Search

Decompression surgery (DS) is a standard treatment for chronic nerve compression injuries; however, the mechanisms underlying its effects remain unclear. Here, we investigated the effects of DS on messenger RNA (mRNA) expression of tumor necrosis factor-α (TNF-α) and T cell recruitment in a rat sciatic nerve (SN) chronic constriction injury (CCI) model. Male Wistar rats were subjected to CCI to establish a model of SN injury (CCI group). DS, in which all ligatures were removed, was performed 3 days after CCI surgery (CCI + dec group). Mechanical sensitivity was assessed using the von Frey test 3, 7, and 14 days after the CCI surgery. Gene expression of Tnfa, Cd3, Cxcl10, and immunolocalization of TNF-α and the pan T cell marker, CD3, was evaluated using quantitative polymerase chain reaction (qPCR) and immunohistochemistry, respectively. In addition, the effects of TNF-α on Cxcl10 expression and CXCL10 protein production were evaluated using qPCR and enzyme-linked immunosorbent assay in SN cell culture. Rats that received DS had significantly higher withdrawal threshold levels than those in the CCI group. In addition, Tnfa, Cd3, and Cxcl10 mRNA expression increased following CCI. DS suppressed this elevated expression, with the CCI + dec group showing significantly reduced expression levels compared to the CCI group. Furthermore, TNF-α induced Cxcl10 expression and CXCL10 protein production in SN cell culture. Therefore, DS reduced TNF-α expression and T cell recruitment in the rat SN CCI model. These observations may partly explain the mechanism underlying the therapeutic effects of DS.

Certain glycolipids have immunomodulatory potential by activating natural killer T (NKT) cells, a unique T cell subset. Invariant NKT (iNKT) cells recognize α-galactosylceramide (α-GalCer) and synthetic derivatives presented by CD1d molecules and secrete large amounts of cytokines that modulate immune functions. Some iNKT cell ligands induce distinct reactions biased toward either Th1 or Th2 immune responses, while others show mixed responses. We describe the methods for activating iNKT cells by the ligands as represented by α-GalCer using in vitro assays, such as cell-free or co-culture with antigen-presenting cells. In addition, α-GalCer/CD1d multimer can be used to specifically detect iNKT cells using flow cytometry. α-GalCer is also utilized to activate systemic iNKT cells in vivo, which leads to the production of high levels of cytokines in sera. Collectively, α-GalCer and its derivatives activate iNKT cells that modulate immune balance, and we need to understand the characteristics of these ligands for developing their utility.

The pathophysiology of early-stage hip osteoarthritis (EOA) is not fully understood. Although a previous study in an age-unmatched cohort reported that the number of macrophages was increased in knee EOA compared to late OA (LOA), it remained unclear whether increased macrophages in EOA accurately reflect EOA pathology. We investigated the differences in

REV7 is a multitasking protein involved in replication past DNA lesions, cell cycle regulation, and gene expression. REV7 is highly expressed in the adult testis and plays an essential role in primordial germ cell maintenance in mice. In this study, we analyzed whether REV7 can be a molecular target for the treatment of testicular germ cell tumors (TGCTs), in which acquired chemoresistance is a major cause of treatment failure. Strong expression of REV7 was detected in human TGCT tissues by immunohistochemistry. REV7 depletion in the TGCT cell lines suppressed cell proliferation and increased sensitivity to cisplatin and doxorubicin. cDNA microarray analysis revealed that REV7 depletion downregulated genes in the DNA repair gene set and upregulated genes in the apoptosis gene set. REV7 depletion-provoked chemosensitivity was associated with DNA double-strand break accumulation and apoptosis activation. In addition, inactivation of REV7 in cisplatin-resistant TGCT cells recovered chemosensitivity at almost equal levels as parental cells in vitro and in vivo. Our results indicate that inactivation of REV7 enhances chemosensitivity and overcomes chemoresistance in TGCT cells, suggesting REV7 as a potential therapeutic target in chemoresistant TGCTs.

Uveitis is a sight-threatening intraocular inflammatory disease that accounts for almost 10% of blindness worldwide. NF-κB signaling plays pivotal roles in inflammatory diseases. We have reported that IMD-0354, which inhibits NF-κB signaling via selective blockade of IKK-β, suppresses inflammation in several ocular disease models. Here, we examined the therapeutic effect of IMD-0354 in an experimental autoimmune uveoretinitis (EAU) model, a well-established animal model for endogenous uveitis in humans. Systemic administration of IMD-0354 significantly suppressed the clinical and histological severity, inflammatory edema, and the translocation of NF-κB p65 into the nucleus of retinas in EAU mice. Furthermore, IMD-0354 treatment significantly inhibited the levels of several Th1/Th17-mediated pro-inflammatory cytokines in vitro. Our current data demonstrate that inhibition of IKKβ with IMD-0354 ameliorates inflammatory responses in the mouse EAU model, suggesting that IMD-0354 may be a promising therapeutic agent for human endogenous uveitis.

Macrophages, particularly M1 macrophages, produce proinflammatory cytokines and contribute to the degenerative process in injured intervertebral discs (IVDs). We previously showed that macrophages in both intact and injured IVDs increased following IVD injury. Resident macrophages and macrophages recruited from the peripheral blood have distinct roles in tissue. However, it remains to be determined whether increased macrophages derive from resident or recruited macrophages. We investigated the origin of M1 macrophages in injured IVDs using green fluorescent protein (GFP) transgenic bone marrow chimeric mice. The M1 macrophage marker, CD86, increased in both disc-derived resident macrophages and bone marrow-derived macrophages (BMMs) after lipopolysaccharide/interferon γ stimulation in vitro. Following IVD injury, the proportion of cells positive for the CD86 ligand, the F4/80 antigen, and the surface glycoprotein CD11b (CD86+ CD11b+ F4/80+) significantly increased in GFP+ populations at days 3, 7, and 14. In contrast, CD86+ CD11b+ F4/80+ cells in GFP- populations significantly increased on day 3, and thereafter decreased on days 7 and 14. The proportion of CD86+ CD11b+ F4/80+ cells in the GFP+ populations was significantly higher than that in the GFP- populations at days 1, 3, 7, and 14. Monocyte chemoattractant protein-1 expression in disc-derived macrophages, but not in BMMs, increased following interleukin-1β stimulation. Our results suggest M1 macrophages following IVD injury originate from recruited macrophages. Resident macrophages may behave differently in IVD injury. The role of resident macrophages needs to be clarified. Further investigation is needed.

Obesity is accompanied by and accelerated with chronic inflammation in adipose tissue, especially visceral adipose tissue (VAT). This low-level inflammation predisposes the host to the development of metabolic disease, most notably type 2 diabetes. We have focused on the capacity of glycolipid-reactive, CD1d-restricted natural killer T (NKT) cells to modulate obesity and its associated metabolic sequelae. We previously reported that CD1d knockout (KO) mice are partially protected against the development of obesity-associated insulin resistance, and these findings were recapitulated in mice with an adipocyte-specific CD1d deficiency, suggesting that NKT cell-adipocyte interactions play a critical role in exacerbating disease. However, many other CD1d-expressing cells contribute to the in vivo responses of NKT cells to lipid antigens. In the present study, we examined the role of CD1d expression by macrophages (Mϕ) in the development of obesity-associated metabolic inflammation using LysMcre-cd1d1

The pathophysiology of early-stage hip osteoarthritis (EOA) is not fully understood. Although a previous study in an age-unmatched cohort reported that the number of macrophages was increased in knee EOA compared to late OA (LOA), it remained unclear whether increased macrophages in EOA accurately reflect EOA pathology. We investigated the differences in CD14 expression levels between EOA and LOA using age-unmatched and -matched cohorts. Synovial tissues were obtained from 34 EOA (Tönnis grades 0 and 1) and 80 LOA (Tönnis grades 2 and 3) patients. To correct for differences in demographics between patients with LOA and EOA, we also created propensity score-matched cohorts (16 EOA and 16 LOA). CD14 expression and its association with pain was estimated in LOA and EOA before and after propensity matching. We performed flow cytometry on tissues from the 16 patients, with 8 from each group, to assess for CD14+ subsets in the cells. The CD14 expression in EOA was higher than that in LOA both before and after propensity matching. The proportion of CD14high subsets in EOA was higher than that in LOA. The CD14 expression was associated with pain in EOA before matching. However, no difference was observed between the pain and CD14 expression after matching in EOA. The increased CD14 expression and the proportion of CD14high subsets may be important features associated with hip EOA pathology. To accurately compare early and late OA, the analysis of a propensity score-matched cohort is necessary.

Expression of CD163, a scavenger receptor specifically expressed by monocytes and macrophages, is elevated in the synovial tissue of patients with knee osteoarthritis (OA) compared with healthy controls. However, the association between CD163 expression in the synovium and pain in OA patients is unclear. We investigated the correlation between synovial CD163 expression and resting and active pain levels in patients with hip osteoarthritis (HOA). To investigate the possible contribution of CD163+ subsets to pain pathogenesis, we compared pain-related cytokine expression and M1/M2 macrophage marker expression in CD163+ and CD163- cells. We performed flow cytometric analysis to study the CD163+ cell population. We also examined pain-related cytokine expression and M1/M2 macrophage marker expression on CD163+ CD14high and CD163+ CD14low cells using cell sorting. Synovial CD163 expression significantly correlated with resting pain levels (p = 0.006; R = 0.321), but not active pain levels (p = 0.155; R = 0.169). Expression of the M1 macrophage marker CD80 was significantly higher in CD163+ than CD163- cells (p = 0.010), as was the expression of M2 macrophage markers CD206 and IL10 (CD206, p = 0.014; IL10, p = 0.005), and TNFA and IL1B (TNFA, p = 0.002; IL1B, p = 0.001). TNFA expression was significantly higher in CD163+ CD14low than CD163+ CD14high cells, while IL1B, IL10, and CD206 expression were comparable among these subsets. Our findings suggest that CD163 expression is associated with higher resting pain scores. As TNF-α plays a role in the pain process, CD163+ CD14low cells expressing TNFA may be a potent contributor to the pathogenesis of resting pain in HOA.

Macrophages produce proinflammatory cytokines in injured intervertebral discs (IVDs). We recently showed that macrophage-derived inflammatory cytokines contribute to the production of pain-related factors. However, the mechanism by which macrophages are recruited to injured IVDs has not been fully clarified. Here, we examined the expression dynamics of the chemokine CCL2 in a mouse IVD injury model and the mechanisms of its regulation. The percentage of macrophages increased from day 1 after injury and persisted up until day 28. At 1 and 3 days after injury, the expression of both Ccl2 messenger RNA (mRNA) and CCL2 protein was elevated in the IVD injury group, after which expression decreased to basal levels. Consistent with the increase in CCL2 expression, Ccr2 and Tnfa expression and various types of macrophages were also immediately elevated following disc injury. Further, tumor necrosis factor-α (TNF-α) stimulated Ccl2 mRNA and CCL2 protein expression in IVD cells in vitro. The expressions of M1 (Cd86 and Nos2) and M2a (Ym1) macrophage markers were all significantly elevated from day 1 following injury in injured compared with control mice. Meanwhile, the expression of Cd206 (M2a and M2c marker) was significantly elevated on days 3, 7, 14, and 28 following injury. These results suggest that in IVD injury, TNF-α stimulates CCL2, which, in turn, mediates the recruitment of macrophages with the recruited macrophages subsequently differentiating into M1 and M2 subtypes. CCL2 signaling may, therefore, play an important role in IVD pathology via macrophage recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:895-901, 2020.

Osteoarthritis (OA) is a chronic degenerative musculoskeletal disease that causes articular cartilage degeneration and chronic pain. Research into OA animal models suggests that elevated NGF levels in the synovium contribute to pain and central sensitization (CS). However, it is unclear whether synovial NGF contributes to CS in patients with OA. We investigated the association between synovial NGF expression and clinical assessments of pain and CS in hip OA (hOA) patients. We also aimed to identify which cells in the synovium of hOA patients express NGF. Sixty-six patients who received total hip replacement and a diagnosis of hOA were enrolled. We measured NGF mRNA expression in synovial samples obtained from 50 patients using qPCR and analyzed the correlation of NGF expression with the CS inventory (CSI) score and Japanese Orthopaedic Association (JOA) score, a clinical scoring system for OA. To identify the synovial cells expressing NGF, we analyzed NGF mRNA expression in CD14+ and CD14- cells, which represent macrophage-rich and fibroblast-rich fractions, respectively, extracted from 8 patients. To further identify which macrophage subtypes express NGF, we examined NGF mRNA expression in CD14high and CD14low cells sorted from 8 patients. Synovial NGF mRNA expression was negatively correlated with JOA score but positively correlated with CSI score (JOA pain, r = − 0.337 , P = 0.017 ; CSI score, r = 0.358 , P = 0.011 ). Significantly greater levels of NGF were observed in CD14- cells compared to CD14+ cells ( P = 0.036 ) and in CD14high cells compared to CD14low cells ( P = 0.008 ). In conclusion, synovial NGF expression is correlated with the degree of pain and CS in hOA patients. NGF is predominantly expressed in synovial fibroblasts. Further, CD14high synovial macrophages expressed higher levels of NGF. Our results may provide a novel NGF-targeted therapeutic strategy for hOA pain.

Experimental autoimmune uveoretinitis (EAU) in mice provides a useful platform to study the pathogenesis and experimental therapeutics of human uveitis. One often used EAU model employs C57BL/6 (B6) mice sensitized with a peptide residue having 1 to 20 amino acids of human interphotoreceptor retinoid binding protein (hIRBP1-20). The model using the B6 background has permitted a liberal use of genetically engineered strains and has provided insights for understanding uveoretinitis. However, this is usually acute/monophasic and does not represent human uveoretinitis that is characterized as a chronic/recurrent disease. Several chronic/recurrent EAU models have been developed; of these, we employed administration of staphylococcal enterotoxin B (SEB) for relapse in the present study, and found that recurrence was induced at day 24 after primary immunization, which is thought to be the convalescent phase. We reported the activation of invariant natural killer T (iNKT)-cells upon primary immunization of the EAU model mice with the ligand RCAI-56, which was found to mitigate the disease in our previous study. Here, we first attempted to ameliorate EAU in the relapse model using a preventive regimen by activating iNKT cells at the same time relapse induction (day 24) or in a regimen after 3 days of relapse induction (day 27). The preventive as well as post-inductive regimens were successful in reducing histopathological scores by inhibiting the Ag-specific Th17-biased response. Collectively, activation of iNKT cells may be useful to mitigate the relapse response of EAU induced with SEB.

Introduction Studies have identified the presence of M1 and M2 macrophages (Mϕ) in injured intervertebral discs (IVDs). However, the origin and polarization-regulatory factor of M2 Mϕ are not fully understood. TGF-β is a regulatory factor for M2 polarization in several tissues. Here, we investigated the source of M2 Mϕ and the role of TGF-β on M2 polarization using a mice disc-puncture injury model. Methods To investigate the origin of M2 macrophages, 30 GFP chimeric mice were created by bone marrow transplantation. IVDs were obtained from both groups on pre-puncture (control) and post-puncture days 1, 3, 7, and 14 and CD86 (M1 marker)- and CD206 (M2 marker)-positive cells evaluated by flow cytometry ( n = 5 at each time point). To investigate the role of TGF-β on M2 polarization, TGF-β inhibitor (SB431542) was also injected on post-puncture days (PPD) 5 and 6 and CD206 expression was evaluated on day 7 by flow cytometry ( n = 5) and real time PCR ( n = 10). Results The proportion of CD86+ Mϕ within the GFP+ population was significantly increased at PPD 1, 3, 7, and 14 compared to control. CD206-positive cells in GFP-populations were significantly increased on PPD 7 and 14. In addition, the percentage of CD206-positive cells was significantly higher in GFP-populations than in GFP+ populations. TGF-β inhibitor reduced CD206-positive cells and Cd206 expression at 7 days after puncture. Conclusion Our findings suggest that M2 Mϕ following IVD injury may originate from resident Mϕ. TGF-β is a key factor for M2 polarization of macrophages following IVD injury.

Background In 2003, a perinatal helicopter air ambulance service was introduced for remote areas of Wakayama and Mie prefectures, Japan. However, its long-term impact on perinatal medicine has not yet been analyzed. Methods A retrospective observational study was conducted on helicopter air ambulance cases recorded between January 2003 and December 2016 at Wakayama Medical University Hospital (WMUH). Results During that period, 61 pregnant mothers were transferred by helicopter air ambulance to WMUH. Between 2003 and 2009, the mean period from transfer to birth was 0.6 weeks, whereas between 2008 and 2016, this increased to 1.6 weeks, and the survival rate of infants who were born after transfer did not differ significantly (84.2% [32/38] and 82.1% [23/28]). Seventy-three neonates were transferred. The number transferred between 2003 and 2009 was 46, whereas this decreased to 27 between 2010 and 2016. The neonatal mortality rate in south Wakayama plus south Mie gradually decreased. The reasons for the longer period from transfer to birth, and the decrease in the rate of very low birth weight infants after transfer may be developments in the management of threatened premature labor, and the earlier transfer of such cases by regional obstetricians. The reasons for the decline in neonatal transfer cases may have included the development of fetal diagnostic techniques and improved efficiency of neonatal ground-transport in the South Wakayama region. Conclusions The helicopter air ambulance is an important form of medical transportation in the south Kii peninsula. This article is protected by copyright. All rights reserved.

iNKT cells play important roles in immune regulation by bridging the innate and acquired immune systems. The functions of iNKT cells have been investigated in mice lacking the Traj18 gene segment that were generated by traditional embryonic stem cell technology, but these animals contain a biased T cell receptor (TCR) repertoire that might affect immune responses. To circumvent this confounding factor, we have generated a new strain of iNKT cell-deficient mice by deleting the Traj18 locus using CRISPR/Cas9 technology, and these animals contain an unbiased TCR repertoire. We employed these mice to investigate the contribution of iNKT cells to metabolic disease and found a pathogenic role of these cells in obesity-associated insulin-resistance. The new Traj18-deficient mouse strain will assist in studies of iNKT cell biology.

Experimental autoimmune uveoretinitis (EAU) represents an experimental model for human endogenous uveitis, which is caused by Th1/Th17 cell-mediated inflammation. Natural killer T (NKT) cells recognize lipid antigens and produce large amounts of cytokines upon activation. To examine the role of NKT cells in the development of uveitis, EAU was elicited by immunization with a peptide from the human interphotoreceptor retinoid-binding protein (hIRBP1-20) in complete Freund's adjuvant and histopathology scores were evaluated in C57BL/6 (WT) and NKT cell-deficient mice. NKT cell-deficient mice developed more severe EAU pathology than WT mice. When WT mice were treated with ligands of the invariant subset of NKT cells (α-GalCer or RCAI-56), EAU was ameliorated in mice treated with RCAI-56 but not α-GalCer. IRBP-specific Th1/Th17 cytokines were reduced in RCAI-56-treated compared with vehicle-treated mice. Although the numbers of IRBP-specific T cells detected by hIRBP3-13/I-Ab tetramers in the spleen and the draining lymph node were the same for vehicle and RCAI-56 treatment groups, RORγt expression by tetramer-positive cells in RCAI-56-treated mice was lower than in control mice. Moreover, the eyes of RCAI-56-treated mice contained fewer IRBP-specific T cells compared with control mice. These results suggest that invariant NKT (iNKT) cells suppress the induction of Th17 cells and infiltration of IRBP-specific T cells into the eyes, thereby reducing ocular inflammation. However, in sharp contrast to the ameliorating effects of iNKT cell activation during the initiation phase of EAU, iNKT cell activation during the effector phase exacerbated disease pathology. Thus, we conclude that iNKT cells exhibit dual roles in the development of EAU.

Nerve growth factor (NGF) is increased in intervertebral discs (IVDs) after disc injury and anti-NGF therapy improves low back pain in humans. Furthermore, M1 and M2 macrophage subtypes play a role in degenerative IVD injury. We examined M1 and M2 macrophage markers and NGF and cytokine expression in IVD-derived cells from control and IVD-injured mice for 28 days following injury. Ngf messenger RNA (mRNA) expression was increased 1 day after injury in injured compared with control mice, and persisted for up to 28 days. Flow cytometric analysis demonstrated that the proportion of F4/80+ CD11b+ cells was significantly increased from 1 day after injury for up to 28 days in injured compared to control mice. mRNA expression of M1 macrophage markers Tnfa, Il1b, and Nos2 was significantly increased 1 day after injury in injured compared to control mice, before gradually decreasing. At 28 days, no significant difference was observed in M1 markers. The M2a marker, Ym1, was significantly increased 1 day after injury in injured compared with control mice, while M2a and M2c markers Tgfb and Cd206 were significantly increased 7, 14, and 28 days after injury. Tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) stimulated Ngf mRNA and NGF protein expression in IVD cells. Our results suggest that TNF-α and TGF-β may stimulate NGF production under inflammatory and non-inflammatory conditions following IVD injury. As TNF-α and TGF-β are produced by M1 and M2 macrophages, further investigations are needed to reveal the role of macrophages in NGF expression following IVD injury. Our results may aid in developing treatments for IVD-related LBP pathology. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1798-1804, 2019.

Adipose tissue contains various types of immunocompetent cells, and these cells of innate and adaptive immunity control adipose tissue inflammation that blunts insulin sensitivity. Recent studies have shown that adipocytes express CD1d and present lipid antigen(s) to activate natural killer T (NKT) cells. The function of adipocytes is in turn modulated by cytokines that NKT cells produce to alter the expression of anti-inflammatory adipokine(s) and the production of inflammatory and chemoattractant cytokines. These in vitro studies imply that the interaction between adipocytes and NKT cells might affect the development of not only obesity but also obesity-related diseases. To test the importance of the interaction between NKT cells and adipocytes, we examined whether an adipocyte-specific CD1d deletion affected the development of obesity, which had been demonstrated with B6.CD1d−/− (CD1d KO). We found that the interaction is indeed important to induce adipose tissue inflammation and insulin resistance in response to lipid excess. In this commentary, the advances and controversies on NKT cells and obesity are discussed based on our recent report that NKT cells play a pivotal role in the regulation of adipose tissue by communicating with adipocytes via CD1d.

Until now, metal allergies have been regarded as a Th1-type immune response. However, because the contribution of a Th2-type immune response has been suggested by clinical findings, we previously examined the Th2-type immune response during the development of metal allergies using a GATA-3 transgenic (GATA-3 Tg) mouse model. As a result, a Th2-type immunization reaction was suggested to be involved in the early phase of metal allergies. Recently, the involvement of NKT cells in metal allergies has been suggested. We examined this possibility using the activation of NKT cells and an NKT cell-deficient mouse model to determine the contribution of NKT cells to nickel allergy in the present study. In NKT cell-deficient mice, ear swelling was remarkably increased, compared with that in control mice. Also, in mice that had been treated with α-galactosylceramide (α-GalCer) to activate NKT cells, the ear swelling response was remarkably inhibited, compared with that in untreated mice. These facts show that NKT cells are involved in the inhibition of nickel allergy-induced ear swelling responses.

Nickel is a component of several alloy types that are widely used in our environment, including several dental alloy types that cause intraoral metal contact allergy. However, metal-specific immune responses in the oral mucosa have not been elucidated because a suitable animal model has not been established. In this study, we established a novel murine model of nickel-induced intraoral metal contact allergy and aimed to elucidate the immune response in terms of T-cell receptor repertoire and cytokine profiles in inflamed oral mucosa. The intraoral metal contact allergy model was induced by two sensitizations of nickel plus lipopolysaccharide solution into the postauricular skin followed by a single nickel challenge of the buccal mucosa. Cytokine expression profiles and T-cell phenotypes were determined by quantitative polymerase chain reaction. T cells accumulated in the cervical lymph nodes and inflamed oral mucosa were characterized by analyzing their T-cell receptor α- and β-chain repertoires, and the nucleotide sequences of complementary determining region 3. Significant swelling and pathological features were histologically evident at 1 day after challenge in mice with nickel allergy. At 1 day after the challenge, CD8-positive T cells producing high levels of T helper 1 type cytokines had accumulated in the allergic oral mucosa. At 7 days after the challenge, excessive nickel allergy in the oral mucosa was suppressed by regulatory T cells. Characterization of the T-cell receptor repertoire in nickel allergic mice revealed the presence of natural killer T cells and T cells bearing Trav6-6-Traj57 at 1 day after the challenge. Our murine model of nickel-induced intraoral metal contact allergy showed that natural killer T cells and T cells bearing Trav6-6-Traj57 might be involved in the immune responses of nickel-induced intraoral metal contact allergy.

Natural killer T cells (NKT cells) are comprised of several subsets. However, the possible differences in their developmental mechanisms have not been fully investigated. To evaluate the dependence of some NKT subpopulations on nuclear factor-κB-inducing kinase (NIK) for their generation, we analysed the differentiation of NKT cells, dividing them into subsets in various tissues of alymphoplasia (aly/aly), a mutant mouse strain that lacks functional NIK. The results indicated that the efficient differentiation of both invariant NKT (iNKT) and non-iNKT cells relied on NIK expression in non-haematopoietic cells; however, the dependence of non-iNKT cells was lower than that of iNKT cells. Especially, the differentiation of CD8(+) non-iNKT cells was markedly resistant to the aly mutation. The proportion of two other NKT cell subsets, NK1.1(+) γδ T cells and NK1.1(-) iNKT cells, was also significantly reduced in aly/aly mice, and this defect in their development was reversed in wild-type host mice given aly/aly bone marrow cells. In exerting effector functions, NIK in NKT-αβ cells appeared dispensable, as NIK-deficient NKT-αβ cells could secrete interleukin-4 or interferon-γ and exhibit cytolytic activity at a level comparable to that of aly/+ NKT-αβ cells. Collectively, these results imply that the NIK in thymic stroma may be critically involved in the differentiation of most NKT cell subsets (although the level of NIK dependence may vary among the subsets), and also that NIK in NKT-αβ cells may be dispensable for their effector function.

Summary Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)-associated pain. Although recent studies suggest that tumour necrosis factor (TNF)-α and interleukin (IL)-1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF-α and IL-1β in freshly isolated CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL-1β and TNF-α on NGF mRNA expression in cultured CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF-α and IL-1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF-α and IL-1β mRNA levels in CD14-positive cells from the SYN of OA patients was significantly higher than that in CD14-negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14-positive and -negative cells with IL-1β and TNF-α enhanced NGF mRNA and protein levels. Expression of NGF, IL-1β and TNF-α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL-1β and TNF-α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.

Evaluation of: Lynch L, Nowak M, Varghese B et al. Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production. Immunity 37(3), 574–587 (2012). Recent studies have revealed that not only macrophages but also T lymphocytes play active roles in the development of obesity in either an aggravating or ameliorating direction. NKT cells, a subset of innate T lymphocytes, recognize lipid antigens in the context of a nonpolymorphic MHC class I-like molecule, CD1d, implying that NKT cells are likely to participate in the process. Lynch et al. demonstrated that obese patients showed reduced NKT cell count and the reduction of body fat through therapeutic intervention induced the recovery of NKT cells. They attempted to test whether the manipulation of NKT cells (deficiency and stimulation with ligands, among others) could affect adiposity in vivo and demonstrated that NKT cells did indeed play ameliorating roles in the development of obesity, ...

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F2 animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b(+)Gr1(+) cells in spleens and peripheral blood was increased, and CD11b(+)Gr1(+) cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b(+)Gr1(+) cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b(+)Gr1(+) cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F2 mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b(+)Gr1(+) cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b(+)Gr1(+) cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.

It has been shown that CD1d expression and glycolipid-reactive, CD1d-restricted NKT cells exacerbate the development of obesity and insulin resistance in mice. However, the relevant CD1d-expressing cells that influence the effects of NKT cells on the progression of obesity remain incompletely defined. In this study, we have demonstrated that 3T3-L1 adipocytes can present endogenous ligands to NKT cells, leading to IFN-γ production, which in turn, stimulated 3T3-L1 adipocytes to enhance expression of CD1d and CCL2 and decrease expression of adiponectin. Furthermore, adipocyte-specific CD1d deletion decreased the size of the visceral adipose tissue mass and enhanced insulin sensitivity in mice fed a high-fat diet (HFD). Accordingly, NKT cells were less activated, IFN-γ production was significantly reduced and levels of adiponectin were increased in these animals as compared with control mice on HFD. Importantly, macrophage recruitment into the adipose tissue of adipocyte-specific CD1d-deficient mice was significantly blunted. These findings indicate that interactions between NKT cells and CD1d-expressing adipocytes producing endogenous NKT cell ligands play a critical role in the induction of inflammation and functional modulation of adipose tissue that leads to obesity.

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α(-/-)) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α(-/-) DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α(-/-) DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α(-/-) DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α(-/-) DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.

Cysteine proteases (CPs) with N-succinyl-Leu-Tyr-4-methylcoumaryl-7-amide (Suc-LY-MCA) cleavage activity were investigated in green and senescent leaves of spinach. The enzyme activity was separated into two major and several faint minor peaks by hydrophobic chromatography. These peaks were conventionally designated as CP1, CP2 and CP3, according to their order of elution. From the analyses of molecular mass, subunit structure, amino acid sequences and cDNA cloning, CP2 was a monomer complex (SoCP-CPI) (51 kDa) composed of a 41-kDa core protein, SoCP (Spinacia oleracea cysteine protease), and 14-kDa cystatin, a cysteine protease inhibitor (CPI), while CP3 was a trimer complex (SoCP-CPI)(3) (151 kDa) of the same subunits as SoCP-CPI and showed a wider range of specificity toward natural substrates than SoCP-CPI. Trimer (SoCP-CPI)(3) was irreversibly formed from monomers through association. The results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that mRNAs of CPI and SoCP are hardly expressed in green leaves, but they are coordinately expressed in senescent leaves, suggesting that these proteases involve in senescence. Purified recombinant CPI had strong inhibitory activity against trimer SoCP, (SoCP)(3) , which had a cystatin deleted with K(i) value of 1.33 × 10(-9) M. After treatment of the enzyme with a succinate buffer (pH 5) at the most active pH of the enzyme, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and activity analyses showed that cystatin was released from both monomer SoCP-CPI and trimer (SoCP-CPI)(3) complexes with a concomitant activation. Thus, the removal of a cystatin is necessary to activate the enzyme activity.

Thin films of Si-added-MoSi2 were deposited on Si3N4 or alumina substrate using an RF magnetron sputtering with a target made of MoSi2 and Si powders. The thin film consisted of a mixture of Si and MoSi2. The resistance of MoSi2-thin-film heaters fabricated using Si3N4 substrate heated for a long time in air increased with increasing heating-time at temperatures near 420°C due to generation of MoO3, which is an insulator, in thin film due to the oxidation of Mo in MoSi2. On the other hand, oxidation-resistant capability of thin-film heater fabricated by adding Si to MoSi2 was drastically improved compared with MoSi2 thin-film heater, because Si particles or the Si layer on a MoSi2 particle prevented the oxidation of MoSi2. The oxidation-resistant capability was excellent in air at high temperatures near 480°C for a long heating-time exceeding 950h.

Thin-film heaters made of molybdenum silicide (MoSi2) were fabricated by RF magnetron sputtering, and the heating characteristics of these heaters in a high vacuum were evaluated. The crystal structure of thin-film was hexagonal contrary to the tetragonal one of the target. MoSi2 thin-film heaters deposited outside an alumina crucible showed almost linear resistance-temperature (R-T ) characteristics. However, resistance decreased due to repeated heatings. The heaters generated almost no contamination in vacuum. The temperature of the crucible reached 1000°C at an electric power of 190 W.

Summary To understand more clearly the link between osteoarthritis and hyperlipidaemia, we investigated the inflammatory macrophage subsets and macrophage-regulated matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS4) in synovial (ST) and adipose tissues (AT) of osteoarthritic mice with hyperlipidaemia (STR/Ort). CD11c+F4/80+CD11b+ macrophage populations in the ST and AT of 9-month-old STR/Ort and C57BL/6J mice were characterized and compared by flow cytometry and real-time polymerase chain reaction (PCR) analyses. Expression of tumour necrosis factor (TNF)-α, MMP-3 and ADAMTS4, and the response of these factors to anionic liposomal clodronate induced-macrophage depletion were also evaluated by real-time PCR. Expression of TNF-α in CD11c+ cells, which were isolated by magnetic beads, was compared to CD11c– cells. In addition, the effect of TNF-α on cultured synovial fibroblasts and adipocytes was investigated. CD11c+F4/80+CD11b+ macrophages were increased in ST and AT of STR/Ort mice. The CD11c+ cell fraction highly expressed TNF-α. Expression of TNF-α and MMP3 was increased in ST and AT, and was decreased upon macrophage depletion. TNF-α treatment of cultured synovial fibroblasts and adipocytes markedly up-regulated MMP-3. CD11c+F4/80+CD11b+ macrophages were identified as a common inflammatory subset in the AT and ST of STR/Ort mice with hyperlipidaemia. The induction of inflammation in AT and ST may be part of a common mechanism that regulates MMP3 expression through TNF-α. Our findings suggest that increased numbers of CD11c+ macrophages and elevated levels of TNF-α and MMP-3 in AT and ST may explain the relationship between hyperlipidaemia and OA.

Thin-film heaters made of molybdenum silicide (MoSi2) were fabricated by RF magnetron sputtering, and the heating characteristics of these heaters in a high vacuum were evaluated. The crystal structure of thin-film was hexagonal contrary to the tetragonal one of the target. MoSi2 thin-film heaters deposited outside an alumina crucible showed almost linear resistance-temperature (R-T ) characteristics. However, resistance decreased due to repeated heatings. The heaters generated almost no contamination in vacuum. The temperature of the crucible reached 1000°C at an electric power of 190 W.

Leucine aminopeptidase (LAP) is a terminal enzyme catalyzing a cycle of protein turnover. The normal properties of LAP from plants show a hexameric structure, thermostability over 60 °C and optimum pH of approximately 9.5. We isolated three types of LAP from etiolated barley seedlings, which were designated as LAP 1, LAP 2 and LAP 3. All of the subunit compositions were 57 kDa of monomeric structure. The thermostability of LAP 1, LAP 2 and LAP 3 was indicated by the enzymes having 50% of maximum activities, 51, 54 and 56 °C, respectively. The optimum pH was 7.0 for LAP 1 and LAP 2, and 8.0 for LAP 3. LAP activity was found almost equally in leaf tissue, coleoptile and roots from etiolated and green seedlings. All LAPs were sensitive to p-chloromercuribenzoic acid. LAPs from barley seedlings provide novel properties compared to LAPs from other plant species.