-derived growth factor (PDGF)-A, and PDGFR-α genes. PDGF-A-positive staining was observed in both fibroblasts and granules of tryptase-positive MCs. These results suggest that MC-derived tryptase plays a role in the fibrosis of adipose tissue due to lymphedema directly or in cooperation with other mediators.

Involvement of Glycogen Synthase Kinase 3ß (GSK3ß) in Formation of Phosphorylated Tau and Death of Retinal Ganglion Cells of Rats Caused by Optic Nerve Crush.

Tauopathy is a neurodegenerative condition associated with oligomeric tau formation through abnormal phosphorylation. We previously showed that tauopathy is involved in death of retinal ganglion cells (RGCs) after optic nerve crush (ONC). It has been proposed that glycogen synthase kinase 3ß (GSK3ß) is involved in the hyperphosphorylation of tau in Alzheimer's disease. To determine the roles of GSK3ß in tauopathy-related death of RGCs, lithium chloride (LiCl), a GSK3ß inhibitor, was injected intravitreally just after ONC. The neuroprotective effects of LiCl were determined by counting Tuj-1-stained RGCs on day 7. Changes of phosphorylated (ser 396) tau in the retina were determined by Simple Western analysis (WES) on day 3. Retinal GSK3ß levels were determined by immunohistochemistry (IHC) and an ELISA. There was a 1.9- and 2.1-fold increase in the levels of phosphorylated tau monomers and dimers on day 3 after ONC. LiCl significantly suppressed the increase in the levels of phosphorylated tau induced by ONC. GSK3ß was mainly present in somas of RGCs, and ELISA showed that retinal levels increased to 2.0-fold on day 7. IHC showed that the GSK3ß expression increased over time and remained in RGCs that were poorly stained by Tuj-1. The GSK3ß and tau expression was colocalized in RGCs. The number of RGCs decreased from 1881 ± 188 (sham control) to 1150 ± 192 cells/mm2 on day 7, and LiCl preserved the levels at 1548 ± 173 cells/mm2. Accordingly, GSK3ß may be a promising target for some optic nerve injuries.

Increased Chymase-Positive Mast Cells in High-Grade Mucoepidermoid Carcinoma of the Parotid Gland.

It has long been known that high-grade mucoepidermoid carcinoma (MEC) has a poor prognosis, but the detailed molecular and biological mechanisms underlying this are not fully understood. In the present study, the pattern of chymase-positive mast cells, as well as chymase gene expression, in high-grade MEC was compared to that of low-grade and intermediate-grade MEC by using 44 resected tumor samples of MEC of the parotid gland. Chymase expression, as well as chymase-positive mast cells, was found to be markedly increased in high-grade MEC. Significant increases in PCNA-positive cells and VEGF gene expression, as well as lymphangiogenesis, were also confirmed in high-grade MEC. Chymase substrates, such as the latent transforming growth factor-beta (TGF-ß) 1 and pro-matrix metalloproteinase (MMP)-9, were also detected immunohistologically in high-grade MEC. These findings suggested that the increased chymase activity may increase proliferative activity, as well as metastasis in the malignant condition, and the inhibition of chymase may be a strategy to improve the poor prognosis of high-grade MEC of the parotid gland.

Chymase Inhibition Resolves and Prevents Deep Vein Thrombosis Without Increasing Bleeding Time in the Mouse Model.

Background Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life-threatening cardiovascular disease in North America. Post-DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT. It has recently been shown that mast cells play a crucial role in a DVT murine model. The underlying mechanism involved in the prothrombotic properties of mast cells, however, has yet to be identified. Methods and Results C57BL/6 mice and mouse mast cell protease-4 (mMCP-4) genetically depleted mice (mMCP-4 knockout) were used in 2 mouse models of DVT, partial ligation (stenosis) and ferric chloride-endothelial injury model of the inferior vena cava. Thrombus formation and impact of genetically repressed or pharmacologically (specific inhibitor TY-51469) inhibited mMCP-4 were evaluated by morphometric measurements of thrombi immunochemistry (mouse and human DVT), color Doppler ultrasound, bleeding times, and enzymatic activity assays ex vivo. Recombinant chymases, mMCP-4 (mouse) and CMA-1 (human), were used to characterize the interaction with murine and human plasmin, respectively, by mass spectrometry and enzymatic activity assays. Inhibiting mast cell-generated mMCP-4, genetically or pharmacologically, resolves and prevents venous thrombus formation in both DVT models. Inferior vena cava blood flow obstruction was observed in the stenosis model after 6 hours of ligation, in control- but not in TY-51469-treated mice. In addition, chymase inhibition had no impact on bleeding times of healthy or DVT mice. Furthermore, endogenous chymase limits plasmin activity in thrombi ex vivo. Recombinant mouse or human chymase degrades/inactivates purified plasmin in vitro. Finally, mast cell-containing immunoreactive chymase was identified in human DVT. Conclusions This study identified a major role for mMCP-4, a granule-localized protease of chymase type, in DVT formation. These findings support a novel pharmacological strategy to resolve or prevent DVT without affecting the coagulation cascade through the inhibition of chymase activity.

Plasma Chymase Activity Reflects the Change in Hemodynamics Observed after the Surgical Treatment of Patent Ductus Arteriosus in Dogs.

Chymase is a protease stored in mast cell granules that produces angiotensin II (ANG II) from angiotensin I (ANG I) and is associated with tissue injury, inflammation, and remodeling, especially involving the cardiovascular system. As cardiovascular events occur, chymase is activated by degranulation to the extracellular matrix. Although chymase has been suggested to be associated with cardiovascular disease progression, there are not enough reports in veterinary medicine. Patent ductus arteriosus (PDA) is a common congenital cardiac disease in veterinary medicine. Almost all cases of PDA can be treated surgically to prevent the development of congestive heart disease and/or pulmonary hypertension. The aims of the present study were to measure chymase activity before and after PDA occlusions, and to investigate the relationships between the congestive and hemodynamic states of PDA and chymase activity. In the present study, 17 puppies diagnosed with PDA were included and all puppies completely recovered to the level of healthy dogs. Chymase activity significantly decreased at 2 months after the operation, along with the echocardiography parameters of congestion. Therefore, plasma chymase activity may be useful as a novel predictor for understanding the hemodynamics of PDA in veterinary medicine.

Pathophysiological Role of Chymase-Activated Matrix Metalloproteinase-9.

Chymase present in mast cells can directly form matrix metalloproteinase (MMP)-9 from proMMP-9. Chymase-activated MMP-9 has been reportedly closely related to the pathogenesis of various diseases, and inflammation-related diseases in particular. Upregulated chymase and MMP-9 have been observed in tissues from patients and animal models of aortic aneurysm, inflammatory gastrointestinal and hepatic diseases, acute pancreatic failure, atopic dermatitis and rheumatoid arthritis. Chymase at these regions is only derived from mast cells, while MMP-9 is derived from macrophages and neutrophils in addition to mast cells. Chymase inhibitors attenuate MMP-9 formation from pro-MMP-9, and ameliorate the development and progression of these disorders, along with reduction in inflammatory cell numbers. MMP-9 activated by chymase might also be involved in angiogenesis in the tumor environment. Development of angiogenesis around several cancers is closely related to the expression of chymase and MMP-9, and postoperative survival curves have revealed that patients with a higher number of chymase positive cells have lower survival rates. In this review, we wanted to clarify the role of chymase-activated MMP-9, which might become an important therapeutic target for various inflammatory disorders.

Guidelines on the hygienic management of wild meat in Japan.

The density and distribution of wild ungulates, especially sika deer (Cervus nippon) and wild boar (Sus scrofa), have increased across the Japanese archipelago in the last two decades. The tradition of consuming wild game meat has been inherited in limited areas in Japan, but recently, the use of wild animals for food has increased. Game meat has become popular at local restaurants and retail meat shops. However, fundamental knowledge of game meat hygiene and health risks has not been fully established among hunters, meat processors, restaurant operators, and consumers. Moreover, game meat-borne illnesses have been reported occasionally. Unlike domesticated livestock, wild animals are not inspected for diseases when being butchered for food, and the meat obtained is at a high risk of being unhygienic. The Guidelines on the hygienic management of wild meat were established in 2014 to ensure the safety of wild meat in Japan. This study describes the situation regarding wild animals and game meat in Japan.

Downregulation of miR-122-5p Activates Glycolysis via PKM2 in Kupffer Cells of Rat and Mouse Models of Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) has pathological characteristics similar to those of alcoholic hepatitis, despite the absence of a drinking history. The greatest threat associated with NASH is its progression to cirrhosis and hepatocellular carcinoma. The pathophysiology of NASH is not fully understood to date. In this study, we investigated the pathophysiology of NASH from the perspective of glycolysis and the Warburg effect, with a particular focus on microRNA regulation in liver-specific macrophages, also known as Kupffer cells. We established NASH rat and mouse models and evaluated various parameters including the liver-to-body weight ratio, blood indexes, and histopathology. A quantitative phosphoproteomic analysis of the NASH rat model livers revealed the activation of glycolysis. Western blotting and immunohistochemistry results indicated that the expression of pyruvate kinase muscle 2 (PKM2), a rate-limiting enzyme of glycolysis, was upregulated in the liver tissues of both NASH models. Moreover, increases in PKM2 and p-PKM2 were observed in the early phase of NASH. These observations were partially induced by the downregulation of microRNA122-5p (miR-122-5p) and occurred particularly in the Kupffer cells. Our results suggest that the activation of glycolysis in Kupffer cells during NASH was partially induced by the upregulation of PKM2 via miR-122-5p suppression.