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.

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.

Anisotropy and corotation of galactic cosmic rays.

The intensity of Galactic cosmic rays is nearly isotropic because of the influence of magnetic fields in the Milky Way. Here, we present two-dimensional high-precision anisotropy measurement for energies from a few to several hundred teraelectronvolts (TeV), using the large data sample of the Tibet Air Shower Arrays. Besides revealing finer details of the known anisotropies, a new component of Galactic cosmic ray anisotropy in sidereal time is uncovered around the Cygnus region direction. For cosmic-ray energies up to a few hundred TeV, all components of anisotropies fade away, showing a corotation of Galactic cosmic rays with the local Galactic magnetic environment. These results have broad implications for a comprehensive understanding of cosmic rays, supernovae, magnetic fields, and heliospheric and Galactic dynamic environments.

Estrogen-receptor-alpha-positive extramammary Paget's disease treated with hormonal therapy.

The patient was an 80-year-old man with scrotal and penile extramammary Paget's disease and prostate cancer. Both diseases were in advanced stages. Tumor cells of extramammary Paget's disease strongly expressed estrogen receptor alpha. The patient was concurrently treated with two kinds of hormonal therapy: the anti-estrogen tamoxifen (20 mg/day orally) for extramammary Paget's disease and the anti-androgen bicalutamide (80 mg/day orally) for prostate cancer. The toxicity of the therapy was mild. All of the metastatic lesions remained stable for 2 months after initiation of dual hormonal therapy. During a follow-up period of 22 months, performance status was well maintained for 17 months. Hormonal therapy may be an alternative for selected cases of advanced extramammary Paget's disease.

Observation by an air-shower array in Tibet of the multi-TeV cosmic-ray anisotropy due to terrestrial orbital motion around the Sun.

We report on the solar diurnal variation of the galactic cosmic-ray intensity observed by the Tibet III air shower array during the period from 1999 to 2003. In the higher-energy event samples (12 and 6.2 TeV), the variations are fairly consistent with the Compton-Getting anisotropy due to the terrestrial orbital motion around the Sun, while the variation in the lower-energy event sample (4.0 TeV) is inconsistent with this anisotropy. This suggests an additional anisotropy superposed at the multi-TeV energies, e.g., the solar modulation effect. This is the highest-precision measurement of the Compton-Getting anisotropy ever made.

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.

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.

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.

Systematic search for single nucleotide polymorphisms in the insulin gene: evidence for a high frequency of -23T-->A in Japanese subjects.

It has recently been shown that the A/A genotype at g.-23 of the insulin gene correlates with impaired insulin secretion in response to body weight gain in subjects of European descent. To examine whether there are single nucleotide polymorphisms (SNPs) in the insulin gene associated with type 2 diabetes, all exons with their flanking sequences for 113 Japanese type 2 diabetic patients and 99 nondiabetic control subjects were analyzed using PCR direct sequencing. We have only found g.-23T --> A, 806G --> C, 1128T --> C, and 1141A --> C, which have previously been reported in alpha (A-C-C-C) and beta (T-G-T-A) alleles. The allele frequency of -23T --> A in control Japanese subjects was 97.4%, whereas that in Europeans is about 30%. The A/A genotype was found in 94 of 99 Japanese subjects (94.9%) and the allele frequencies of 806G --> C, 1128T --> C, and 1141A --> C were all 96.5%. The estimated haplotype frequencies were (A-C-C-C) (96.0%), (T-G-T-A) (2.0%), (A-G-T-A) (1.5%), and (T-C-C-C) (0.5%). No association of these SNPs or haplotypes with type 2 diabetes was evident. Thus, the A/A genotype at the g.-23 of insulin gene was generally high in Japanese subjects, which could account for the fact that they typically secrete lower levels of insulin.

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.

The role of C-C chemokines and their receptors in osteoarthritis.

OBJECTIVE: To evaluate the involvement of the chemokine/chemokine receptor system in cartilage degradation in osteoarthritis (OA). METHODS: Expression of the 4 C-C chemokines monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, and their receptors CCR-2 and CCR-5, was assessed in 11 OA patients and 5 normal controls, by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunochemistry, and flow cytometry on untreated or interleukin-1beta (IL-1beta)- and/or tumor necrosis factor alpha (TNFalpha)-stimulated chondrocytes. The effects of these chemokines on the expression of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases were assayed by RT-PCR and ELISA. The effects on proteoglycan synthesis and release were also assayed, using 35S-sulfate incorporation and 35S-proteoglycan release. RESULTS: The C-C chemokines and their receptors CCR-2 and CCR-5 were found to be expressed in normal and OA chondrocytes. However, regulation of chemokine expression by IL-1beta and TNFalpha differed between normal and OA chondrocytes. Intracellular staining revealed that approximately 20% of the chondrocytes contained CCR-2 and CCR-5 in the cytoplasm, whereas cell surface expression was detected less frequently. Interestingly, RANTES induced expression of its own receptor, CCR-5, suggesting an autocrine/paracrine pathway of the chemokine within the cartilage milieu. Finally, addition of MCP-1 or RANTES not only induced MMP-3 expression, but also inhibited proteoglycan synthesis and enhanced proteoglycan release from the chondrocytes. CONCLUSION: The differential expression of chemokines and their receptors under the regulation of IL-1beta and TNFalpha suggests that the cytokine-triggered chemokine system may play a key role in the cartilage degradation of OA, possibly acting in an autocrine/paracrine manner.

Quantitative analysis of the proliferation of epidermal cells using a human skin organ culture system and the effect of DbcAMP using markers of proliferation (BrdU, Ki-67, PCNA).

The process of re-epithelialization of a wound in the epidermis comprises the following steps: proliferation of epidermal basal cells, migration of epidermal cells to the wound surface, and cell differentiation. In the present study, we evaluated the proliferation of epidermal basal cells, an important process in wound healing, in the wound margin using a human skin organ culture system and immunohistochemical labeling with bromodeoxyuridine (BrdU), Ki-67, and proliferating cell nuclear antigen (PCNA), as markers of cell proliferation. Dibutyryl cyclic adenosine monophosphate (DbcAMP) is a derivative of cAMP and has been shown to modulate human keratinocyte proliferation. The proliferation of keratinocytes was promoted by DbcAMP and particularly strong effects in terms of BrdU labeling index, Ki-67-positive ratio, and PCNA-positive ratio, were seen at 10(-5) M. The skin organ culture system presented here uses adult preputial skin and is a simple technique that uses easily available materials. In addition to identifying S phase cells using BrdU as an index of cell proliferation, the immunohistochemical method for evaluating the expression of Ki-67 and PCNA is very simple. Accordingly, the method described here seems to be useful for evaluating cell dynamics in wound healing.

Identification of novel C253Y missense and Y864X nonsense mutations in the insulin receptor gene in type A insulin-resistant patients.

Mutations in the insulin receptor gene have been reported in cases of type A insulin resistance. We report herein two cases of type A insulin resistance, which involve some novel mutations. Case 1 is a heterozygote of the C253Y missense mutation and case 2 is a heterozygote of the Y864X nonsense mutation. In the C253Y missense mutation in exon 3, a cysteine residue is replaced with tyrosine in the cysteine-rich domain of the alpha subunit. The Y864X in exon 13 results in a truncated receptor, which is devoid of most of the beta subunit. This mutant receptor could not be expressed on a cell membrane since the transmembrane domain is missing. Other significant mutations were not found for the entire coding regions and splice/donor sites.