Osteoinductive potential of recombinant BMP-9 in bone defects of mice treated with antiresorptive agents.

The aim of the present study was to investigate the effects of recombinant human (rh)BMP-9 on bone regenerative potential in a mouse model of antibody-mediated antiresorptive therapy (AMART). A monoclonal anti-murine receptor activator of nuclear factor-kappa B ligand (RANKL) antibody (mAb) was used to create an AMART model in mice. rhBMP-9 combined with collagen membrane was implanted in calvarial defects in mAb-treated mice. After 4 weeks, the bone formative potential in the defects was evaluated by micro-computed tomography and histological approaches. The groups implanted with rhBMP-9-containing collagen membranes demonstrated substantial osteopromotive potential, with significantly greater new bone volume (Sham + BMP-9 group; 0.86 ± 0.29 mm3 and mAb + BMP-9 group; 0.64 ± 0.16 mm3) than control PBS-membranes (Sham + PBS group; 0.44 ± 0.29 mm3 and mAb + PBS group; 0.24 ± 0.12 mm3) in both sham and mAb-treated mice. In line with in vivo study, bone marrow cells isolated from both sham and mAb-treated mice confirmed greater osteogenic potential upon stimulation with rhBMP-9 in vitro. These findings suggest for the first time that local rhBMP-9 administration might be a strategy to accelerate bone regeneration in the context of AMART.

Double sentinel lymph node mapping with indocyanine green and 99m-technetium-tin colloid in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.

Estimation of kidney injury molecule-1 (Kim-1) in patients with lupus nephritis.

OBJECTIVE: Biomarkers of disease activity in lupus nephritis (LN) are needed. Ideally, such biomarkers would be capable of detecting early sub-clinical disease and could be used to gauge response to therapy, thus obviating the need for serial renal biopsies. Much of the focus in the search for LN biomarkers has been on the measurement of urinary chemokines and cytokines in LN patients. However, these have yet to be widely implemented in clinical practice. Kidney injury molecule-1 (Kim-1) is expressed in damaged tubules, but whether urinary (u) and tubular (t)-Kim-1 could serve as a biomarker of active LN is unknown. To investigate the disease activity and histological findings in LN, we evaluated u-Kim-1 levels and t-Kim-1 cells in patients with systemic lupus erythematosus (SLE). METHOD: We measured u-Kim-1 levels and stained t-Kim-1 expression in 57 patients with LN using an ELISA and immunohistochemistry staining. Patients were classified into two groups (active LN, n = 37; inactive LN, n = 20) based on the presence of active renal disease according to the renal SLE disease activity index. correlations of clinical, laboratory data, and histological findings with urinary and t-Kim-1 expression were assessed. RESULT: The u-Kim-1 levels were significantly correlated with the expression of t-Kim-1 (R = 0.64; P = 0.004) in the SLE patients. The active LN patients exhibited elevated u-Kim-1 levels compared to the inactive LN patients. The number of t-Kim-1 cells was also correlated with histological findings (both glomerular and interstitial inflammation). The u-Kim-1 levels were also correlated with proteinuria and tubular damage in the active LN group. The number of t-Kim-1 cells at baseline was significantly correlated with the estimated glomerular filtration rate (R = 0.72; P = 0.005) and serum creatinine (R = 0.53; P = 0.005) after 6-8 months of treatment. CONCLUSION: These data suggest the potential use of the u-Kim-1 levels to screen for active LN and for the estimation of t-Kim-1 expression in renal biopsies to predict renal damage, ongoing glomerular nephritis and tubulointerstitial inflammation, and tubular atrophy.

Hydrogen-fluorine exchange in NaBH4-NaBF4.

Hydrogen-fluorine exchange in the NaBH4-NaBF4 system is investigated using a range of experimental methods combined with DFT calculations and a possible mechanism for the reactions is proposed. Fluorine substitution is observed using in situ synchrotron radiation powder X-ray diffraction (SR-PXD) as a new Rock salt type compound with idealized composition NaBF2H2 in the temperature range T = 200 to 215 °C. Combined use of solid-state (19)F MAS NMR, FT-IR and DFT calculations supports the formation of a BF2H2(-) complex ion, reproducing the observation of a (19)F chemical shift at -144.2 ppm, which is different from that of NaBF4 at -159.2 ppm, along with the new absorption bands observed in the IR spectra. After further heating, the fluorine substituted compound becomes X-ray amorphous and decomposes to NaF at ~310 °C. This work shows that fluorine-substituted borohydrides tend to decompose to more stable compounds, e.g. NaF and BF3 or amorphous products such as closo-boranes, e.g. Na2B12H12. The NaBH4-NaBF4 composite decomposes at lower temperatures (300 °C) compared to NaBH4 (476 °C), as observed by thermogravimetric analysis. NaBH4-NaBF4 (1:0.5) preserves 30% of the hydrogen storage capacity after three hydrogen release and uptake cycles compared to 8% for NaBH4 as measured using Sievert's method under identical conditions, but more than 50% using prolonged hydrogen absorption time. The reversible hydrogen storage capacity tends to decrease possibly due to the formation of NaF and Na2B12H12. On the other hand, the additive sodium fluoride appears to facilitate hydrogen uptake, prevent foaming, phase segregation and loss of material from the sample container for samples of NaBH4-NaF.

Activation of OASIS family, ER stress transducers, is dependent on its stabilization.

Endoplasmic reticulum (ER) stress transducers transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. BBF2 human homolog on chromosome 7 (BBF2H7) and old astrocyte specifically induced substance (OASIS), ER-resident transmembrane proteins, have recently been identified as novel ER stress transducers that have roles in chondrogenesis and osteogenesis, respectively. However, the molecular mechanisms that regulate the activation of BBF2H7 and OASIS under ER stress conditions remain unresolved. Here, we showed that BBF2H7 and OASIS are notably unstable proteins that are easily degraded via the ubiquitin-proteasome pathway under normal conditions. ER stress conditions enhanced the stability of BBF2H7 and OASIS, and promoted transcription of their target genes. HMG-CoA reductase degradation 1 (HRD1), an ER-resident E3 ubiquitin ligase, ubiquitinated BBF2H7 and OASIS under normal conditions, whereas ER stress conditions dissociated the interaction between HRD1 and BBF2H7 or OASIS. The stabilization of OASIS in Hrd1(-/-) cells enhanced the expression of collagen fibers during osteoblast differentiation, whereas a knockdown of OASIS in Hrd1(-/-) cells suppressed the production of collagen fibers. These findings suggest that ER stress stabilizes OASIS family members and this is a novel molecular mechanism for the activation of ER stress transducers.

A nationwide survey for prevalence of hepatitis E virus antibody in qualified blood donors in Japan.

BACKGROUND AND OBJECTIVES: In previous studies, we reported the transmission of hepatitis E virus (HEV) by transfusion, and the frequent detection of HEV markers in Japanese blood donors with elevated ALT levels. For the current study, we carried out a nationwide survey of the prevalence of IgG anti-HEV in qualified blood donors throughout Japan. MATERIALS AND METHODS: The 12,600 samples from qualified blood donors were collected from seven blood centres (1800 per centre) representing nearly all regions of Japan. Samples were from age- and sex-matched blood donors who tested negative for all the current blood screening tests. The samples were screened using the in-house IgG anti-HEV ELISA. Sequentially, the positive samples were tested by the commercial IgG anti-HEV ELISA. RESULTS: Of 12,600 samples, 431 (3·4%) were regarded as positive for IgG anti-HEV. The prevalence of IgG anti-HEV was higher in eastern Japan (5·6%) than in western Japan (1·8%) (P<0·001), and was also age-dependent and higher in men (3·9%) than in women (2·9%) (P=0·002). CONCLUSION: The spread of the domestic infection of HEV was observed in qualified blood donors in Japan. A higher prevalence of IgG anti-HEV was observed in male donors, older donors and in donors residing in eastern Japan. Further studies are necessary to clarify the potential risk of transfusion-transmission of HEV in Japan.

Relationship of Torque teno virus to chicken anemia virus.

This chapter examines the correlation between Torque teno virus (TTV) and chicken anemia virus (CAV). Each has a circular single-stranded (ss)DNA genome with every one of its known open reading frames (ORF) on its antigenomic strand. This structure is distinct from those of circoviruses. The genomic sizes of TTV and CAV are different, 3.8 kb and 2.3 kb, respectively. While the spectrum of the TTV genome is enormously diverse, that of the CAV genome is quite narrow. Although a 36-nt stretch near the replication origin of TA278 TTV possesses more than 80% similarity to that of CAV, the sequence of the other genomic regions does not exhibit a significant similarity. Nevertheless, the relative allocation of ORFs on each frame in these viruses mimics each other. Three or more messenger RNA (mRNAs) are generated by transcription in both of them. The structural protein with the replicase domain is coded for by frame 1 in each virus, and a nonstructural protein with a phosphatase domain is coded for by frame 2. A protein on frame 3 in each virus induces apoptosis in transformed cells. Recently, we confirmed that apoptin is necessary for the replication of CAV. TTV has been proposed to constitute a new family, Anelloviridae. Considering these similarities and dissimilarities between CAV and TTV, it seems more reasonable to place CAV, the only member of genus Gyrovirus, into Anelloviridae together with TTV, or into a new independent family.

Serum and monoclonal immunoglobulin E antibodies from NC/Nga mice with severe atopic-like dermatitis recognize an auto-antigen, histone H3.

BACKGROUND: NC/Nga mice are known to show a spontaneous outbreak of atopic-like dermatitis accompanied by a marked elevation in serum IgE levels when reared in a conventional environment. The specific effects of such a strong serum IgE response on the development of the dermatitis and specific antigens recognized by the IgE antibodies are still uncertain. OBJECTIVE AND METHODS: To characterize the IgE of NC/Nga mice, we established IgE-secreting hybridoma clones from spleen cells of NC/Nga mice spontaneously developing dermatitis and identified variable-region genes and specific antigens of the IgE monoclonal antibodies (mAbs). Serum polyclonal IgE, as well as IgG1 and IgG2a, specific for the identified antigen were also analysed. RESULTS: Four IgE-producing hybridoma clones were established. Variable-region nucleotide sequences of the IgE mAbs showed that these clones did not necessarily share common germline gene segments (V, D or J) for each variable region, and several somatic mutations had occurred in the V gene segments. Through antigen screening, histone H3 was identified to be an auto-antigen recognized by three of the four IgE mAbs. Serum IgE as well as IgG1 specific for histone H3 were almost undetectable in 6-week-old mice, but rapidly increased by 10-12 weeks of age. This age-dependent increase in the serum anti-histone H3 IgE was roughly in parallel with the onset of dermatitis, and slightly preceding total IgE elevation. The serum-specific IgE level correlated well with a dermatitis-severity score of each mouse at 12-16 weeks of age, and weakly with the severity of ear erosion of each mouse over 28 weeks of age. Furthermore, immunologically detectable histone-H3 antigens were observed in skin tissue sections from the dermatitis sites. CONCLUSION: In NC/Nga mice, anti-histone H3 auto-antibodies may contribute, at least in part, to the considerably elevated serum IgE and might play some roles in the development and exacerbation of dermatitis.

Effects of bosentan on the skin lesions: an observational study from a single center in Japan.

Effects of a dual endothelin receptor antagonist, bosentan on peripheral circulatioin and skin lesions as well as pulmonary arterial hypertension (PAH) were investigated in Japanese patients with connective tissue diseases (CTD). Fifteen patients with PAH associated with CTD [systemic sclerosis (SSc) 13, mixed connective tissue disease (MCTD) 2] were treated with bosentan for 40-96 weeks, and changes of exercise capacity (6-min walk distance and Borg's dyspnea scale), cardio-pulmonary hemodynamics (right ventricular pressure, specific activity scale and cardiac index), Raynaud's phenomenon, digital ulcers and dermal sclerosis were observed. Bosentan improved exercise capacity, had a positive effect on hemodynamic parameters, and was well tolerated as previously reported. After a median 8 weeks of treatment, 13 out of 15 patients had improved Raynaud's phenomenon. Digital ulcers also improved after a median 12 weeks' treatment in all of 8 patients. Modified Rodnan total skin score decreased from 21.0 +/- 5.9 to 11.5 +/- 3.9 in diffuse cutaneous SSc and from 17.0 +/- 6.5 to 9.5 +/- 4.5 in limited cutaneous SSc after 24 months' treatment, reaching significance after 6 months in both groups. These data suggest that bosentan is effective for both PAH and peripheral vascular diseases in Japanese patients with CTD. The pathological background to the improvement in dermal sclerosis observed in this study should be further investigated.

Endoplasmic reticulum stress response in dendrites of cultured primary neurons.

The endoplasmic reticulum (ER) is an organelle in which secretory and transmembrane proteins are folded or processed, and is susceptible to various stresses that provoke the accumulation of unfolded proteins in the ER lumen. Recently, ER stress has been reported to be linked to neuronal death in various neurodegenerative diseases. Neurons contain the ER not only in the soma, but also in the dendrites, thus presenting a different case to non-neuronal cells. The ER in the dendrites has potential functions in local protein synthesis and sorting of synthesized proteins to postsynaptic membranes. It raises the possibility that ER stress could occur locally in the dendrites. Here we showed that ER stress sensors, inositol-requiring 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) exist in the ER of both soma and dendrites in primary mouse neurons, and that under ER stress conditions, GRP78/BiP and phosphorylated eIF2alpha are induced. Furthermore, XBP1 mRNA was localized in the proximal dendrites where IRE1 was rapidly phosphorylated in response to ER stress. These results indicate that the ER in dendrites could respond to ER stress and retain the capacity of protein quality control.

Detection of p16 promoter methylation in the serum of oral cancer patients.

P16 promoter methylation occurs frequently in oral squamous cell carcinoma (OSCC). For the early detection of tumour-related aberrant DNA, we examined p16 methylation using the methylation-specific polymerase chain reaction (MSP) in tumour and serum samples of 17 OSCC patients. Aberrant p16 methylation was detected in 11 (64.7%) cases of primary OSCC. Of these 11 patients, 6 (54.5%) showed the same alteration in their serum. No methylation was found in control groups. Interestingly, DNA was detected in the serum of 3 out of 4 patients with recurrence. These results suggest that the MSP may be a sensitive and useful method for detecting recurrent OSCC.

Response of diffuse sclerosing osteomyelitis of the mandible to alendronate: follow-up study by 99mTc scintigraphy.

We report a case of diffuse sclerosing osteomyelitis of the mandible responded to alendronate, after a poor response to intravenous antibiotics, antibiotic irrigation-perfusion, and decortication. The patient was given an intravenous infusion of 10mg of alendronate. Pain resolved within 24 h. There were no severe adverse events. Increased uptake of 99mTc in the mandible almost completely disappeared 3 months after treatment.

In vivo proliferation of differentiated pancreatic islet beta cells in transgenic mice expressing mutated cyclin-dependent kinase 4.

AIMS/HYPOTHESIS: It has previously been hypothesised that highly differentiated endocrine cells do not proliferate or regenerate. However, recent studies have revealed that cyclin-dependent kinase 4 (CDK4) is necessary for the proliferation of pancreatic islet beta cells. The aim of this study was to determine whether activation of CDK4 can potentially be used as a radical treatment for diabetes without malignant transformation. METHODS: We generated transgenic mice expressing mutant CDK4 under the control of the insulin promoter to examine the effect of activated CDK4 overexpression in the postnatal development of pancreatic islets. RESULTS: In the transgenic mice, total CDK4 protein expression was increased by up to 5-fold, with a concomitant increase in CDK4 activity indicated by the detection of phosphorylated Rb protein in pancreatic islets. Histopathologically, many cells tested positive for proliferating cell nuclear antigen, and pancreatic islets displayed hyperplasia due to the extreme proliferation of beta cells containing a large number of insulin granules. Pancreatic islet alpha, delta and PP cells did not increase. Over an 18-month observation period, the transgenic mice did not develop insulinoma. Levels of expression of GLUT1 and c-myc were comparable to those in the littermates of the transgenic mice. GLUT2 expression was identified in the pancreatic islets of transgenic mice. No significant differences in telomerase activities were detected between transgenic mice and their littermates. Transgenic mice were superior to their littermates in terms of glucose tolerance and insulin secretion in response to the intraperitoneal injection of glucose, and hypoglycaemia was not observed. CONCLUSIONS/INTERPRETATION: Activated CDK4 stimulates postnatal pancreatic beta cell proliferation, during which the highly differentiated phenotypes of pancreatic islet beta cells are preserved without malignant transformation.

Intraoperative real-time genetic diagnosis for sentinel node navigation surgery.

Sentinel node navigation surgery (SNNS) has received considerable attention for its role in deciding whether to perform neck dissection in patients with early oral cancer. However, diagnostic accuracy and its intraoperative availability of results remain important concerns. First, we shortened the examination time required for genetic diagnosis. Second, we assessed the quality of the extracted mRNA. Third, 10 patients with early N0 oral cancer underwent SNNS, using our new technique for genetic diagnosis to determine whether neck dissection was required. The examination time of our one-step reverse-transcriptase polymerase chain reaction method using a minicolumn and LightCycler was successfully shortened to 2 h, permitting intraoperative genetic diagnosis. The extracted mRNA was of high quality. Six sentinel nodes in four patients were diagnosed to be metastatic on genetic diagnosis; these patients underwent neck dissection. The other six patients avoided unnecessary surgery. We conclude that intraoperative genetic diagnosis of micrometastasis holds promise of being a sensitive method that can be used to support SNNS.

Use of the curved linear-array echo endoscope to identify gastrorenal shunts in patients with gastric fundal varices.

BACKGROUND AND STUDY AIMS: : Balloon-occluded retrograde transvenous obliteration (B-RTO) has emerged as an effective, minimally invasive treatment for fundal varices. B-RTO requires a spontaneously developed gastrorenal shunt as a pathway for the balloon catheter to reach the fundal varices. We used a curved linear-array (CLA) echo endoscope in patients with fundal varices to identify gastrorenal shunts, and compared the detection rate with the gold standard, contrast-enhanced computed tomography (CECT). PATIENTS AND METHODS: A total of 40 patients with fundal varices were examined with both CLA echo endoscopy and CECT. The CECT images were retrospectively and independently evaluated by two gastroenterologists who were unaware of the clinical details, including the results of the CLA echo endoscopy. RESULTS: CLA echo endoscopy identified gastrorenal shunts in 26/40 patients with fundal varices. It visualized the shunt in a longitudinal direction and provided images of the connections of the shunt at both ends, the fundal varices and the left renal vein/branch of the inferior adrenal vein. The kappa index for CLA echo endoscopy and CECT for the identification of gastrorenal shunt was 0.9 (95 % CI, 0.6 to 1.0). When the cutoff point for the diameter of the gastrorenal shunt detected by the CLA echo endoscope was set at equal to or greater than 5 mm, the kappa index was 1.0 (95 % CI, 0.7 to 1.0). CONCLUSIONS: These results suggest that CLA echo endoscopy can successfully identify gastrorenal shunt and provide detailed morphological information. It also efficiently identifies patients suitable for B-RTO, particularly in cases of acute bleeding. It also has considerable potential for providing detailed information with regard to the treatment of gastric varices.

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure.

Suppressed expression of transgenes in vivo is the major obstacle in the gene therapy. For the long-term expression, we utilized a chromatin insulator from sea urchin arylsulfatase (Ars) gene locus (Ars insulator, ArsI), which has been shown to epigenetically regulate gene expression across species. ArsI was able to prevent silencing of the transgene in a myeloid cell line, HL-60, and a murine embryonic stem cell line, CCE, in an orientation-dependent manner, but not in Huh-7, K562 and MCF-7 cells, indicating that the effect of ArsI on gene silencing was cell type dependent. Although anti-silencing effect of ArsI was almost equivalent to that of chicken beta-globin insulator, incorporation of ArsI into lentiviral vector had little effect on the virus titer compared with chicken beta-globin insulator. Clonal analysis of transduced HL-60 cells revealed that ArsI protects the lentiviral vector from position effects regardless of its orientation. Furthermore, chromatin immunoprecipitation assays revealed that a high acetylation level was observed in the promoter of the insulated vector, whereas that of ArsI was independent of its anti-silencing capacity. In addition to it having little deteriorative effect on the virus titer, the identified anti-silencing effect of ArsI suggested its possibility for application in gene therapy.

Transcriptional activation of cyclin-dependent kinase inhibitor, p21waf1 gene by treatment with a differentiation inducing agent, vesnarinone in a human salivary gland cancer cell line.

Recently, a new concept for cancer therapy termed "tumor dormancy therapy" has been proposed. The concept of this therapy is to prolong the survival time of cancer patients while maintaining their quality of life. We have been developing a differentiation-inducing therapy, which is included in the tumor dormancy therapy, for salivary gland cancer. In this study, we examined the effect of a differentiation-inducing drug, Vesnarinone on the growth of several cancer cells, and examined the molecular mechanism by which Vesnarinone induces the cyclin dependent kinase inhibitor, p21waf1 in the cancer cells. Vesnarinone significantly suppressed the growth of TYS (salivary gland cancer cells), PC3 (prostate cancer cells), and A431 (squamous cell cancer cells). Furthermore, Vesnarinone dose-dependently enhanced the expression of p21waf1 mRNA in TYS cells. Using the luciferase reporter assay it was found that the enhancement of p21waf1 mRNA expression by Vesnarinone was through direct transcriptional activation of the p21waf1 promoter. Thus, analyzing the molecular mechanisms of differentiation inducing drugs may lead to the development of a new therapeutic strategy for several human malignancies, including salivary gland cancer.

Vesnarinone, a differentiation inducing drug, directly activates p21(waf1) gene promoter via Sp1 sites in a human salivary gland cancer cell line.

We previously demonstrated that a differentiation inducing drug, vesnarinone induced the growth arrest and p21(waf1) gene expression in a human salivary gland cancer cell line, TYS. In the present study, we investigated the mechanism of the induction of p21(waf1) gene by vesnarinone in TYS cells. We constructed several reporter plasmids containing the p21(waf1) promoter, and attempted to identify vesnarinone-responsive elements in the p21(waf1) promoter. By the luciferase reporter assay, we identified the minimal vesnarinone-responsive element in the p21(waf1) promoter at -124 to -61 relative to the transcription start site. Moreover, we demonstrated by electrophoretic mobility shift assay that Sp1 and Sp3 transcription factors bound to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone hyperacetylation in TYS cells. These results suggest that vesnarinone directly activates p21(waf1) promoter via the activation of Sp1 and Sp3 transcription factors and the histone hyperacetylation in TYS cells.