CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration.

We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKL-stimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKL-dependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration. INTRODUCTION: We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells. MATERIALS AND METHODS: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay. RESULTS AND CONCLUSIONS: We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA- or CCR1 siRNA-treated cells were used. Moreover, treatment with a Galpha inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.     

Malignant lymphoma occurring subsequent to autoimmune disease]

The authors report six cases of non-Hodgkin's lymphoma (NHL) (3B-cell type, one T-cell type, one non-T non-B cell type, one unclassified type) occurring subsequently to autoimmune diseases. The patients were females aged 43 to 70 (median 61). Rheumatoid arthritis was most frequent as the preceding autoimmune disease, and the intervals from the onset of an autoimmune disease to that of NHL were 10 to 36 years (median 20). Polyclonal hypergammaglobulinemia was seen in 4 cases, lymphocytopenia in 3 cases, and conversion to negative PPD reaction in 2 cases. Only one patient had been given corticosteroids, and immunosuppressive agents may not contribute much to the development of lymphoma in patients with autoimmune diseases.     

Metabolic expression of intrinsic developmental programs for dentine and enamel biomineralization in serumless,chemically-defined,organotypic culture

Summary Biomineralization was investigated using embryonic mouse mandibular first molars (M₁) cultured in the presence or absence of fetal calf serum. Metabolic features including cell division and Ca²⁺ and phosphate incorporation into dentine and enamel extracellular matrices were analyzed. The relative timing and magnitude of DNA synthesis for serumless cultures was comparable toin vivo controls. Isotopic calcium and phosphate incorporation into the mineral phase of dentine and enamel matrices, in the absence of serum, fluctuated during development. Molar tooth morphogenesis, cytodifferentiation, and extracellular matrix formation approximated late crown-stage development in serumless cultures. Von Kossa histochemical staining indicated calcium phosphate salt formation in serumless cultures. Analysis of anhydrous fixation-prepared enamel and dentine representing serumless cultured explants indicated that crystal size and orientation were comparable toin vivo enamel and dentine. In contrast, serum-supplemented cultures showed atypical crystal size and orientation. Calcium/phosphorous (Ca/P) ratio values for serumless cultures after 21 days showed Ca/P enamel values of 2.03 (SD±0.04, p<0.025) and dentine values of 1.89 (SD±0.01, p<0.025). Electron diffraction patterns of enamel and dentine formed in serumless cultures were principally those of highly-ordered crystalline hydroxyapatite. Our results suggest that tissue-specific dentine and enamel biomineralization is regulated by endogenous factors intrinsic to the developmental program of embryonic tooth organs during serumless culture.     

An automated on-line method for simultaneous analysis of phenothiazines in human serum by high-performance liquid chromatography/sonic spray ionization mass spectrometry using backflush column switching

An automated on-line method for simultaneous analysis of five phenothiazine drugs by high-performance liquid chromatography (HPLC)/sonic spray ionization mass spectrometry (SSI-MS) has been established, using backflush column switching. A 400-μl portion of serum sample diluted 81-fold with distilled water was subjected to the on-line system. In the system, an Oasis HLB cartridge was used as the precolumn for extraction; large molecules such as proteins in serum were discarded by use of distilled water containing 0.1% formic acid as a mobile phase. After switching a valve, the analytes trapped in the precolumn were eluted in the backflush mode and separated by a Chromolith Performance RP-18e column, which is composed of C₁₈-bonded monolithic silica. The column effluents were then introduced into the SSI-MS. The present method provided successful separation and determination of six phenothiazines including an internal standard. Satisfactory linearities, reproducibility, and sensitivity were obtained at concentration levels that matched the toxic levels of phenothiazines. All drug peaks appeared within 18 min, and the system could be reequilibrated in only about 8 min for the next run. Because of the simplicity and rapidness of the method, it is likely to be useful in the fields of emergency medicine and forensic toxicology.     

A novel compound heterozygous dysferlin mutation in Miyoshi myopathy siblings responding to dantrolene

Miyoshi myopathy (MM) is an autosomal recessive distal muscular dystrophy characterized by mutations of the dysferlin gene. Although several pairs of homozygous/heterozygous mutations have been reported, few effective treatments of MM are available. We had observed the decreased serum creatine kinase (CK) before and after administration of dantrolene in the elder brother and the increased serum CK before and after discontinuance of the drug on suspicion of drug-induced hepatopathy in the younger sister. We report a novel pair of heterozygous mutations in the 3'-splicing site of exon 26 and the translation site of exon 28 of the dysferlin gene in two siblings, and effective treatment of their MM with dantrolene.     

Inhibition of IgE formation in mice by glycoproteins from corn silk

The inhibitory effects of glycoproteins separated from a hot water extract of corn silk (U-CSE) on the formation of IgE antibodies after primarily and secondarily challenged responses with dinitrophenyl (DNP)-ovalbumin (OVA) antigen in mice were investigated using the passive cutaneous anaphylaxis (PCA) test. When U-CSE was given intranasally or intraperitoneally the day before primary immunization, IgE antibody production was strongly inhibited. Furthermore, it was found that new formation of IgE antibodies was readily inhibited by U-CSE administration in mice with high levels of IgE after primary immunization. It was also found that U-CSE markedly suppressed IgE antibody formation in secondarily challenged responses with the antigen. U-CSE may be clinically applicable to type I allergic diseases.     

Effect of body positions and splints in bioelectrical impedance analysis.

The objective of this study was to evaluate body composition as measured by bioelectric impedance analysis using splints and body positions differing from the standard supine position. Forty-three patients, randomized into two groups of different body positions, and 101 healthy volunteers were prospectively studied. Resistance and reactance of body tissues were measured by bioelectric impedance analysis. Body composition is described by a three-compartment model composed of body fat, body cell mass, and extra cellular mass. The patients were measured in the standard supine position and then randomized into two groups. They were then remeasured with the appropriate splinting device or position change. Volunteers were measured in the standard supine position and all four alternative positions. There was a statistically significant difference demonstrated in whole body resistance, whole body reactance, body cell mass, and the ratio of extracellular mass to body cell mass in some body positions. The percentage of change with different body positions and splints, when compared with the standard supine position, was generally below 2%, a clinically insignificant difference. We conclude that the reliability of resistance and reactance as measured by bioelectric impedance analysis is clinically valid using any of the tested body positions and/or splints. The three-compartment model may be a useful concept to measure body composition changes in both healthy and sick persons.     

Evaluation of the measurement of plasma fructosamine concentration in aged subjects

Recently, plasma fructosamine concentration has been used as an indication of mean plasma glucose level preceding at last 1 to 2 weeks. In the present study, to characterize the clinical significance and problems of plasma fructosamine concentration in aged subjects (greater than or equal to 65 yrs), we determined plasma fructosamine concentration as well as serum albumin, total protein, HbA1, AbA1c and fasting plasma glucose concentrations in 81 (less than 65 yrs) non-diabetic subjects (group A), 161 aged (greater than or equal to 65 yrs) non-diabetic subjects and 26 aged diabetics (group D). Aged non-diabetic subjects were further classified into 75 subjects with good ADL (group B) and 86 with poor ADL (group C). The normal limit of plasma fructosamine concentration (mean +/- 2SD) in group A was 24% higher (3.1 mmol/l) than that in group B (2.5 mmol/l) but the plasma fructosamine/serum albumin ratio (F/ALB) was similar in these two groups. Plasma fructosamine correlated negatively (p less than 0.01) with age. This aging effect was explained by the reduced serum albumin in aged subjects. However, in group C, reduced plasma albumin was not associated with reduced plasma fructosamine. Plasma fructosamine corrected by albumin (F/ALB) is a useful parameter of blood glucose control in aged subjects. In aged subjects with poor ADL, HbA1, HbA1c and plasma glucose should be determined with fructosamine.