Inhibition of fibrotic changes in infrapatellar fat pad alleviates persistent pain and articular cartilage degeneration in monoiodoacetic acid-induced rat arthritis model.

OBJECTIVE: We have reported that fibrotic changes in infrapatellar fat pad (IFP) after acute joint inflammation are closely associated with persistent pain in rats. In this study, to examine the effects of anti-fibrotic treatment on persistent pain, we used C-type natriuretic peptides (CNP) at the recovery phase after acute joint inflammation. DESIGN: Thirty-two male Wistar rats were used in this study. Monoiodoacetic acid (MIA) was injected intra-articularly to induce IFP fibrosis and persistent pain. CNP was injected after acute inflammatory phase in the same knee joint. Time-course pain-avoidance behavior tests and histological analyses were performed to examine the effects of CNP. RESULTS: Histological evaluations indicated that intra-articular injection of CNP inhibited fibrotic changes in IFP after acute inflammation. Incapacitance tests indicated that MIA injection into rat knee joint quickly decreased the percent weight on ipsilateral limb. In the vehicle group, the decrease was maintained up to day 28, suggesting that pain persistence occurred after acute inflammation (Day 0/Day 28, Est Dif -8.15, CI -10.78∼-5.53, Linear mixed-effect model). In contrast, the pain was alleviated in the CNP group after day 14 (Day0/Day 14, -0.51, -2.62-1.59). In addition, we observed significant improvement in the degree of articular cartilage degeneration at day 14 in the CNP group (OARSI score: vehicle 16.14 ± 4.37 vs CNP 6.87 ± 3.44, P < 0.01; Wilcoxon rank sum test). CONCLUSION: Fibrotic changes in IFP may play important roles in both persistent pain and articular cartilage degeneration.

Histopathological analyses of murine menisci: implications for joint aging and osteoarthritis.

OBJECTIVE: To establish a standardized protocol for histopathological assessment of murine menisci that can be applied to evaluate transgenic, knock-out/in, and surgically induced OA models. METHODS: Knee joints from C57BL/6J mice (6-36 months) as well as from mice with surgically-induced OA were processed and cut into sagittal sections. All sections included the anterior and posterior horns of the menisci and were graded for (1) surface integrity, (2) cellularity, (3) Safranin-O staining distribution and intensity. Articular cartilage in the knee joints was also scored. RESULTS: The new histopathological grading system showed good inter- and intra-class correlation coefficients. The major age-related changes in murine menisci in the absence of OA included decreased Safranin O staining intensity, abnormal cell distribution and the appearance of acellular areas. Menisci from mice with surgically-induced OA showed severe fibrillations, partial/total loss of tissue, and calcifications. Abnormal cell arrangements included both regional hypercellularity and hypocellularity along with hypertrophy and cell clusters. In general, the posterior horns were less affected by age and OA. CONCLUSION: A new standardized protocol and histopathological grading system has been developed and validated to allow for a comprehensive, systematic evaluation of changes in aging and OA-affected murine menisci. This system was developed to serve as a standardized technique and tool for further studies in murine meniscal pathophysiology models.

Global methylation levels in peripheral blood leukocyte DNA by LUMA and breast cancer: a case-control study in Japanese women.

BACKGROUND: Global hypomethylation has been suggested to cause genomic instability and lead to an increased risk of cancer. We examined the association between the global methylation level of peripheral blood leukocyte DNA and breast cancer among Japanese women. METHODS: We conducted a hospital-based case-control study of 384 patients aged 20-74 years with newly diagnosed, histologically confirmed invasive breast cancer, and 384 matched controls from medical checkup examinees in Nagano, Japan. Global methylation levels in leukocyte DNA were measured by LUminometric Methylation Assay. Odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between global hypomethylation and breast cancer were estimated using a logistic regression model. RESULTS: Compared with women in the highest tertile of global methylation level, ORs for the second and lowest tertiles were 1.87 (95% CI=1.20-2.91) and 2.86 (95% CI=1.85-4.44), respectively. Global methylation levels were significantly lower in cases than controls, regardless of the hormone receptor status of the cancer (all P values for trend <0.05). INTERPRETATION: These findings suggest that the global methylation level of peripheral blood leukocyte DNA is low in patients with breast cancer and may be a potential biomarker for breast cancer risk.

Probe of the solar magnetic field using the "cosmic-ray shadow" of the sun.

We report on a clear solar-cycle variation of the Sun's shadow in the 10 TeV cosmic-ray flux observed by the Tibet air shower array during a full solar cycle from 1996 to 2009. In order to clarify the physical implications of the observed solar cycle variation, we develop numerical simulations of the Sun's shadow, using the potential field source surface model and the current sheet source surface (CSSS) model for the coronal magnetic field. We find that the intensity deficit in the simulated Sun's shadow is very sensitive to the coronal magnetic field structure, and the observed variation of the Sun's shadow is better reproduced by the CSSS model. This is the first successful attempt to evaluate the coronal magnetic field models by using the Sun's shadow observed in the TeV cosmic-ray flux.

Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production.

The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1-3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2-/- mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2-/- mice (63% decrease, P<0.04). Macrophages from Ucp2-/- mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.

Prognostic significance of FTO genotype in the development of obesity in Japanese: the J-SHIPP study.

OBJECTIVE: Susceptibility of fat mass and obesity-associated (FTO) gene polymorphisms to obesity has been reported in various populations. Polymorphisms in the melanocortin 4 receptor (MC4R) gene were recently explored as another susceptible locus. However, prognostic significance of these genetic variations has not been fully elucidated. Here, we investigated the involvement of FTO rs9939609 and MC4R rs17782313 polymorphisms in the development of obesity. Association with type 2 diabetes mellitus (T2DM) was also investigated. SUBJECTS: We analyzed 2806 community-dwelling middle-aged to elderly subjects (61+/-14 years). Clinical parameters were obtained from the subjects' personal health records, evaluated at their annual medical check-up. RESULTS: FTO genotype was significantly associated with current body mass index (BMI; TT 23.2+/-3.2, TA 23.7+/-3.2, AA 24.4+/-3.2 kg m(-2), P=2.5 x 10(-6)) and frequency of obesity (26.6, 32.0, 43.0% respectively, P=2.0 x 10(-4)). Age- and sex-adjusted odds ratio for obesity was 1.30 (P=0.004) in TA and 2.07 (P=0.002) in AA genotype. During the 9.4 years comprising the follow-up period, 214 new cases of obesity were diagnosed among 1718 subjects whose retrospective data were available. A allele frequency of the FTO genotype was significantly higher in subjects who developed obesity (22.2, 15.8%, P=0.001), Age-, sex- and initial BMI-adjusted odds ratio for the development of obesity was 1.46 (95% confidence interval, 1.04-2.04) (P=0.031). However, association studies and meta-analysis of T2DM did not actively support the involvement of FTO genotype. No significant differences were observed between the MC4R genotype and BMI (P=0.015), and the frequency of obesity (P=0.284). CONCLUSION: FTO genotype is an independent risk factor for future development of obesity.

[Pseudomesotheliomatous carcinoma of the lung].

Pseudomesotheliomatous carcinoma is the lung cancer with marked pleural extension resembling malignant pleural mesothelioma on diagnostic imaging. We report a rare case of pseudomesotheliomatous carcinoma of the lung in a 72-year-old man. The patient had complained of dyspnea and a chest roentgenogram showed right pleural effusion. Computed tomography (CT) of the chest revealed diffuse irregular pleural thickening, which mimicked pleural malignant mesothelioma. Pleural tissue sampling was performed to obtain definitive diagnosis by video-assisted thoracoscopic surgery. At the operation. the tumor was found to have a spread along the pleural surface and primary lesion was not detected in the right lung parenchyma. Immunohistochemically, the tumor was positive for carcinoembryonic antigen (CEA), but negative for calretinin, thrombomodulin, and pulmonary surfactant apoprotein. Final diagnosis was adenocarcinoma of the lung.

Mitogen-activated protein kinase and p70 ribosomal protein S6 kinase are not involved in the insulin-dependent stimulation of cAMP phosphodiesterase kinase in rat adipocytes.

To elucidate the mechanism of anti-lipolytic action of insulin in rat epididymal adipocytes, we explored the potential mechanism that might be involved in the hormone-dependent stimulation of cAMP phosphodiesterase (PDE) kinase. PDE kinase was assayed in a cell-free system. Both wortmannin and LY294002, highly specific inhibitors of phosphatidylinositol 3-kinase, almost completely blocked the hormonal effect not only on PDE kinase but also on mitogen-activated protein (MAP) kinase. Neither PD98059, a specific inhibitor of MAP kinase, nor rapamycin, a potent inhibitor of insulin-dependent stimulation of p70 ribosomal protein S6 kinase (p70S6K), had inhibitory effect on that of PDE kinase. These results are consistent with the view that (i) insulin-activated PDE kinase as well as MAP kinase and p70S6K are localized downstream of phosphatidylinositol 3-kinase, (ii) PDE kinase is distinct from either MAP kinase or p70S6K and (iii) PDE kinase does not exist downstream of either MAP kinase or p70S6K. It is suggested that PDE kinase and MAP kinase or p70S6K may be localized in separate branches of the cascade of insulin action. The branching point of the cascade could be either at or below the level of phosphatidylinositol 3-kinase.

Improvement in insulin resistance and the restoration of reduced phosphodiesterase 3B gene expression by pioglitazone in adipose tissue of obese diabetic KKAy mice.

Phosphodiesterase (PDE) 3B is a key enzyme in the mediation of the antilipolytic action of insulin in adipocytes, and activation of this molecule results in a reduced output of free fatty acids (FFAs). An elevation of serum FFAs is known to cause insulin resistance in skeletal muscle and liver, which could be the primary cause of type 2 diabetes. To elucidate whether PDE3B is involved in this disease, we examined the PDE3B gene expression in epididymal fat tissues of obese insulin-resistant diabetic KKAy mice. We also examined the effect of an insulin-sensitizing drug, pioglitazone, on this gene expression. In adipose tissue of KKAy mice, PDE3B mRNA and its corresponding protein were reduced to 48 and 43% of those in C57BL/6J control mice. Basal and insulin-stimulated membrane-bound PDE activities were also decreased to 50 and 36% of those in the controls, respectively. Pioglitazone increased both PDE3B mRNA and protein levels by 1.8-fold of those in untreated KKAy mice. Basal and insulin-induced membrane-bound PDE activities were also increased by 1.6- and 2.0-fold, respectively. Pioglitazone reduced the elevated levels of serum insulin, glucose, FFAs, and triglyceride in KKAy mice. Thus, the reduced PDE3B gene expression in adipose tissues could be the primary event in the development of insulin resistance in KKAy mice, which was improved by pioglitazone possibly because of the restoration of the reduced PDE3B gene expression.

Activation of mouse phosphodiesterase 3B gene promoter by adipocyte differentiation in 3T3-L1 cells.

Activation of phosphodiesterase (PDE) 3B reduces free fatty acid output from adipocytes. Induction of PDE3B gene expression by adipocyte differentiation could improve insulin resistance. To examine whether the PDE3B promoter is activated by this differentiation, the 5' flanking sequence of the mouse PDE3B gene was isolated. The transcription initiation site was determined to be located 195 bp upstream of the translation start site. No putative binding site for peroxisome proliferator-activated receptor gamma was found within 2 kb upstream of the transcription initiation site. This region had promoter activity, which was further activated on adipocyte differentiation in 3T3-L1 cells.

Association of HLA-DPB1*0501 with early-onset Graves' disease in Japanese.

To investigate the association between HLA antigens and Graves' disease among Japanese, serologic typing and DPB1 genotyping using the PCR-RFLP method have been performed. HLA alleles of 106 patients with Graves' disease were determined, and the frequency of HLA-B46 was found to be significantly increased. Furthermore, the frequencies of HLA antigens were compared between two age groups: early-onset and late-onset patients (under and over 20 years, respectively). It was found that the frequency of DPB1*0501 (88.9%) was significantly increased (pc < 0.004) in the early-onset group as compared with the healthy controls (55.0%) but not in the late-onset group (60.7%). On the other hand, a significant increase of HLA-B46 was observed in the late-onset patients (pc < 0.0004). These results suggest that the genetic background of Japanese patients with early-onset Graves' disease is different from late-onset patients. Namely, the HLA-DP allele (DPB1*0501) and the HLA-B allele (B46) are primarily involved in the pathogenesis of early-onset and late-onset Graves' disease in Japanese, respectively.

Adipocyte-specific reduction of phosphodiesterase 3B gene expression and its restoration by JTT-501 in the obese, diabetic KKAy mouse.

OBJECTIVE: Phosphodiesterase (PDE) 3B is a key enzyme involved in the anti-lipolytic action of insulin in adipocytes. PDE3B activation results in a reduced output of free fatty acids (FFA), whereas elevated serum FFA is known to cause insulin resistance. We have recently reported that reduced PDE3B gene expression is restored by treatment with pioglitazone, in the adipose tissues of obese, insulin-resistant diabetic KKAy mice. To determine whether the altered PDE3B gene expression is specific for adipocytes, the expression of this gene in liver and epididymal fat tissues of KKAy mice was examined. The effect of JTT-501, another peroxisome proliferator-activated receptor (PPAR)gamma ligand, which is different from thiazolidinedione, was also examined. METHODS: PDE3B mRNA and protein were quantified by an RNase protection assay and Western blotting respectively. Membrane-bound PDE activities were also measured. RESULTS: In adipose tissues of KKAy mice, PDE3B mRNA, protein and membrane-bound PDE activity were reduced to 47%, 57% and 51% respectively relative to those in C57BL/6J control mice. JTT-501 increased PDE3B mRNA, protein and membrane-bound PDE activity by 2.2-, 1.6- and 1.7-fold respectively over those of untreated KKAy mice. In the liver, PDE3B gene expression remained unchanged in KKAy mice, and was not affected by JTT-501. JTT-501 reduced the elevated levels of serum insulin, glucose, FFA and triglyceride in KKAy mice. CONCLUSIONS: PDE3B gene expression was specifically reduced in the adipose tissues of KKAy mice. JTT-501 restored this reduced gene expression with an accompanying improvement in elevated serum FFA and insulin resistance.

Clinical evaluation of upper mediastinal dissection for differentiated thyroid carcinoma.

BACKGROUND: An extensive upper mediastinal dissection in advanced differentiated thyroid carcinoma is occasionally required. This investigation was undertaken to clarify the indications for mediastinal lymph node dissection and the route of upper mediastinal metastases. METHODS: Twenty-one patients with differentiated thyroid cancer, who underwent their first radical operations with mediastinal dissection through a partial midline sternotomy, were enrolled in this study. Of 21 patients, 10 (48%) were found to have mediastinal lymph node metastases. RESULTS: The tumor size in the group with metastatic disease was much bigger than that in the group without metastatic disease. Histologic type and age were similar between the two groups. The extent of cervical lymph node metastases was more significant in the group with metastatic disease; in particular, all 10 patients showed more than two metastatic nodes along the internal jugular vein of the tumor-free side. CONCLUSIONS: This study indicates that metastases to the internal jugular chain on the side contralateral to the primary tumor would be an extremely important factor for indication of extensive upper mediastinal lymph node dissection after median partial sternotomy in patients with differentiated thyroid carcinoma.

A calcineurin inhibitor, FK506, blocks voltage-gated calcium channel-dependent LTP in the hippocampus.

The effects of FK506, an immunosuppressant and protein phosphatase 2B (calcineurin) inhibitor, on the voltage-gated calcium channel (VGCC)-dependent long-term potentiation (LTP) were investigated in the CA1 region of mice hippocampal slices. VGCC-dependent LTP was induced either by a brief application of a potassium channel blocker tetraethyleneanmonium (TEA), or by a strong tetanic stimulation under the blockade of NMDA-receptors. FK506 (1-50 microM) produced dose-dependent inhibition on TEA-induced LTP. Cyclosporin A (CysA 50 microM), another calcineurin inhibitor, showed a similar inhibitory effect on TEA-induced LTP. FK506 (10 microM) also blocked the strong tetanus-induced LTP, but had no effect on the post-tetanic potentiation. By using a subthreshold weak tetanic stimulation protocol, we also found that low concentration of FK506 (1 microM) produced neither inhibition nor potentiation on VGCC-dependent LTP. These results showed FK506 and CysA exerted inhibitory effects on VGCC-dependent LTP, and suggest that calcineurin is involved in the processes of this kind of synaptic plasticity.

Effects of vasopressin and involvement of receptor subtypes in the rat central amygdaloid nucleus in vitro.

Effects of arginine-vasopressin (AVP) on neurons in the central amygdaloid nucleus (ACe) were investigated with rat brain slice preparations using extracellular recording methods. Of 160 ACe neurons tested, 70 cells (44%) were excited and 9 cells (6%) were inhibited by bath application of AVP at 3 x 10(-7) M. The excitatory effects of AVP were dose-dependent and the threshold concentration was approximately 10(-10) to 10(-9) M. The excitatory effects of AVP persisted under blockade of synaptic transmission by perfusing with Ca2+-free and high-Mg2+ medium, whereas the inhibitory effects were abolished by synaptic blockade. AVP-induced effects were mimicked by a V1-receptor agonist and completely blocked by a selective V1-antagonist. V2-agonist produced no effects on ACe neurons and V2-antagonist had no effect on AVP-induced excitation. These results showed that the excitatory effect of AVP on ACe neurons was produced by a direct action through the V1-receptors, whereas the inhibitory response of ACe neurons to AVP seemed to be produced by an indirect action. The results of this study suggest that AVP is involved in the amygdala-relevant functions as a neurotransmitter or a neuromodulator.

Significance of values of thyrotropin binding inhibitor immunoglobulins and appearance of intrathyroidal lymphocytes at subtotal thyroidectomy for Graves' disease.

This study was undertaken to investigate whether thyrotropin binding inhibitor immunoglobulins (TBII) values at and after subtotal thyroidectomy correlated with the outcome or the histologic grade applied to the appearance of intrathyroidal lymphocytes at the time of surgical treatment. In addition, the main reason for the study was to determine whether or not it was possible to predict the outcome after the operation by the data obtained. There was no relation between the TBII results and the grade of the appearance of intrathyroidal lymphocytes at the time of operation or the TBII postoperative results (whether positive or negative) and the final outcome. However, it was of interest that patients with a recurrence of hyperthyroidism had the TBII values of more than 50 percent at the time of surgical treatment, and also manifested continuously positive TBII values after the operation. They also had moderate grades of lymphocytic infiltration and lymph follicle formation in the surgical specimen. It seemed impossible to predict the outcome of each instance in accordance with TBII values and the grade of the appearance of intrathyroidal lymphocytes at the time of the operation. However, it might be possible to predict at least the recurrence of hyperthyroidism by the consideration of changes of TBII values postoperatively.

Adrenoceptors, uncoupling proteins, and energy expenditure.

Interest in the biology of adipose tissue has undergone a revival in recent years with the discovery of a host of genes that contribute to the regulation of satiety and metabolic rate. The catecholamines have long been known to be key modulators of adipose tissue lipolysis and the hydrolysis of triglyceride energy stores. However, more recent efforts to understand the role of individual adrenergic receptor subtypes expressed in adipocytes and their signal transduction pathways have revealed a complexity not previously appreciated. Combined with this interest in the modulation of adipocyte metabolism is a renewed focus upon brown adipose tissue and the mechanisms of whole body thermogenesis in general. The discovery of novel homologs of the brown fat uncoupling protein (UCP) such as UCP2 and UCP3 has provoked intensive study of these mitochondrial proteins and the role that they play in fuel metabolism. The story of the novel UCPs has proven to be intriguing and still incompletely understood. Here, we review the status of adipose tissue from inert storage depot to endocrine organ, interesting signal transduction pathways triggered by beta-adrenergic receptors in adipocytes, the potential of these receptors for discriminating and coordinated metabolic regulation, and current views on the role of UCP2 and UCP3 based on physiological studies and gene knockout models.

Phosphodiesterase 3B gene expression is enhanced in the liver but reduced in the adipose tissue of obese insulin resistant db/db mouse.

Phosphodiesterase (PDE) 3B, when activated by insulin, causes a decrease in intracellular cAMP concentration. The activation of this enzyme results in the reduced output of free fatty acids (FFA) from adipocytes, and an increased lipogenesis in liver. We have recently shown that PDE3B gene expression is reduced in adipose tissues of KKAy mice. We intend to further elucidate the regulation of PDE3B in liver as well as adipose tissues in relation to the insulin resistant state. We examined PDE3B gene expression in liver and adipose tissues of obese, insulin-resistant diabetic db/db mice and also checked the effect of an insulin-sensitizing drug, troglitazone, on this gene expression. In the liver of db/db mice, PDE3B mRNA, its corresponding protein, and the associated catalytic activity were all increased by 2.1, 1.9 and 1.6-fold, respectively, over those in db/+ control mice. Histological examination revealed substantial triglyceride storage in the liver of db/db mice. Conversely, in the adipose tissue of db/db mice, PDE3B mRNA, protein, and its associated activity were all decreased by 0.38, 0.33 and 0.36-fold, respectively. Troglitazone, which has no effect on PDE3B in liver, increased the expression of this gene in adipocytes. This increase is associated with a reduction in the elevated levels of serum insulin, glucose, FFA and triglycerides. The reduced PDE3B gene expression in adipose tissues, which results in the elevation of serum FFA, could be the primary event in the development of insulin resistance in db/db mice. The enhanced PDE3B gene expression may correlate with changes in triglyceride storage in the liver of these mice.

Osteoarthritic articular chondrocytes stimulate autologous T cell responses in vitro.

OBJECTIVE: To clarify the presence of specific T cell immune response to autologous chondrocytes in patients with osteoarthritis (OA). METHODS: Peripheral blood mononuclear cells obtained from OA or post-traumatic patients were co-cultured with irradiated autologous chondrocytes, and their proliferative response was assessed using 3H-thymidine incorporation. Expression of HLA-class II molecules was also assessed on chondrocytes by immunohistochemistry or flow cytometry. RESULTS: T cell responses to autologous chondrocytes in OA yielded a significantly greater mean stimulation index (6.35 +/- 1.63) compared to controls (1.21 +/- 0.09, p < 0.01). This response was partially blocked by antibodies against HLA class I, class II, CD4 or CD8. Increased expression of HLA-DP, -DQ, and -DR was observed. CONCLUSION: This study showed the autologous T cell-stimulating property of OA chondrocytes in vitro. The elucidation of the autoimmune responses may contribute to the understanding of immune-mediated mechanisms in OA.