Peptidylarginine deiminase is involved in maintaining the cornified oral mucosa of rats.

BACKGROUND AND OBJECTIVE: Epithelial cells derived from different regions exhibit marked differences in their differentiation capacity, allowing them to provide a suitable protective barrier. We aimed to clarify the role of peptidylarginine deiminase (PAD) in modifying the key epidermal proteins filaggrin (FLG) and keratin 1 (K1) during stratification of the rat palate and buccal mucosa. MATERIAL AND METHODS: We performed immunofluorescence, immunoblotting, PAD activity assays and 2-dimensional electrophoresis, and developed an organotypic culture model. RESULTS: PAD1 expression was highest in the palate, whereas PAD2, PAD3 and PAD4 expression was highest in the skin, suggesting the tissue-specific expression of PAD isozymes that leads to differences in calcium dependency. Immunoblotting showed that the FLG monomer, as well as its degradation products and precursor (proFLG), were most abundantly expressed in the skin but had low expression in the palate, whereas only faint proFLG expression was detected in the buccal mucosa. FLG and K1 were colocalized with PAD1 and were likely to be citrullinated in the cornified layers of the skin; this colocalization was not detected on the palatal surface, and dot-like presence of proFLG that might be citrullinated and that of PAD1 were found in the granules of the palate. Organotypic models derived from the rat palate revealed that PAD inhibition reduced the breakdown of FLG, increased its association with K1 together with epithelial compaction, and decreased permeability in a dye permeability assay. Conversely, PAD stimulation had the opposite effects. CONCLUSION: Citrullination is likely a protein modification that plays an important role in maintaining the structure and function of oral cornified mucosa in a way that is distinctly different from that of the skin.

Clinical efficacy and treatment persistence of monthly minodronate for osteoporotic patients unsatisfied with, and shifted from, daily or weekly bisphosphonates: the BP-MUSASHI study.

UNLABELLED: This multi-center, prospective, open-label, observational study evaluated the effects of once-monthly minodronate (50 mg) on treatment persistence, bone turnover markers, bone mineral density, low back pain, and upper gastrointestinal symptoms in outpatients with osteoporosis previously treated with daily or weekly bisphosphonate products. INTRODUCTION: The purposes of this study were to investigate the effects of once-monthly oral minodronate (MIN 50 mg) on bone turnover markers and bone mineral density, low back pain, and upper gastrointestinal symptoms, as well as preference for and treatment persistence of MIN 50 mg among Japanese osteoporosis patients currently treated with daily or weekly bisphosphonates. METHODS: Study patients were allocated based on their preference to either the Switch group (patients willing to switch over to MIN 50 mg) or the Continue group (patients wanting to continue their current therapies). Patients' treatment persistence and satisfaction levels with the therapies were assessed using a self-administered questionnaire. The study endpoints were serum TRACP-5b, serum P1NP, bone mineral density, upper gastrointestinal symptoms, and low back pain. RESULTS: In total, 264 and 133 patients were allocated into the Switch and Continue groups, respectively. Approximately, 65 % of patients were willing to switch to MIN 50 mg, with the predominant reason being "less frequent dosing more convenient." Treatment persistence was significantly higher in the Switch group (MIN 50 mg) than the Continue group. Almost all patients with abnormal bone metabolism markers demonstrated normalization after switchover. MIN 50 mg alleviated low back pain and upper gastrointestinal symptoms induced by prior bisphosphonate use. CONCLUSIONS: MIN 50 mg alleviates low back pain, reduces bone turnover markers and increases bone density, and induces fewer upper gastrointestinal symptoms after switchover from prior bisphosphonate products, and therefore, it may provide patients with a more convenient treatment option and enhance long-term treatment persistence.

Deiodinase 2 upregulation demonstrated in osteoarthritis patients cartilage causes cartilage destruction in tissue-specific transgenic rats.

OBJECTIVE: Chondrocyte hypertrophy followed by cartilage destruction is a crucial step for osteoarthritis (OA) development, however, the underlying mechanism remains largely unknown. The objectives of this study are to identify the gene that may cause cartilage hypertrophy and to elucidate its role on OA pathogenesis. DESIGN: Gene expression profiles of cartilages from OA patients and normal subjects were examined by microarray analysis. Expression of deiodinases, enzymes for regulation of triiodothyronine (T3) biosynthesis, in human and rat articular cartilage (AC) were examined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Rat ACs and chondrocytes were treated with T3 to investigate its role on chondrocyte hypertrophy and inflammatory reaction. Cartilage-specific Type II deiodinase (DIO2) transgenic rats were generated using bacterial artificial chromosome harboring the entire rat Col2a1 and human DIO2 gene. An experimental OA model was created in the animal to examine the role of DIO2 on cartilage degeneration. RESULTS: DIO2 is highly expressed in OA patient AC compared to normal control. In rat AC, DIO2 is specifically expressed among deiodinases and dominantly expressed the same as in brown adipose tissue. T3 induces hypertrophic markers in articular chondrocyte and cartilage explant culture, and enhances the effect of IL-1α on induction of cartilage degrading enzymes. Importantly, cartilage-specific DIO2 transgenic rats are more susceptible to knee joint destabilization and develop severe AC destruction. CONCLUSION: Our findings demonstrate that upregulated expression of DIO2 in OA patient cartilage might be responsible for OA pathogenesis by enhancing the chondrocyte hypertrophy and inflammatory response.

Identification of Hic-5 as a novel regulatory factor for integrin αIIbß3 activation and platelet aggregation in mice.

BACKGROUND: Integrin αIIbß3 plays key roles in platelet aggregation and subsequent thrombus formation. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbß3 at its cytoplasmic strand. OBJECTIVES: Hic-5 function in αIIbß3 activation and subsequent platelet aggregation remains unknown. To address this question, platelets from Hic-5(-/-) mice were analyzed. METHODS AND RESULTS: Hic-5(-/-) mice displayed a significant hemostatic defect and resistance to thromboembolism, which were explained in part by weaker thrombin-induced aggregation in Hic-5(-/-) platelets. Mechanistically, Hic-5(-/-) platelets showed limited activation of αIIbß3 upon thrombin treatment. Morphological alteration in Hic-5(-/-) platelets after thrombin stimulation on fibrinogen plates was also limited. As a direct consequence, the quantity of actin co-immunoprecipitating with the activated αIIbß3 was smaller in Hic-5(-/-) platelets than in wild-type platelets. CONCLUSION: We identified Hic-5 as a novel and specific regulatory factor for thrombin-induced αIIbß3 activation and subsequent platelet aggregation in mice.

Creation of an improved mutant TNF with TNFR1-selectivity and antagonistic activity by phage display technology.

Tumor necrosis factor-alpha (TNF), which binds two types of TNF receptors (TNFR1 and TNFR2), regulates the onset and exacerbation of autoimmune diseases such as rheumatoid arthritis and Crohn's disease. In particular, TNFR1-mediated signals are predominantly related to the induction of inflammatory responses. We have previously generated a TNFR1-selective antagonistic TNF-mutant (mutTNF) and shown that mutTNF efficiently inhibits TNFR1-mediated bioactivity in vitro and attenuates inflammatory conditions in vivo. In this study, we aimed to improve the TNFR1-selectivity of mutTNF This was achieved by constructing a phage library displaying mutTNF-based variants, in which the amino acid residues at the predicted receptor binding sites were substituted to other amino acids. From this mutant TNF library, 20 candidate TNFR1-selective antagonists were isolated. Like mutTNF, all 20 candidates were found to have an inhibitory effect on TNFR1-mediated bioactivity. However, one of the mutants, N7, displayed significantly more than 40-fold greater TNFR1-selectivty than mutTNF. Therefore, N7 could be a promising anti-autoimmune agent that does not interfere with TNFR2-mediated signaling pathways.

Encephalomyelitis of cattle caused by Akabane virus in southern Japan in 2006.

Six calves, aged between 55 days and 15 months, were presented between September and November 2006 with neurological signs including limb weakness and circling. Microscopical examination of the brain and spinal cord revealed the presence of non-suppurative encephalitis in all animals. Perivascular cuffing of lymphocytes and macrophages and diffuse gliosis was prominent in the cerebrum and degeneration and/or necrosis of neurons with vacuolation of the neuropil was present in the brainstem. Neuronal necrosis and neuronophagia were noted in the ventral horn of the spinal cord. The distribution of the lesions was closely related to the clinical signs displayed by each calf. Five calves presenting with astasia with low head carriage or torticollis had lesions throughout the central nervous system (CNS). The oldest calf displayed astasia caused by weakness of the "hindlimb" one word and had lesions largely restricted to the caudal spinal cord. Akabane virus (AKAV) antigens were detected immunohistochemically within neurons and axons in lesional tissue. Virus was not isolated from CNS tissue but the AKAV S gene was detected in this tissue from five calves by reverse transcriptase polymerase chain reaction (RT-PCR). It is suggested that AKAV infection is likely to have occurred during the early life period in the calves of this study.

Clinical evaluation of polypropylene glycol-based caries detecting dyes for primary and permanent carious dentin.

OBJECTIVE: The aim of this study was to compare the clinical efficacy of new caries detecting dye Caries Check Blue (CCB) with Caries Check (CC) and Caries Detector (CD) using a laser fluorescence device (DIAGNOdent). METHOD: Primary and permanent teeth with dentin caries were stained with polypropylene glycol (MW=300) based new caries detecting dyes CCB, CC, or propylene glycol (MW=76) based CD. In the CCB and CC groups, stained dentin was completely removed. In the CD groups, pink-stained dentin was retained according to the manufacturers' instructions. Cavities before and after caries removal were measured with the DIAGNOdent. Data were analyzed using ANOVA and Fisher's PLSD multiple comparison test at alpha=0.05. Regression analyses were performed between DIAGNOdent readings and scores obtained from the clinical parameters. RESULTS: The DIAGNOdent readings after caries removal were: primary-CCB (13.2+/-10.4), primary-CC (14.3+/-16.7), primary-CD (9.0+/-5.2), permanent-CCB (22.7+/-13.4), permanent-CC (10.6+/-6.8) and permanent-CD (9.7+/-9.0). Significant differences were identified between the permanent-CCB and all other groups. Correlation coefficients between DIAGNOdent readings and clinical parameters were low. CONCLUSIONS: When dentin stained with Caries Check Blue or Caries Check was completely removed, the DIAGNOdent readings were higher than those recorded when palely-stained pink dentin was retained with the Caries Detector, with significant difference observed for the permanent-CCB group. Caries Check Blue may be used clinically to avoid excessive removal of caries-affected or sound dentin in permanent teeth but not in primary teeth.

Prognostic importance of vascular endothelial growth factor and its receptors in the uterine sarcoma.

Vascular endothelial growth factor (VEGF) and its receptors play an important role in tumor progression; however, there is no report regarding this factor in uterine sarcoma. Thirty-nine patients with uterine sarcoma, 14 carcinosarcomas, 4 endometrial stromal sarcomas, and 21 leiomyosarcomas, were studied. By immunohistochemical staining, VEGF was not detected in normal uterine smooth muscle, but VEGF receptor-1 (flt-1) and VEGF receptor-2 (flk-1) were observed in 14 and 4 of 14 normal smooth muscles, respectively. Of 39 sarcomas, 25 expressed VEGF, and 38 and 34 sarcomas expressed flt-1 and flk-1 at various intensities, respectively. The staining intensity of VEGF, flt-1, and flk-1 was significantly higher in sarcoma than in normal uterine smooth muscle, but that of phospho-flt-1 (p-flt-1) was significantly lower in sarcoma than in normal uterine smooth muscle. When sarcomas were divided into two groups according to staining intensity, a significant difference in survival curves was observed in only p-flt-1 of leiomyosarcoma (P= 0.008), and in all sarcomas, a lower survival curve was also observed in the high staining intensity group than in the low staining intensity group, although there was no significant difference (P= 0.102). In conclusion, VEGF and its receptors are suggested to be involved in progression of uterine sarcoma, but only the p-flt-1 level significantly affected the survival of leiomyosarcoma patients.

Possible involvement of urokinase-type plasminogen activator release from human peripheral blood lymphocytes in the pathophysiology of chronic allograft nephropathy.

BACKGROUND: Little is known of the fibrinolytic host immune mechanisms responsible for induction of chronic allograft nephropathy (CAN), defined as a loss in glomerular filtration rate (GFR) caused by tubular atrophy and interstitial fibrosis, often with fibrous intimal thickening in the small arteries. However, chronic rejection has been reported to be associated with decreased activity of the fibrinolytic system. In our previous study, [Deamino-Cys1, D-Arg8]-vasopressin (dDAVP) induced urokinase-type plasminogen activator (uPA) release from human peripheral T lymphocytes via arginine vasopressin (AVP) V2-receptor-mediated reaction enhanced by an AVP V1-receptor antagonist. Therefore, we examined the level of uPA released from peripheral T lymphocytes by AVP in transplant patients with CAN in comparison with control groups. PATIENTS AND METHODS: In this study, we evaluated in vitro uPA releasing activity of lymphocytes obtained from renal allograft patients with well-functioning grafts (n = 9), CAN (n = 5), or acute rejection episodes (n = 5) compared with lymphocytes from healthy volunteers with normal renal function (n = 12) or patients with renal insufficiency (n = 5). RESULTS: Lymphocytes prepared from patients with chronic allograft nephropathy showed a significantly lower increase in uPA release induced by the combination of the V1-receptor antagonist and dDAVP compared with those from the other groups. CONCLUSION: This finding suggested that a decrease in uPA release from human peripheral blood lymphocytes by AVP-related peptides may be potentially involved in the pathophysiology of CAN.

Simplified method for cryopreservation of islets using hydroxyethyl starch and dimethyl sulfoxide as cryoprotectants.

Cryopreservation is an ideal method for long-term storage of human islets. Dimethyl sulfoxide (DMSO) has been used as an intracellular cryoprotectant. However, because of its toxicity, DMSO has to be added stepwise and diluted stepwise with sucrose. We combined hydroxyethyl starch (HES) as an extracellular cryoprotectant with DMSO to simplify the freeze-thawing procedure. Islets were isolated from the pancreas of beagle dogs by an automated digestion method and Ficoll purification. After overnight culture, the islets were cryogeneically stored using cooling by a programmed freezing system. After 4-week storage in liquid nitrogen, the container was rapidly thawed in a 37 degrees C water bath. The function of the islets was assessed upon static incubation immediately after thawing, showing a recovery rate of 71.16% +/- 20.14% and a stimulation index of 1.80 +/- 0.78. In conclusion use of HES allowed a decrease in DMSO concentration and simplified the freeze-thawing procedure for islets.

Prolongation of islet allograft survival in mice by combined treatment with pravastatin and low-dose cyclosporine.

Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, is known to have suppressive effects on immune and inflammatory cells. We have previously shown in mice and dogs that this agent prevents primary nonfunction of islet iso- and autografts by reducing inflammation at the graft site. The present study was designed to further investigate whether pravastatin has a synergistic effect with cyclosporine (Cs) to prolong islet allograft survival in mice. Unpurified 3000 BALB/c newborn islets were transplanted under the renal capsule of a streptozotocin-diabetic C57BL/6 mouse. Pravastatin and Cs were administered for 10 days starting on the day of grafting (day 0). Five groups were set up based on the treatment protocol: group 1, treatment with 40 mg/kg pravastatin; group 2, 30 mg/kg Cs; group 3, 50 mg/kg Cs; group 4, 40 mg/kg pravastatin and 30 mg/kg Cs; group 5, vehicle alone. Graft survival was indicated by blood glucose levels sustained at <200 mg/dl, and graft rejection by >250 mg/dl for 2 consecutive days. Hyperglycemia persisted in six of the eight (75%) mice and grafts were rejected in 3.6 +/- 0.5 days (mean +/- SD) in group 5. In group 1, grafts were also rejected in 3.8 +/- 0.8 days, but blood glucose was transiently <200 mg/dl in three of the five mice. Despite Cs, grafts were rejected between 7 and 15 days (10.3 +/- 2.4 days) in group 2. Among six mice in group 3, one maintained euglycemia for >60 days, the other rejected the graft on day 15, and the remaining four died with functioning grafts between 9 and 13 days due to Cs toxicity. A combination of a low dose of Cs and pravastatin (group 4) prolonged graft survival for >19 days in five of the eight mice, and for 7-13 days in the remaining three mice. Histological examination of the grafts in this group showed significantly reduced local inflammation. Results indicate a synergistic effect of pravastatin and Cs on prevention of islet allograft rejection.

Increased islet viability by addition of beraprost sodium to collagenase solution.

The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 nM was added to the collagenase solution. After purification, the islet yield was 434,561 +/- 35.691 islet number expressed as 150 microm equivalent size (IEQ)/pancreas or 8,799 +/- 345 IEQ/g of pancreas in the BPS group and 349,987 +/- 52,887 IEQ/pancreas or 7,998 +/-1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 +/- 0.7% in the BPS group and 82.0 +/-0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number x % viability) was 384,586 +/- 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 +/- 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 +/- 38.5 versus 92.3 +/- 25.6 microU/IEQ; P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves beta cell function.

Involvement of platelet-derived growth factor and histocompatibility of DRB 1 in chronic renal allograft nephropathy.

BACKGROUND: The role of activated T cells in graft arteriosclerosis, which is observed in chronic renal allograft nephropathy, and the involvement of major histocompatibility complex (MHC) incompatibility remain to be determined. We examined the effect of T lymphocytes that were obtained from renal transplant patients undergoing chronic rejection treated with cyclosporine (CsA) on platelet-derived growth factor (PDGF)-induced proliferation of cultured human vascular smooth muscle cells (SMC) and compared the proliferation activity of T lymphocytes with MHC incompatibility, especially DRB 1 mismatch. METHODS: Renal transplant patients with continued allograft function, who survived more than 1 year after transplantation, were recruited. Chronic rejection was documented by graft-biopsy findings together with increasing serum creatinine levels (10-20% per year). After the incubation of supernatant (conditioning medium) of cultured T cells from CsA-treated renal transplant patients with chronic rejection (n=18) and with normal renal function (n=14) as control, normal subjects (n=11) and chronic hemodialysis (HD) patients (n=5) with cultured SMC in the presence or absence of PDGF, DNA synthesis (3H-thymidine uptake) of SMC was examined. The in vitro effects of CsA on DNA synthesis of cultured SMC were also evaluated. RESULTS: The supernatant of cultured T cells from renal transplant patients with chronic rejection stimulated PDGF-induced DNA synthesis of SMC in a dose-dependent manner, showing significant enhancement as compared with control transplant patients, normal subjects, and chronic HD patients. The supernatant itself did not significantly stimulate DNA synthesis of SMC. No significant in vitro stimulation of CsA on DNA synthesis was observed. The supernatant of T cells obtained from recipients undergoing chronic rejection with two DRB 1 mismatches showed significantly higher enhanced activity of PDGF-induced DNA synthesis than the supernatant from those recipients without mismatch of DRB 1. On the other hand, no significant correlation of the enhanced activity by T cell supernatant to HLA A and B mismatch numbers was observed. CONCLUSIONS: Growth factor-promoting factors(s) derived from activated T cells associated with MHC class II DR expression, which promotes PDGF-induced proliferation of SMC, exists in renal transplant patients with chronic renal allograft nephropathy, and is probably involved in arteriosclerosis of the graft kidney.

A simple titration method for determining Chlamydia infectivity using an image analyzing system.

In the biological study of Chlamydia, it is very important to determine the infectivity titer of the organism. For many years researchers used the serial dilution method to determine this titer. This method consists of diluting the material to be examined, inoculating suitable dilutions into susceptible cell cultures and cultivating them. The number of inclusions formed in host cells can be calculated with the naked eye under a microscope. The precision and accuracy of this method, however, depend on the number of inclusions per field and the number of fields counted. In this report, we present a simple and rapid method for counting a large number of inclusions using an image analysis system and an appropriate number of samples, and propose a sampling method based on a statistical analysis of the data obtained with 84 microscopic fields.

Medical versus surgical androgen suppression therapy for prostate cancer: a 10-year longitudinal cost study.

PURPOSE: We provide a relative cost comparison of medical versus surgical androgen suppressive therapy for prostate cancer. MATERIALS AND METHODS: Comparison is based on a cohort of 96 patients who began androgen suppressive therapy for prostate cancer between 1988 and 1990. Patients were followed until death or the end point of study in June 2000 at which time 15% were alive. Current Medicare orchiectomy reimbursements were compared to 1999 wholesale drug costs. RESULTS: For an individual patient the cost of luteinizing hormone releasing hormone (LH-RH) agonist treatment surpassed the cost of surgery at less than 4.2 to 5.3 months, and for combined androgen blockade (LH-RH agonists and nonsteroidal antiandrogens) at less than 2.7 to 3.4 months. For 5 (5.2%) patients on combined androgen blockade and 6 (6.3%) on LH-RH agonists alone, medical therapy would have had a cost advantage over bilateral orchiectomy. For the androgen suppression cohort the cost of LH-RH agonist treatment was 10.7 to 13.5 times and combined androgen blockade was 17.3 to 20.9 times the cost of bilateral orchiectomy. Urology resource use comparisons are provided. These findings significantly underestimate the cost advantage of surgery. A seventh of the patients were alive at study end point, and prostate specific antigen induced stage shifting and changes in practice patterns resulted in earlier and more frequent androgen suppressive treatment. CONCLUSIONS: Except for patients with short anticipated survivals current medical androgen suppressive treatment options are more costly than bilateral orchiectomy. There is a need for a cost comparable medical option to orchiectomy.

Validity of cadmium concentration in rice as the "dose" of the dose-response relationship between cadmium intake and renal dysfunction.

It is well known that cadmium (Cd) causes renal dysfunction such as increase of beta(2)-microglobulin excretion into urine. Although Cd in rice seems to be one of the largest sources of total Cd intake in Japan, there are very few studies that have epidemiologically clarified the relationship between Cd concentration in rice (Cd-R) and renal dysfunction, because such studies are basically ecological studies, in which confounding factors are difficult to take into consideration. To derive safety levels for foodstuff from Cd-R, it is essential to evaluate the effect of confounding factors. Thus, we investigated the dose-response relationship between renal dysfunction and not only Cd-R but also confounding factors, and we tried to determine whether Cd-R is an adequate indicator of "dose" in the dose-response relationship between Cd intake and renal dysfunction. In 1971, Cd-R data were obtained from rice samples collected by the Environment Agency, Government of Japan in the Fuchu area of Toyama Prefecture, which is known as a place where many itai-itai disease patients were found, and medical data were collected during 1979-1984 by Toyama Prefecture. First, the dose-response relationship between Cd-R and renal dysfunction was analyzed using the data from the Fuchu area. Second, to investigate the effect of confounding factors, analysis using the data from both the Fuchu area and an unpolluted area with environmental factors different from those of the Fuchu area was performed. The results showed that the cause of renal dysfunction could not be explained by Cd-R alone, and confounding factors were not negligible. Although it is difficult to clarify precisely the confounding factors from the available data, it is concluded that deriving a safety level for foodstuffs using only the Cd-R level as a reference is not appropriate.