Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration.

Role of inter-alpha-trypsin inhibitor heavy chain 4 and its citrullinated form in experimental arthritis murine models.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is a major protein in serum and reported to be upregulated at the onset of rheumatoid arthritis (RA). Its citrullinated form, cit-ITIH4, is specifically found in the serum and synovial fluid of patients with RA. However, the detailed function of ITIH4 in arthritis remains unknown. The aim of this study was to clarify the role of ITIH4 and cit-ITIH4 using experimental arthritis models. ITIH4 and cit-ITIH4 expression was examined in steady-state mice and two different arthritis models, and their pathological effects were examined in Itih4-deficient mice. In naïve C57BL/6 (WT) mice, ITIH4 was expressed as mRNA in the liver and the lung and was expressed as protein in serum and hepatocytes. In K/BxN serum transferred arthritis (K/BxN-STA) and collagen-induced arthritis (CIA), ITIH4 and cit-ITIH4 in sera were increased before the onset of arthritis, and cit-ITIH4 was further increased at the peak of arthritis. In Itih4-deficient mice, citrullinated proteins in serum and joints, especially 120 kDa protein, were clearly diminished; however, there was no significant difference in arthritis severity between WT and itih-/- mice either in the K/BxN-STA or CIA model. CIA mice also exhibited pulmonary lesions and itih4-/- mice tended to show enhanced inflammatory cell aggregation compared to WT mice. Neutrophils in the lungs of itih4-/- mice were significantly increased compared to WT mice. In summary, ITIH4 itself did not alter the severity of arthritis but may inhibit autoimmune inflammation via suppression of neutrophil recruitment.

Renal Function in Aged C57BL/6J Mice Is Impaired by Deposition of Age-Related Apolipoprotein A-II Amyloid Independent of Kidney Aging.

Spontaneous and age-related amyloidosis has been reported in C57BL/6J mice. However, the biochemical characteristics of age-related amyloidosis remain unclear. Herein, the age-related prevalence of amyloidosis, the types of amyloid fibril proteins, and the effects of amyloid deposition were investigated in renal function in C57BL/6J mice. The results obtained revealed a high incidence of amyloidosis in C57BL/6J mice originating from The Jackson Laboratory as well as the deposition of large amounts of amyloid in the glomeruli of aged mice. The amyloid fibril protein was identified as wild-type apolipoprotein A-II (ApoA-II). Induction of amyloid deposition in 40-week-old mice, equivalent to that of spontaneous development in 80-week-old mice, to rule out the effects of aging, revealed subsequent damage to kidney function by amyloid deposits. Furthermore, amyloid deposition in the mesangial region decreased podocyte density, compromised foot processes, and led to the accumulation of fibroblast growth factor 2 in glomeruli. Collectively, these results suggest that ApoA-II deposition is a general pathology in aged C57BL/6J mice and is dependent on supplier colonies. Therefore, the effects of age-related amyloid deposition need to be considered in research on aging in mice.

Effects of heated tobacco product aerosol extracts on DNA methylation and gene transcription in lung epithelial cells.

AIMS: Smoking causes DNA methylation (DNAm) alterations that lead to lung cancer development. Although the use of heated tobacco products (HTPs) has recently increased, their impact on health remains unclear. This study aimed to evaluate the effects of HTPs on DNAm and gene transcription in human lung epithelial cells in vitro. MAIN METHODS: Human lung adenocarcinoma (A549) cells with type II alveolar epithelial characteristics were treated with aerosol extracts of two HTPs or a smoke extract of combustible reference cigarette (RC). Global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels were quantified using dot blot analysis. Furthermore, reduced representation of bisulfite sequencing, DNA microarray, and quantitative PCR analyses were performed to determine CpG methylation and gene transcription changes induced by HTP and RC. KEY FINDINGS: Global 5-mC and 5-hmC levels were decreased by the RC extract but not the HTP extracts. However, an HTP extract altered the CpG methylation pattern, and Gene Ontology enrichment analysis of the differentially methylated regions of the RC and HTP groups showed a similar pattern. The HTP extract affected gene expression, albeit to a lesser extent than the RC extract. In particular, the HTP extract markedly affected the mRNA expression and promoter methylation of cytochrome P450 family 1 subfamily A member 1 (CYP1A1), which is associated with carcinogenic risk. SIGNIFICANCE: The study results suggest that HTPs as well as conventional combustible cigarettes can alter CpG methylation and gene transcription in lung epithelial cells.

Accuracy of Therapeutic Drug Monitoring of Teicoplanin at the Onset of Febrile Neutropenia.

Background and Objectives: Teicoplanin (TEIC) is an effective drug for patients with febrile neutropenia (FN); however, it has been reported that these patients may have increased TEIC clearance compared with patients who do not have FN. The purpose of this study was to study therapeutic drug monitoring in patients with FN when the TEIC dosing design was based on the population mean method. Materials and Methods: Thirty-nine FN patients with hematological malignancy were included in the study. To calculate the predicted blood concentration of TEIC, we used the two population pharmacokinetic (population PK) parameters (parameters 1 and 2) reported by Nakayama et al. and parameter 3, which is a modification of the population PK of Nakayama et al. We calculated the mean prediction error (ME), an indicator of prediction bias, and the mean absolute prediction error (MAE), an indicator of accuracy. Furthermore, the percentage of predicted TEIC blood concentration within 25% and 50% of the measured TEIC blood concentration was calculated. Results: The ME values were -0.54, -0.25, and -0.30 and the MAE values were 2.29, 2.19, and 2.22 for parameters 1, 2, and 3, respectively. For all of the three parameters, the ME values were calculated as minus values, and the predicted concentrations tended to be biased toward smaller values relative to the measured concentrations. Patients with serum creatinine (Scr) < 0.6 mg/dL and neutrophil counts < 100/µL had greater ME and MAE values and a smaller percentage of predicted TEIC blood concentration within 25% of measured TEIC blood concentrations compared with other patients. Conclusions: In patients with FN, the accuracy of predicting TEIC blood concentrations was good, with no significant differences between each parameter. However, patients with a Scr < 0.6 mg/dL and a neutrophil count < 100/µL showed slightly inferior prediction accuracy.

Determination of tissue-specific interaction between vitamin C and vitamin E in vivo using senescence marker protein-30 knockout mice as a vitamin C synthesis deficiency model.

Vitamin E (α-tocopherol; VE) is known to be regenerated from VE radicals by vitamin C (L-ascorbic acid; VC) in vitro. However, their in vivo interaction in various tissues is still unclear. Therefore, we alternatively examined the in vivo interaction of VC and VE by measurement of their concentrations in various tissues of senescence marker protein-30 (SMP30) knockout (KO) mice as a VC synthesis deficiency model. Male SMP30-KO mice were divided into four groups (VC+/VE+, VC+/VE-, VC-/VE+ and VC-/VE-), fed diets with or without 500 mg/kg VE and given water with or without 1·5 g/l VC ad libitum. Then, VC and VE concentrations in the plasma and various tissues were determined. Further, gene expression levels of transporters associated with VC and VE, such as α-tocopherol transfer protein (α-TTP) and sodium-dependent vitamin C transporters (SVCTs), were examined. These results showed that the VE levels in the VC-depleted (VC-/VE+) group were significantly lower than those in the VC+/VE+ group in the liver and heart; the VC levels in the VE-depleted (VC+/VE-) group were significantly lower than those in the VC+/VE+ group in the kidneys. The α-TTP gene expression in the liver and kidneys was decreased by VC and/or VE depletion. Moreover, SVCT1 gene expression in the liver was decreased by both VC and VE depletion. In conclusion, these results indicate that VC spares VE mainly in the liver and heart and that VE spares VC in the kidneys of SMP30-KO mice. Thus, interaction between VC and VE is likely to be tissue specific.

Specific Increase in Joint Neutrophil Extracellular Traps and Its Relation to Interleukin 6 in Autoimmune Arthritis.

Neutrophils and their extracellular traps have been shown to play an important role in the pathogenesis of rheumatoid arthritis (RA), but the detailed mechanisms in joints are still unclear, and their regulation remains to be solved. Here, we explored neutrophil extracellular trap (NET)osis in experimental models of arthritis and further investigated the effects of interleukin-6 (IL-6) inhibition in neutrophils and NETosis. In skins of peptide GPI-induced arthritis (pGIA), citrullinated protein was detected as well as citrullinated histone expression in immunized skin but this was not specific to pGIA. Citrullinated histone expression in pGIA joints was specific to pGIA and was merged with neutrophil elastase, suggesting NETosis. Neutrophils in joints tend to upregulate IL-6 receptors when compared with bone marrow neutrophils. Administration of mouse anti-IL-6 receptor antibodies in pGIA suppressed arthritis in association with a decrease in neutrophil infiltration and NETosis in joints. In the plasma of RA patients, citrullinated protein was significantly reduced after tocilizumab treatment. Our results suggest that IL-6 enhances neutrophil chemotaxis and NETosis in inflammatory joints and could be the source of citrullinated proteins.

Neuroprotective and Anti-Inflammatory Effects of Evernic Acid in an MPTP-Induced Parkinson's Disease Model.

Oxidative stress, mitochondrial dysfunction, and neuroinflammation are strongly associated with the pathogenesis of Parkinson's disease (PD), which suggests that anti-oxidative and anti-inflammatory compounds might provide an alternative treatment for PD. Here, we evaluated the neuroprotective effects of evernic aid (EA), which was screened from a lichen library provided by the Korean Lichen Research Institute at Sunchon National University. EA is a secondary metabolite generated by lichens, including Ramalina, Evernia, and Hypogymnia, and several studies have described its anticancer, antifungal, and antimicrobial effects. However, the neuroprotective effects of EA have not been studied. We found that EA protected primary cultured neurons against 1-methyl-4-phenylpyridium (MPP+)-induced cell death, mitochondrial dysfunction, and oxidative stress, and effectively reduced MPP+-induced astroglial activation by inhibiting the NF-κB pathway. In vivo, EA ameliorated MPTP-induced motor dysfunction, dopaminergic neuronal loss, and neuroinflammation in the nigrostriatal pathway in C57BL/6 mice. Taken together, our findings demonstrate that EA has neuroprotective and anti-inflammatory effects in PD models and suggest that EA is a potential therapeutic candidate for PD.

Age-related dysfunction of p53-regulated phagocytic activity in macrophages.

Aging promotes polarization of M2-like macrophages to M1-like macrophages and reduces their phagocytic ability. However, the molecular mechanisms underlying these aging-related changes remain poorly understood. Here, we demonstrate that p53 regulates phagocytic activity in macrophages from young mice but not in those from old ones. Macrophages from both old and young mice expressed functional p53 to induce target genes including p21 and Mdm2. In macrophages from young mice, chemically induced p53 decreased phagocytic activity and c-Myc levels, with the latter change reducing M2-related genes. However, in macrophages from old mice, phagocytic activity and c-Myc expression were independent of p53 activity. Furthermore, c-Myc suppression did not affect M2-related genes in old-mouse macrophages. These results demonstrate that dysregulation of p53 function is a molecular mechanism underlying reduced phagocytic activity in aged-mouse macrophages.

Vitamin C and vitamin E double-deficiency increased neuroinflammation and impaired conditioned fear memory.

BACKGROUND: Vitamin C (l-ascorbic acid, VC) and vitamin E (α-tocopherol, VE) play important physiological roles as endogenous antioxidants in many tissues and organs. However, their roles in the brain remain entirely elusive. We established senescence marker protein 30 (SMP30)/α-tocopherol transfer protein (αTTP) double knockout (DKO) mice as a novel VC and VE double-deficiency model and examined the effect of VC and VE double-deficiency on brain functions. METHODS: DKO and wild-type (WT) mice were divided into the following two groups: mice in the CE (+) group were supplied with sufficient amounts of VC and VE and mice in the CE (-) group were deficient in both VC and VE. After 8 weeks of CE (+) or CE (-) treatments, a battery of behavioral experiments was conducted to analyze cognitive functions, including memory, through the Morris water maze and Pavlovian fear conditioning tasks. RESULTS: The plasma VC and VE levels in DKO-CE (-) mice and VE level in WT-CE (-) mice were almost completely depleted after 8 weeks of the deficient treatment. The behavioral study revealed that the general behaviors, including locomotor activity and anxiety level, were not influenced by the CE (-) treatment in DKO and WT mice. However, in the Pavlovian fear conditioning task, DKO-CE (-) mice showed impaired conditioned fear memory compared with that of DKO-CE (+) mice. Furthermore, increased mRNA expression was observed in inflammatory-related genes, such as IL-6, TNFα, F4/80, and Mcp-1, in the hippocampus of DKO-CE (-) mice. CONCLUSIONS: The findings of this study provide evidence that VC and VE deficiency led to impaired conditioned fear memory possibly caused by neuroinflammation in the brain.

High dietary vitamin C intake reduces glucocorticoid-induced immunosuppression and measures of oxidative stress in vitamin C-deficient senescence marker protein 30 knockout mice.

Vitamin C (VC) is a vital micronutrient for humans and some other mammals and also has antioxidant activity. Stress-induced elevation of glucocorticoid production is well known to cause immunosuppression. The present study evaluated the effect of high VC intake on glucocorticoid-induced immune changes in mice. Senescence marker protein 30 knockout mice with genetic VC deficiency were fed a diet containing the recommended VC content (20 mg/kg per d; 0·02 %VC group) or a high VC content (200 mg/kg per d; 0·2 %VC group) for 2 months, then dexamethasone was given by intraperitoneal injection. After administration of dexamethasone, the plasma ascorbic acid concentration decreased significantly in the 0·02 %VC group and was unchanged in wild-type C57BL/6 mice on a VC-deficient diet (wild-type group), while it was significantly higher in the 0·2 %VC group compared with the other two groups. In the 0·02 %VC and wild-type groups, dexamethasone caused a significant decrease in the cluster of differentiation (CD)4+ and CD8+ T cells among splenocytes as well as a significant decrease in IL-2, IL-12p40 and interferon-γ protein production by splenocytes and a significant decrease in T-cell proliferation among splenocytes. In the 0·2 %VC group, these dexamethasone-induced immunosuppression improved when compared with the other two groups. In addition, reduction in the intracellular levels of ascorbic acid, superoxide dismutase and glutathione in splenocytes by dexamethasone as well as elevation in thiobarbituric acid-reactive substances were significantly suppressed in the 0·2 %VC group. These findings suggest that high dietary VC intake reduces glucocorticoid-induced T-cell dysfunction by maintaining intracellular antioxidant activity.

Peptidyl arginine deiminase inhibition suppresses arthritis via decreased protein citrullination in joints and serum with the downregulation of interleukin-6.

Objective: To explore the relevance of citrullinated proteins and anti-citrullinated protein antibodies (ACPA) via protein arginine deiminase (PAD) inhibition in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA).Methods: Cl-amidine, a PAD inhibitor, was injected into pGIA. Clinical scores and histopathological findings of ankle joints were assessed. Serum ACPA titers were analyzed using ELISA. Citrullinated protein expression in joints and sera were examined with immunohistochemistry and Western blot analysis, respectively. Serum levels of IL-6, TNFα, and IL-1ß were measured with cytometric bead array (CBA). Gene expression levels of IL-6 and TNFα in joints, lymph nodes, and spleens were analyzed with quantitative PCR. GPI-specific productions of IFNγ and IL-17 from T cells in lymph nodes were evaluated.Results: Cl-amidine treatment significantly reduced arthritis severity while ACPA titers tended to be lower, but not significantly different compared to the control. Citrullinated proteins in joints and sera from treated mice were clearly decreased. With Cl-amidine treatment, serum IL-6 levels were significantly decreased, and IL-6 and TNFα gene expression were significantly reduced in joints. IL-17 production from GPI-specific T cells tended to be lower in Cl-amidine-treated mice, but not significantly different.Conclusion: Our results suggested that PAD-mediated citrullinated protein was involved in the pathogenesis of arthritis via IL-6.

Age-dependent changes in vancomycin-induced nephrotoxicity in mice.

Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic that is commonly used to eradicate methicillin-resistant gram-positive cocci, despite its nephrotoxic side effects. Elderly people are particularly susceptible to developing VCM-induced nephrotoxicity. However, the precise mechanism by which VCM induces nephrotoxicity in elderly people is not completely understood. Therefore, we investigated VCM-induced nephrotoxicity in mice of different ages. VCM was injected intraperitoneally into mice at 1, 3, 6, 12, and 24 months of age at a dosage of 400 mg/kg body weight for 3 and 14 days. Twenty-four hours after the last injection, we examined plasma creatinine levels and histopathological alterations in the kidneys. VCM administration increased plasma creatinine levels, and these values gradually increased to higher levels with aging. The histological examination revealed renal tubular degeneration, such as brush-border atrophy, apoptosis/necrosis of the tubular epithelium, and epithelial desquamation, that gradually became more severe with aging. Furthermore, immunohistochemical staining with anti-CD10 and anti-single-stranded DNA antibodies revealed damaged renal proximal tubules with marked dilatation, as well as numerous apoptotic cells, and these features increased in severity in 12- and 24-month-old mice receiving VCM. Based on these results, aged mice were highly susceptible to kidney damage induced by VCM administration. In addition, proximal tubular epithelial cells likely underwent apoptosis after the administration of VCM. This report is the first to document VCM-induced nephrotoxicity in mice of different ages. Thus, this mouse model could be useful for understanding the mechanisms of VCM-induced nephrotoxicity in the elderly.

Hypoxia-induced production of peptidylarginine deiminases and citrullinated proteins in malignant glioma cells.

BACKGROUND: Recently, it has been reported that hypoxia highly enhances expression of peptidylarginine deiminase (PAD) 4 and production of citrullinated proteins in some tumor cells. However, little is known about malignant gliomas on this issue. Therefore, we here investigated whether expression of PADs was induced by hypoxia and whether PADs citrullinated intracellular proteins if induced using U-251 MG cells of a human malignant glioma cell line. METHODS: Expression of PADs in U-251 MG cells, cultured under hypoxia or normoxia for 24 h, was investigated by quantitative polymerase chain reaction (qPCR). Citrullination of proteins in the cells and the cell lysates incubated for 48 h with or without Ca2+ was detected by western blotting. Citrullinated proteins were identified by mass spectrometry. RESULTS: The mRNA levels of PAD1, 2, 3, and 4 were up-regulated by hypoxia in a hypoxia-inducible factor-1-dependent manner in U-251 MG cells. In spite of the increased expression, intracellular proteins were not citrullinated. However, the induced PADs citrullinated U-251 MG cell-derived proteins when the cells were lysed. Multiple proteins citrullinated by hypoxia-induced PADs were identified. In addition, the extracellular domain of vascular endothelial growth factor receptor 2 was citrullinated by human PAD2 in vitro. CONCLUSION: Our data may contribute to understanding of pathophysiology of malignant gliomas from the aspects of protein citrullination.

Hydrogen-rich pure water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice.

Chronic obstructive pulmonary disease (COPD) is predominantly a cigarette smoke (CS)-triggered disease with features of chronic systemic inflammation. Oxidants derived from CS can induce DNA damage and stress-induced premature cellular senescence in the respiratory system, which play significant roles in COPD. Therefore, antioxidants should provide benefits for the treatment of COPD; however, their therapeutic potential remains limited owing to the complexity of this disease. Recently, molecular hydrogen (H2) has been reported as a preventive and therapeutic antioxidant. Molecular H2 can selectively reduce hydroxyl radical accumulation with no known side effects, showing potential applications in managing oxidative stress, inflammation, apoptosis, and lipid metabolism. However, there have been no reports on the efficacy of molecular H2 in COPD patients. In the present study, we used a mouse model of COPD to investigate whether CS-induced histological damage in the lungs could be attenuated by administration of molecular H2. We administered H2-rich pure water to senescence marker protein 30 knockout (SMP30-KO) mice exposed to CS for 8 weeks. Administration of H2-rich water attenuated the CS-induced lung damage in the SMP30-KO mice and reduced the mean linear intercept and destructive index of the lungs. Moreover, H2-rich water significantly restored the static lung compliance in the CS-exposed mice compared with that in the CS-exposed H2-untreated mice. Moreover, treatment with H2-rich water decreased the levels of oxidative DNA damage markers such as phosphorylated histone H2AX and 8-hydroxy-2'-deoxyguanosine, and senescence markers such as cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 1, and ß-galactosidase in the CS-exposed mice. These results demonstrated that H2-rich pure water attenuated CS-induced emphysema in SMP30-KO mice by reducing CS-induced oxidative DNA damage and premature cell senescence in the lungs. Our study suggests that administration of molecular H2 may be a novel preventive and therapeutic strategy for COPD.

Induction of peptidylarginine deiminase 2 and 3 by dibutyryl cAMP via cAMP-PKA signaling in human astrocytoma U-251MG cells.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium-dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U-251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U-251MG cells. The addition of dbcAMP in a dose-dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose-dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP-dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP-induced PAD2 and PAD3 mRNA is mediated by the cAMP-PKA signaling pathway in U-251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP-PKA signaling pathway in U-251MG cells. © 2016 Wiley Periodicals, Inc.

Carbamylated albumin is one of the target antigens of anti-carbamylated protein antibodies.

Objectives: Anti-carbamylated protein (anti-CarP) antibodies are detected in RA patients. Fetal calf serum is used as an antigen source in anti-CarP ELISA, and the precise target antigens have not been found. We aimed to identify the target antigens of anti-CarP antibodies. Methods: Western blotting of anti-CarP antibodies was conducted. Anti-carbamylated human albumin (CarALB) antibody was detected by in-house ELISA for 493 RA patients and 144 healthy controls (HCs). An inhibition ELISA of anti-CarP antibodies by CarALB and citrullinated albumin (citALB) was performed using eight RA patients' sera. Serum CarALB was detected by liquid chromatography-tandem mass spectroscopy (LC/MS/MS), and the serum MPO concentration was measured by ELISA. Results: We focused on carbamylated albumin because it corresponded to the size of the thickest band detected by western blotting of anti-CarP antibodies. Anti-CarALB antibody was detected in 31.4% of RA patients, and the correlation of the titres between anti-CarALB and anti-CarP was much closer than that between anti-citALB and anti-CCP antibodies (ρ = 0.59 and ρ = 0.16, respectively). The inhibition ELISA showed that anti-CarP antibodies were inhibited by CarALB, but not by citALB. CarALB was detected in sera from RA patients by LC/MS/MS. The serum MPO concentration was correlated with disease activity and was higher in RA patients with anti-CarALB antibody than in those without. Conclusion: We found that carbamylated albumin is a novel target antigen of anti-CarP antibodies, and it is the first reported target antigen that has not been reported as the target of ACPA.

Abnormal lipid/lipoprotein metabolism and high plasma testosterone levels in male but not female aromatase-knockout mice.

Sex steroid hormones, such as estrogen and testosterone, are believed to play important roles in lipid metabolism. To elucidate the effects of estrogen depletion on lipid metabolism in male and female mice, we used aromatase-knockout (ArKO) mice, in which Cyp19 gene disruption prevented estrogen synthesis in vivo. These mice were divided into the following 4 groups: male and female ArKO mice and male and female wild-type (WT) mice. These mice were fed a normal-fat diet (13.6% fat) ad libitum. At 159 days after birth, the mice were tested for liver and plasma lipid content and hepatic hormone receptor- and lipid/lipoprotein metabolism-related gene expression. Interestingly, we found that hepatic steatosis was accompanied by markedly elevated plasma testosterone levels in male ArKO mice but not in female ArKO mice. Plasma lipoprotein profiles exhibited concurrent decreases in LDL- and small dense LDL-triglyceride (TG) levels in male ArKO mice. Moreover, male mice, but not female mice, exhibited marked elevations in androgen receptor (AR), sterol regulatory element-binding protein 1 (SREBP1), and CD36 expression. These results strongly suggest that Cyp19 gene disruption, which induces a sexually dimorphic response and high plasma testosterone levels in male mice, also induces hepatic steatosis.

Age-related changes of vitamin E: α-tocopherol levels in plasma and various tissues of mice and hepatic α-tocopherol transfer protein.

PURPOSE: Despite numerous studies on the RRR- and all-rac-α-tocopherol isoform of vitamin E (VE) during aging, this relationship has not been examined in specific tissues. Since α-tocopherol is the most abundant of VE's eight isoforms, and VE is an important antioxidant that impacts the aging process, we analyzed α-tocopherol levels in plasma and tissues of mice at progressive ages. Moreover, we examined protein and mRNA expression levels of hepatic α-tocopherol transfer protein (α-TTP), which specifically binds α-tocopherol, during aging. METHODS: The α-tocopherol levels in plasma, liver, cerebrum, hippocampus, cerebellum, heart, kidney, epididymal adipose tissue, testis, pancreas, soleus muscle, plantaris muscle, and duodenum from male C57BL/6NCr mice at 3, 6, 12, 18, and 24 months of age were determined by HPLC and fluorescence detection. Also, hepatic α-TTP protein and mRNA expression levels were analyzed by Western blot and qPCR, respectively. RESULTS: Tissue-specific, age-related changes of α-tocopherol levels normalized by tissue weight were observed in the liver, cerebrum, hippocampus, cerebellum, heart, kidney, and epididymal adipose tissue. Specifically, α-tocopherol levels in epididymal adipose tissue increased greatly as mice aged from 6 to 24 months. Although hepatic α-TTP protein levels also showed age-related changes, α-TTP mRNA expression levels measured after overnight fasting were not altered. CONCLUSIONS: In this study, we determined that α-tocopherol levels and hepatic α-TTP protein levels of mice undergo significant tissue-specific, age-related changes. This is the first report to investigate VE in terms of the α-tocopherol levels in plasma and various tissues of mice and hepatic α-TTP protein levels during aging.

Time-Dependent Alterations of Vancomycin-Induced Nephrotoxicity in Mice.

Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic that is commonly used against methicillin-resistant, Gram-positive cocci despite the nephrotoxic side effects. VCM-induced nephrotoxicity has been reported in 5-28% of recipient patients. Therefore, renal failure induced by VCM has become an important clinical problem. However, the exceedingly complex mechanism of VCM-induced nephrotoxicity is not fully understood. Therefore, this study was designed to clarify time-dependent alterations of VCM-induced nephrotoxicity in mice as a step toward decreasing the risks of kidney injury associated with VCM therapy. VCM was injected intraperitoneally into mice at a dose of 400 mg/kg body weight at 24-h intervals for 3, 5, 7, and 14 d. At 24 h after the last injection, we examined histopathological alterations of the kidney as well as blood biochemistry. VCM administration resulted in a decrease of body weight and increase of kidney weight. Histological examination revealed renal damage such as dilated proximal tubules with occasional casts and interstitial fibrosis in VCM-treated mice. Furthermore, immunohistochemical staining with anti-CD10 and anti-single-stranded DNA antibodies highlighted damaged renal proximal tubules with marked dilatation as well as numerous apoptotic cells as early as day 4 of VCM-treatment. The severity of symptoms progressed until day 15. These results suggest that VCM-induced renal damage and incipient renal failure begin soon after the start of treatment and progressively worsen. This is the first report describing the time-dependence of VCM-induced nephrotoxicity in mice and depicting a model that clarifies the mechanisms of this tissue damage.