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.

Overexpression of bone morphogenetic protein-3b (BMP-3b) in adipose tissues protects against high-fat diet-induced obesity.

BACKGROUND: Bone morphogenetic protein-3b (BMP-3b) is a member of the transforming growth factor-ß superfamily and has several activities that differ from those of other BMPs. We previously found that BMP-3b is highly expressed in adipocytes, its level is increased during obesity, and it inhibits adipogenesis by suppressing peroxisome proliferator-activated receptor γ (PPARγ) in vitro. However, the function of BMP-3b in adipose tissues in vivo remains unknown. METHODS: To determine the role of BMP-3b overexpression in adipose tissues in vivo, we generated transgenic mice (BMP-3b Tg) by using a conditional overexpression approach in fatty acid-binding protein 4-expressing adipocytes. We examined BMP-3b Tg mice fed a high-fat diet to elucidate the effects of BMP-3b on obesity. Adipocyte function was evaluated as expression of adipogenic and lipogenic markers in adipose tissue. We also performed glucose and insulin tolerance tests (GTT and ITT, respectively), and biochemical analysis of serum and measured energy expenditure by indirect calorimetry. RESULTS: BMP-3b Tg mice fed a high-fat diet showed decreases in weight gain, fat-pad mass and adipocyte area, compared with wild-type mice. The adipose tissues of BMP-3b Tg mice showed downregulated expression of PPARγ and its target gene encoding fatty acid translocase/CD36. In addition, BMP-3b Tg mice had decreased blood glucose levels on GTT and ITT, and their serum leptin levels were decreased and adiponectin concentrations were increased. These changes in BMP-3b Tg mice were accompanied by increased energy expenditure, indicated as increased locomotor activity and oxygen consumption. CONCLUSIONS: These results provide in vivo evidence that BMP-3b regulates adipocyte function to cause an anti-obesity effect.

Bone morphogenetic protein-3b (BMP-3b) is expressed in adipocytes and inhibits adipogenesis as a unique complex.

BACKGROUND: Bone morphogenetic protein-3b (BMP-3b) is a member of the transforming growth factor-ß (TGF-ß) superfamily. BMP-3b regulates osteogenesis and has critical roles in developing embryos. BMP-3b is expressed not only in the bone and developing embryos but also in adipose tissues. However, the functions of BMP-3b in adipose tissue are still unknown. METHODS: BMP-3b expression was quantified in various adipose tissues and in the adipose-derived stromal-vascular fraction (SVF) and mature adipocyte fraction (AD.F) of mice. We also used 3T3-L1 preadipocytes to analyze the expression, function and molecular forms of BMP-3b. In order to determine the effects of BMP-3b on the adipogenesis of 3T3-L1 cells, BMP-3b siRNA-mediated knockdown and gene overexpression studies were performed, and a conditioned medium (CM) containing the BMP-3b protein was added to 3T3-L1 cell cultures. Adipocyte differentiation was evaluated by measuring the expression of adipogenic markers or by Oil Red O staining. The molecular form of BMP-3b that was secreted from the 3T3-L1 cells was analyzed by western blotting. RESULTS: BMP-3b is expressed in all adipose tissues and is expressed at higher levels in preadipocytes than in mature adipocytes. In mesenteric adipose tissue, BMP-3b expression was increased in diet-induced obesity (DIO) mice as compared with that in control mice. BMP-3b was also expressed highly in 3T3-L1 cells. We showed that siRNA-mediated knockdown of endogenous BMP-3b expression in 3T3-L1 cells enhanced adipogenesis. Conversely, overexpressing BMP-3b inhibited adipocyte differentiation. We also showed that addition of CM containing the BMP-3b protein inhibited the differentiation of 3T3-L1 cells, and that this inhibitory effect was abolished by removing BMP-3b with an anti-BMP-3b antibody. Furthermore, BMP-3b was secreted from adipocytes as a unique non-covalent complex. CONCLUSION: These data suggest that BMP-3b is secreted from adipocytes and is involved in adipocyte differentiation.

Rat aortic smooth-muscle cell proliferation is bidirectionally regulated in a cell cycle-dependent manner via PACAP/VIP type 2 receptor.

In the cardiovascular system, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been well characterized as potent vasodepressors or vasodilators. However, their pathophysiological implication in proliferation of vascular smooth muscle cells has not yet been elucidated. In the present study, we have first identified PACAP/VIP type 2 receptor as a dominant type in rat vascular smooth muscle cell (VSMC) by RT-PCR. PACAP and VIP increased cyclic AMP accumulation with similar potency. In 24-h [3H]thymidine incorporation assay, PACAP or VIP exhibited a suppressive effect on the DNA synthesis of rat VSMC stimulated by serum when added at the late G1 phase. In contrast, when added at G0/G1 phase of the cell cycle, PACAP or VIP enhanced the serum-induced DNA synthesis. In 24-h incubation, PACAP alone has little mitogenic activity. However, when incubated up to 48 h, PACAP stimulated significantly the DNA synthesis and the cell proliferation of rat VSMC. These results suggest that PACAP and VIP regulate the proliferation of rat VSMC by enhancing or suppressing in a cell cycle-dependent manner and induce delayed mitogenesis and cell proliferation.

Chromosomal sublocalization and microsatellite DNA polymorphism of human adrenomedullin gene.

Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues such as the heart, kidney and vascular cells. We have cloned and sequenced genomic DNA encoding the human AM gene. In this study, we determined that the AM gene was located in the short arm of chromosome 11 (p15.1-3). The 3'-end of the gene is flanked by a microsatellite marker of cytosine adenine (CA) repeats. Moreover, we analyzed this DNA variation in the AM gene in the general Japanese population. Genomic DNA was obtained from the peripheral leukocytes of healthy normotensive subjects, 327 men and 149 women, aged 51 +/- 8 years (mean +/- SD). The genomic DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined via polyacrylamide gel electrophoresis (PAGE). Plasma AM concentration was measured by RIA and compared with respect to the number of CA repeats adjacent to the AM gene. In Japanese, four types of alleles with different CA-repeat numbers; 11, 13, 14 and 19, appear to exist. The frequencies of these alleles were as follows: 11 repeats, 28.8%; 13 repeats, 33.1%; 14 repeats, 35.0% and 19 repeats, 3.1%. This DNA variation does not seem to affect the transcription of the AM gene, because plasma concentrations of AM were not significantly different between the genotypes.

[An optimum dose-finding study of HR810 (CPR) in chronic respiratory tract infections].

In order to determine the optimal dose of cefpirome sulfate (HR810, CPR) against respiratory tract infections (RTI), an optimal dose-finding study was conducted on cases of chronic RTI, and the clinical properties of the drug were compared with those of ceftazidime (CAZ). Inpatients with chronic RTI were randomly assigned to 3 groups: an HR 0.5 g group, receiving 0.5 g X 2/day of CPR an HR 1.0 g group, receiving 1.0 X 2/day of CPR and a CAZ group, receiving 1.0 g X 2/day of CAZ. As a rule, the drugs were administered by intravenous drip infusion for 14 days, after which period clinical efficacy, bacteriological response, safety, and utility were investigated. Of the total 121 cases, 106 were subject to analysis of clinical efficacy, including 38 cases in the HR 0.5 g group, 32 in the HR 1.0 g group, and 36 in the CAZ group. Efficacy rates in the assessment by the committee were 84.2% for the HR 0.5 g group, 75.0% for the HR 1.0 g group, and 86.1% for the CAZ group, without any significant difference between the 3 groups. The bacterial elimination rates were 73.9%, 75.0% m and 88.5%, respectively, without any significant difference between the 3 groups. Associated reactions were noted in 2 of 36 cases in the HR 1.0 g group (eruption and diarrhea), but not in the other 2 groups. The incidence of abnormal clinical laboratory findings was 23.1% in the HR 0.5 g group, 22.2% in the HR 1.0 g group, and 22.5% in the CAZ group, without any significant difference between the 3 groups. Utility rates were 84.2% for the HR 0.5 g group, 74.2% for the HR 1.0 g group, and 86.1% for the CAZ group, without any significant difference between the 3 groups. The HR 0.5 g and 1.0 groups showed no difference in clinical efficacy, bacteriological response, safety, and utility against RTI, and the results of both groups were about equal to those of the CAZ group.

[A comparative study between cefpirome (CPR) and ceftazidime (CAZ) in respiratory tract infections].

Efficacy and safety of a new injectable cephem antibiotic, cefpirome sulfate (hereafter, CPR), against respiratory tract infections were examined and compared with those of a control drug, ceftazidime (hereafter, CAZ). As a rule, CPR 0.5 g twice a day, 1.0 g twice a day, or CAZ 1.0 g twice a day (hereafter CPR 0.5 g group, CPR 1.0 g group, and CAZ group) was administered for 14 days and the following results were obtained. 1. The total number of cases was 470 (155 cases in the CPR 0.5 g group, 160 cases in the CPR 1.0 g group, and 155 cases in the CAZ group). Among them 390 cases were subjected to analyses of clinical efficacy by the efficacy evaluation committee (131 cases in the CPR 0.5 g group, 131 cases in the CPR 1.0 g group and 128 cases in the CAZ group). 2. Efficacy rates determined by the efficacy evaluation committee were 82.4% (108/131) for the CPR 0.5 g group, 81.7% (107/131) for the CPR 1.0 g group, and 83.6% (107/128) for the CAZ group. Efficacy rates determined by the physician in charge were 82.0% (105/128) for the CPR 0.5 g group, 80.5% (99/123) for the CPR 1.0 g group, and 88.5% (108/122) for the CAZ group. No statistically significant difference was observed among the 3 groups. In evaluation of equivalency, clinical efficacy for the CPR 0.5 g group and the CPR 1.0 g group determined by the clinical efficacy evaluation committee was proved to be statistically equivalent to that for the CAZ group. 3. In patients with pneumonia, efficacy rates determined by the efficacy evaluation committee were 87.1% (61/70) for the CPR 0.5 g group, 80.7% (71/88) for the CPR 1.0 g group, and 78.9% (56/71) for the CAZ group. Efficacy rates determined by the physician in charge were 85.3% (58/68) for the CPR 0.5 g group, 80.7% (67/83) for the CPR 1.0 g group, and 86.2% (56/65) for the CAZ group and no statistically significant difference was observed among the 3 groups. In patients with chronic respiratory tract infection, efficacy rates determined by the efficacy evaluation committee were 77.0% (47/61) for the CPR 0.5 g group, 83.7% (36/43) for the CPR 1.0 g group, and 89.5% (51/57) for the CAZ group. Efficacy rates determined by the physician in charge were 78.3% (47/60) for the CPR 0.5 g group, 80.0% (32/40) for the CPR 1.0 g group, and 91.2% (52/57) for the CAZ group. No statistically significant difference was observed among the 3 groups.(ABSTRACT TRUNCATED AT 400 WORDS)

[A case of pneumonia caused by Chlamydia pneumoniae, strain TWAR].

A case of pneumonia caused by C. pneumoniae, strain TWAR is described in this paper. A 65 year-old male with a persistent dry cough was admitted to our division for left lower lobe infiltrates of the chest X-ray. The serum antibody titers against mycoplasma and some viruses were not elevated, but the serum antibody titers against TWAR reached the maximum level (IgG X 1024, IgA X 256) using microplate immunofluorescence antibody technique (MFA). Isolation of TWAR was tried by BAL and nasophalingial swabs, but were not successful. TBLB from Lt. S10 revealed TWAR inclusion bodies within alveolar epithelial cells using TWAR specific monoclonal antibody (Washington Research Foundation).

[Double-blind comparative study of roxithromycin (RU 28965) and midecamycin acetate (MOM) in the treatment of pneumonia].

The clinical efficacy and safety of Roxithromycin (RU) were compared with those of Midecamycin acetate (MOM) in patients with pneumonia in a double blind study. RU and MOM were administered orally for 14 days with daily doses of 300 mg (150 mg b.i.d.) and 600 mg (200 mg t.i.d.), respectively. The following results were obtained. 1. RU and MOM were administered to a total of 204 patients (RU: 101, MOM: 103). The clinical efficacy was judged in 150 patients (RU: 70, MOM: 80), with 54 of the patients excluded from the total by the committee. 2. The clinical efficacy rates were 81.4% for RU and 70.0% for MOM on the basis of the committee's judgement. There was no significant difference between the two groups. In the evaluation of the clinical efficacy by the doctors in charge, the efficacy rates were 81.4% for RU and 67.5% for MOM, which constitutes a significant difference between the two groups (p less than 0.05). 3. No significant difference was found between the two drugs in bacteriological efficacy. 4. No significant differences were observed in either the incidence of side effects between RU (4.3%) and MOM (4.0%) or in abnormal changes in the laboratory findings. 5. Regarding the clinical usefulness judged by the committee, RU showed a significantly higher rate than MOM (79.2% vs. 67.9%). There was no significant difference in the judgement by the doctors in charge. From the above results, it was concluded that a daily dosage of 300 mg of RU was equal in usefulness to 600 mg daily of MOM in the treatment of mild to moderate pneumonia.

[A study of antichlamydial effect of rokitamycin].

MICs of a new macrolide antibiotic, rokitamycin (RKM), for Chlamydia psittaci and Chlamydia trachomatis were determined. Meanwhile, the organisms were observed under the electron microscope for morphologic changes with the addition of RKM. 1. MICs of RKM for C. psittaci MP and 3 strains of C. psittaci isolated from budgerigars kept by patients ranged from 0.05 to 0.10 microgram/ml, and those for 3 strains of C. trachomatis B, E and L2 ranged from 0.20 to 0.39 microgram/ml. These MICs were higher than MICs of minocycline (MINO), doxycycline and rifampicin, but lower than MICs of erythromycin and midecamycin against these organisms. 2. The addition of MINO or RKM to C. psittaci Izawa and C. trachomatis L2 at concentrations twice as high as MICs resulted in no formation of elementary body or intermediate form inside the inclusion body, and abnormal enlargement of reticulate body containing irregularly distributed cytoplasmic components.

[A clinical evaluation of fluconazole in the treatment of deep mycosis].

Fluconazole is a novel triazole antifungal agent developed by Pfizer Inc. and available in both oral and intravenous forms. It is characterized by a long serum half-life of 25 to 30 hours and good absorbability into tissues. In the present study, fluconazole was given to 12 patients with deep mycosis orally, intravenously or by local infusion. The patients included 4 cases of candidemia, 1 case each of candidemia and candiduria, candiduria, esophageal candidiasis, Candida hepatic abscess, pulmonary cryptococcosis and septicemia due to unspecified yeasts and 2 cases of pulmonary aspergillosis. Clinical efficacies of fluconazole against these infections were excellent in 2 cases, good in 8 and fair in 2. None of the patients reported any side effects. From the results of the study, fluconazole appears to be a useful and safe drug for the treatment of deep seated mycosis.

[A case of tuberculous pleurisy associated with myoclonus and Quincke's edema due to isoniazid and isoniazid sodium methanesulfonate].

A case of tuberculous pleurisy associated with myoclonus and Quincke's edema due to isoniazid (INH) and isoniazid sodium methanesulfonate (IHMS) was reported. A 75-year-old man was admitted to our division because of chest discomfort and the left chest pain of one month's duration. A conventional chest roentgenogram revealed pleural effusion in the left thoracic cavity. The pleural specimen obtained from the left parietal pleura revealed caseating granuloma. Myoclonus suddenly appeared two months after the administration of antituberculous drugs for tuberculous pleurisy. Therefore, INH was discontinued. Three days later the patient's myoclonus disappeared and nine days later IHMS was newly administered. The patient abruptly developed myoclonus and Quincke's edema. IHMS was discontinued and 30 mg of prednisolone was simultaneously given. Two days later myoclonus disappeared and two days more later Quincke's edema was improved. The lymphocyte stimulation test using IHMS was positive. At that time, levels of serum vitamin B6 were within normal levels. These results suggest that myoclonus may result from epileptogenic action caused by INH or IHMS, and Quincke's edema may result from hypersensitive reaction associated with IHMS.

Bone morphogenetic protein-3b (BMP-3b) gene expression is correlated with differentiation in rat calvarial osteoblasts.

BMP-3b (also called GDF-10) is a novel BMP-3-related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor beta1 (TGF-beta1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-beta1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts.