Hydroxychloroquine modulates elevated expression of S100 proteins in systemic lupus erythematosus.

OBJECTIVES: We investigated the effect of hydroxychloroquine (HCQ) on S100A8 and S100A9 serum levels in systemic lupus erythematosus (SLE) patients with low disease activity receiving immunosuppressants. METHODS: SELENA-SLEDAI, Cutaneous Lupus Erythematous Disease Area and Severity Index (CLASI) and serum levels of complement factors, anti-dsDNA antibodies, and white blood cell, lymphocyte, and platelet counts were used to evaluate disease activity, cutaneous disease activity, and immunological activity, respectively. Serum S100A8 and S100A9 were measured at HCQ administration and after 3 or 6 months using ELISA. RESULTS: S100A8 and S100A9 serum levels were elevated at baseline and the magnitude of decrease from baseline at 3 and 6 months after HCQ administration was greater in patients with renal involvement than in those without (baseline: S100A8, p = 0.034; S100A9, p = 0.0084; decrease: S100A8, p = 0.049; S100A9, p = 0.023). S100 modulation was observed in patients with (n = 17; S100A8, p = 0.0011; S100A9, p = 0.0002) and without renal involvement (n = 20; S100A8, p = 0.0056; S100A9, p = 0.0012), and was more apparent in patients with improved CLASI activity scores (improved: S100A8, p = 0.013; S100A9, p = 0.0032; unimproved: S100A8, p = 0.055; S100A9, p = 0.055). No associations were observed for immunological biomarkers. CONCLUSION: HCQ may improve organ involvement in SLE by modulating S100 protein levels, especially in patients with renal or skin involvement.

Microgaps and Demineralization Progress around Composite Restorations.

This study investigated the influence of adhesives and marginal sealing on demineralization progress using optical coherence tomography (OCT). Cavities (4 × 2 mm) were prepared in bovine incisors and restored using Clearfil SE Protect (SP), Bond Force (BF), Scotchbond Universal (SB), or G-Bond Plus (GB), followed by Estelite Flow Quick flowable composite. The control group received no adhesive (n = 10). After 3-d incubation in artificial saliva and 10,000 thermal cycles, gaps at enamel and dentin margins were measured at 8 locations on cross-sectional images obtained from each restoration using swept-source OCT at 1310-nm wavelength. Specimens were demineralized using acidified gel (pH = 4.5) for 5 wk and scanned every week to monitor the lesion progress at the same marginal locations. Repeated-measures analysis of variance showed that demineralization period and adhesive type and their interaction had a significant effect on the lesion size in both substrates (P < 0.001). SP, BF, and SB had significantly lower enamel and dentin initial gaps than the control and GB (P < 0.05). Enamel lesion progress was slower in the fluoride-releasing adhesives SP and BF and significantly different from SB, GB, and the control (P < 0.001). SP and BF dentin lesions were significantly different from GB and the control (P < 0.001), but not from SB (P > 0.05). A significant positive correlation (P < 0.05) was found between initial gap length and formed lesion size in both substrates, which was stronger in enamel (r = 0.63) than dentin (r = 0.35). Microgaps forming at the margins of restorations depend on adhesives and significantly contribute to the progress of demineralization around the margins, while fluoride release may decrease the rate of progression.

Environmental radioactivity of water samples collected in Higashi-Hiroshima campus, Hiroshima University, Japan.

The relation between concentration of elements and microbial activity in the water samples of Higashi-Hiroshima Campus, Hiroshima University was investigated. Energy dispersive X-ray spectroscopy revealed that microbial mat contains iron, aluminium, silicon and phosphorus. Model experiment revealed that the potassium was adsorbed by living microorganism in the microbial mats, while it was not adsorbed by dead microbial mat. Iron was adsorbed by both living and dead microbial mats. The present results explain the increase in the total ß-radioactivity of water sample in summer and the decrease in winter.

Early vitamin K deficiency bleeding in a neonate associated with maternal Crohn's disease.

We report herein a case of early vitamin K deficiency bleeding (VKDB) in a neonate associated with maternal Crohn's disease. A female neonate was born at 37 weeks' gestation and weighed 2778 g. She developed broad purpura on her back on day 1. Laboratory data showed anemia, prolonged coagulation time and elevated protein induced by vitamin K absence or antagonist-II. Early VKDB has not been reported in a neonate born from mother with active Crohn's disease. It is essential to give vitamin K selectively as soon as possible after birth to prevent early VKDB in neonates.

Linagliptin blocks renal damage in type 1 diabetic rats by suppressing advanced glycation end products-receptor axis.

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. We have recently found that linagliptin, an inhibitor of dipeptidyl peptidase-4 (DPP-4) suppresses the AGE-induced oxidative stress generation and intercellular adhesion molecule-1 (ICAM-1) gene expression in endothelial cells. However, whether linagliptin could have beneficial effects on experimental diabetic nephropathy in a glucose-lowering independent manner remains unknown. To address the issue, this study examined the effects of linagliptin on renal damage in streptozotocin-induced diabetic rats. Serum levels of DPP-4 were significantly elevated in diabetic rats compared with control rats. Although linagliptin treatment for 2 weeks did not improve hyperglycemia in diabetic rats, linagliptin significantly reduced AGEs levels, RAGE gene expression, and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress in the kidney of diabetic rats. Furthermore, linagliptin significantly reduced albuminuria, renal ICAM-1 mRNA levels, and lymphocyte infiltration into the glomeruli of diabetic rats. Our present study suggests that linagliptin could exert beneficial effects on diabetic nephropathy partly by blocking the AGE-RAGE-evoked oxidative stress generation in the kidney of streptozotocin-induced diabetic rats. Inhibition of DPP-4 by linagliptin might be a promising strategy for the treatment of diabetic nephropathy.

Recovery of nanomolecular electronic states from tunneling spectroscopy: LDOS of low-dimensional phthalocyanine molecular structures on Cu(111).

Organic nanomolecules have become one of the most attractive materials for new nanoelectronics devices. Understanding of the electronic density of states around the Fermi energy of low-dimensional molecules is crucial in designing the electronic properties of molecular devices. The low dimensionality of nanomolecules results in new electronic properties owing to their unique symmetry. Scanning tunneling spectroscopy is one of the most effective techniques for studying the electronic states of nanomolecules, particularly near the Fermi energy (±1.5 eV), whereas these molecular electronic states are frequently buried by the tunneling probability background in tunneling spectroscopy, resulting in incorrect determination of the molecular electronic states. Here, we demonstrate how to recover nanomolecular electronic states from dI/dV curves obtained by tunneling spectroscopy. Precise local density of states (LDOS) peaks for low-dimensional nanostructures (monolayer ultrathin films, one-dimensional chains, and single molecules) of phthalocyanine (H2Pc) molecules grown on noble fcc-Cu(111) were obtained.

CCL-1 in the spinal cord contributes to neuropathic pain induced by nerve injury.

Cytokines such as interleukins are known to be involved in the development of neuropathic pain through activation of neuroglia. However, the role of chemokine (C-C motif) ligand 1 (CCL-1), a well-characterized chemokine secreted by activated T cells, in the nociceptive transmission remains unclear. We found that CCL-1 was upregulated in the spinal dorsal horn after partial sciatic nerve ligation. Therefore, we examined actions of recombinant CCL-1 on behavioural pain score, synaptic transmission, glial cell function and cytokine production in the spinal dorsal horn. Here we show that CCL-1 is one of the key mediators involved in the development of neuropathic pain. Expression of CCL-1 mRNA was mainly detected in the ipsilateral dorsal root ganglion, and the expression of specific CCL-1 receptor CCR-8 was upregulated in the superficial dorsal horn. Increased expression of CCR-8 was observed not only in neurons but also in microglia and astrocytes in the ipsilateral side. Recombinant CCL-1 injected intrathecally (i.t.) to naive mice induced allodynia, which was prevented by the supplemental addition of N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801. Patch-clamp recordings from spinal cord slices revealed that application of CCL-1 transiently enhanced excitatory synaptic transmission in the substantia gelatinosa (lamina II). In the long term, i.t. injection of CCL-1 induced phosphorylation of NMDA receptor subunit, NR1 and NR2B, in the spinal cord. Injection of CCL-1 also upregulated mRNA level of glial cell markers and proinflammatory cytokines (IL-1ß, TNF-α and IL-6). The tactile allodynia induced by nerve ligation was attenuated by prophylactic and chronic administration of neutralizing antibody against CCL-1 and by knocking down of CCR-8. Our results indicate that CCL-1 is one of the key molecules in pathogenesis, and CCL-1/CCR-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain.

Estimation of the enamel and dentin mineral content from the refractive index.

Recent advances in the field of optics have enabled accurate and localized measurement of optical properties of biological substrates. This work aimed to elucidate the relationship between the local refractive index (n) and mineral content (MC) of enamel and dentin. De- and remineralized lesions in bovine enamel and dentin blocks were sectioned into 300- to 400-µm-thick slices, and placed on a metal plate to capture images of sound, de- and remineralized regions transversely by optical coherence tomography. Mean n at each depth level of the lesion (20- or 40-µm steps for enamel or dentin) was measured by the optical path length-matching method and used to plot n through lesion depth. The specimens were further polished and processed for transverse microradiography for analysis of MC. The n and MC ranged from 1.52 to 1.63 and 50 to 87 (vol.%) in enamel, and from 1.43 to 1.57 and 11 to 48 (vol.%) in dentin, respectively. Strong, positive linear correlations were found between n and MC (Pearson's r = 0.95 and 0.91 for de- and remineralized enamel, and r = 0.94 and 0.91 for dentin, respectively, p < 0.001). Experimental data were validated with a theoretical calculation of n from MC. De- and remineralization of enamel and dentin resulted in measurable changes of n, and, in turn, MC changes of the tissue could be estimated with good accuracy from this long-known optical property by the new analytical approach. Compositional changes of enamel crystallites after remineralization affect n.

Enamel lesion parameter correlations between polychromatic micro-CT and TMR.

Transverse microradiography (TMR) is considered as the gold standard technique for the evaluation of enamel lesions. Micro-computed tomography (µCT) has the advantage of non-destructive measurements, but the beam-hardening effect with polychromatic x-rays is a major drawback. To date, no study has validated µCT against TMR. The objective of this study was to validate µCT measurements of enamel lesions under various x-ray conditions and software beam-hardening correction (BHC) against TMR. Human molars with natural white-spot lesions were scanned for 5 min by µCT at 100 kV in different conditions: 50 µA (0.5-mm Al filter), 165 µA (0.5-mm Al/0.3-mm Cu), and 200 µA (0.5-mm Al/0.4-mm Cu), with or without BHC. Grayscale values were converted into mineral density values using phantoms. Thin sections at the same positions were then prepared for TMR. Lesion depth (LD; µm) and mineral loss (ΔZ; vol%µm) were compared between µCT and TMR by Pearson's correlations. µCT measurements correlated well with TMR under all conditions (p < 0.001, r > 0.86 for LD and ΔZ), except for 0.5-mm Al without BHC (p > 0.05). Even without BHC, combined Al/Cu filters successfully reduced the beam-hardening effect. µCT can be used as a non-destructive alternative to TMR with comparable parameters for the study of enamel lesions.

Overexpression of RegIV in peritoneal dissemination of gastric cancer and its potential as A novel marker for the detection of peritoneal micrometastasis.

BACKGROUND: Regenerating gene type IV (RegIV) is a candidate marker for cancer and inflammatory bowel disease. In this study, its potential as a novel marker for the detection of gastric cancer peritoneal micrometastases was examined. PATIENTS AND METHODS: RegIV mRNA levels in the peritoneal washes of 95 gastric cancer patients and 22 with benign disease were quantified by real-time RT-PCR. To examine whether expression of RegIV enhance tumorigenicity or not, thirty two mice were injected intraperitoneally or subcutaneously with RegIV transfectants of TMK-1 cells, parental TMK-1 cells, or neomycin control transfectants. RESULTS: RegIV expression was markedly higher in patients with peritoneal metastases compared to those without. The level of RegIV mRNA in gastric cancer patients was related to the extent of wall penetration. A cut-off value for RegIV-positive expression was based on an analysis of negative control patients with benign disease, and gastric cancer patients above the cut-off value constituted the micrometastasis (MM+) group. Based on this criteria, 3 out of 43 T1 or T2 cases were MM+ (93% specificity). Among 15 patients with peritoneal dissemination (7 out of 15 cases were positive by cytology), 14 cases were positive for RegIV expression (93% sensitivity), while analysis of carcinoembryonic antigen (CEA) mRNA failed to detect micrometastases in 4 cases (73% sensitivity). Combined analysis of CEA and RegIV improved the accuracy of diagnosis to 100%. The prognosis of RegIV-positive cases was significantly worse than that of RegIV-negative cases. Multivariate analysis using the Cox proportional hazards model suggested that RegIV may be an independent prognostic factor. Stable expression of RegIV significantly enhanced peritoneal metastasis in an animal model of gastric cancer. CONCLUSION: These findings suggest that RegIV mRNA expression has the potential to serve as a novel marker for detecting peritoneal dissemination in gastric cancer.

Relationship between quantitative assessments of salivary buffering capacity and ion activity product for hydroxyapatite in relation to cariogenic potential.

BACKGROUND: The ion activity product for hydroxyapatite (Ip(HA)) is a comprehensive parameter reflecting pH, calcium and phosphate ion concentration in saliva which govern the degree of saturation with respect to the dissolving tooth mineral. The aim of this study was to evaluate the relationship between quantitative assessments of salivary buffering capacity and Ip(HA) in relation to cariogenic potential. METHODS: Stimulated whole saliva was collected from 33 patients, and the initial pH of samples was measured using a hand-held pH meter. Then samples were titrated with 0.1 N HCl to evaluate buffering capacities and divided into three groups (high, medium and low). After measuring concentrations of calcium and phosphate ions in the samples, Ip(HA) was calculated using the values of the ion concentrations and pH. Differences in the mean pH values, the concentrations of calcium, phosphate ions and log[Ip(HA)] among three groups were analysed using the Kruskal Wallis and the Mann-Whitney non-parametric test, p < 0.05. RESULTS: After HCl 50 microL titration, there were statistical differences of the mean pH and Ip(HA) among each buffering capacity group. Moreover, after 50 microL HCl titration, there was an excellent correlation between the buffer capacity and log[Ip(HA)]. CONCLUSIONS: The pH change for saliva after HCl titration has a significant influence on the rate of Ip(HA).

Influence of salivary macromolecules and fluoride on enamel lesion remineralization in vitro.

The aim of this study was to investigate the influence of salivary macromolecules on enamel lesion remineralization in the presence or absence of fluoride. Paraffin-stimulated whole saliva was centrifuged, and the supernatant was dialyzed in 1,000 molecular-weight cutoff dialysis tubes, first against a phosphate buffer and then against a mineral solution containing Ca and phosphate. Artificial subsurface lesions of human enamel, created in pH 4.5 acetate buffer, were remineralized for 28 days in 4 remineralizing solutions: group C--mineral solution as a control; group S--mineral solution + dialyzed saliva; group F--mineral solution + 1 ppm F; group SF--mineral solution + dialyzed saliva + 1 ppm F. Changes in relative mineral concentration in the lesions were assessed by transverse microradiography. The results showed statistically significant mineral gains in the lesion body in groups C (DeltaZ = 3,254 +/- 1,562% x microm) and SF (DeltaZ = 2,973 +/- 1,349% x microm), but not in groups S (DeltaZ = 5,192 +/- 1,863% x microm) and F (DeltaZ = 4,310 +/- 1,138% x microm) compared with the baseline group (DeltaZ = 5,414 +/- 461% x microm). It was also found that the mineral density at the surface layer in group F (75.0 +/- 15.7%) was greater than that in the baseline group (30.1 +/- 12.3%) with statistical significance, but not in group SF (39.9 +/- 16.5%). It was concluded that the macromolecules inhibited lesion remineralization fundamentally but that these molecules, in the presence of fluoride, seemed to play an important role in the continuation of remineralization by reducing mineral gains at the surface layer.

Frequent silencing of RUNX3 in esophageal squamous cell carcinomas is associated with radioresistance and poor prognosis.

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. RUNX3, a novel tumor suppressor of gastric cancer, functions in transforming growth factor (TGF)-beta-dependent apoptosis. We obtained paired samples from 62 patients with advanced esophageal cancers diagnosed initially as T3 or T4 with image diagnosis; one sample was obtained from a biopsy before presurgical radiotherapy, and the other was resected in surgical specimens after radiotherapy. RUNX3 was repressed in 67.7% cases of the pretreatment biopsy samples and 96.7% cases of the irradiated, resected samples. The nuclear expression of RUNX3 was associated with radiosensitivity and a better prognosis than cytoplasmic or no RUNX3 expression (P<0.003); cytoplasmic RUNX3 expression was strictly associated with radioresistance. RUNX3 was downregulated and its promoter was hypermethylated in all radioresistant esophageal cancer cell lines examined. Stable transfection of esophageal cancer cells with RUNX3 slightly inhibited cell proliferation in vitro, enhanced the antiproliferative and apoptotic effects of TGF-beta and increased radiosensitivity in conjunction with Bim induction. In contrast, transfection of RUNX3-expressing cells with a RUNX3 antisense construct or a Bim-specific small interfering RNA induced radioresistance. Treatment with 5-aza-2'-deoxycytidine restored RUNX3 expression, increased radiosensitivity and induced Bim in both control and radioresistant cells. These results suggest that RUNX3 silencing promotes radioresistance in esophageal cancers. Examination of RUNX3 expression in pretreatment specimens may predict radiosensitivity, and induction of RUNX3 expression may increase tumor radiosensitivity.

In situ tissue engineering of the bile duct using polypropylene mesh-collagen tubes.

Multiple attempts have been made to replace biliary defects with a variety of materials. Recently, successful biliary reconstruction using the Gore-Tex vascular graft has been reported experimentally and clinically. We designed a new artificial bile duct consisting of collagen sponge and polypropylene mesh. We presently evaluated the feasibility of using this prosthesis as a scaffold for bile duct tissue regeneration in a canine model. Our prosthesis, a sponge made from porcine dermal collagen, is reinforced with a polypropylene mesh cylinder. We used the prosthesis to reconstruct the middle portion of the common bile duct in seven beagle dogs to evaluate its efficacy. While one dog died of biliary stricture 8 months after operation, six survived without problems to scheduled time points for tissue evaluation at 1 to 12 months. All prostheses had become completely incorporated into the host. A confluent epithelial lining was observed within 3 months. In cholangiograms the prosthesis displayed long-term patency in the six dogs and provided satisfactory bile drainage for up to 12 months. Our graft thus shows promise for repair of biliary defects and should lead to development of a new treatment for biliary reconstruction.

A clinical study of Japanese patients with ulcer induced by low-dose aspirin and other non-steroidal anti-inflammatory drugs.

BACKGROUND: The incidence and severity of non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastro-duodenal ulcer have not been extensively studied in Japan. AIM: We performed a prospective study to clarify NSAIDs-induced gastro-duodenal injury, focusing especially on low-dose aspirin (L-A). METHODS: Two hundred and thirty-eight patients with bleeding peptic ulcers admitted to our hospital. History of taking NSAIDs and anti-ulcer drugs was obtained from all patients who underwent endoscopic examinations. The lesion scores of patients taking L-A were classified numerically from zero (no lesion) to five (ulcer). RESULTS: The NSAIDs were associated with 28.2% of hemorrhagic ulcers. The rates of patients using L-A, loxoprofen, diclofenac, and combination of two of these drugs were 27, 16, 10 and 9%, respectively. Co-administered anti-ulcer drugs were cytoprotective anti-ulcer drugs (27%), H2 receptor antagonists (16%), PPI (4%), and none (53%). In patients taking L-A, H2 receptor antagonists were used most frequently. The HP was positive in 63% of L-A-induced ulcer cases and in 69% of NSAIDs other than low-dose aspirin-induced ulcer cases. The lesion scores of patients taking L-A with H2 receptor antagonists or PPI were significantly lower than those of patients who were taking only L-A (P < 0.05). CONCLUSIONS: Approximately one-third of hospitalized patients with NSAIDs-induced hemorrhagic ulcer showed an association with L-A. Prospective randomized controlled trials including H2 receptor antagonists are required to establish preventive efforts aimed at L-A-induced gastro-duodenal injury.

Highly efficient transfection of human marrow stromal cells by nucleofection.

Human marrow stromal cells (hMSCs) are an attractive source for autologous cell and gene therapies. In this study, we developed a highly efficient transfection method for hMSCs. Although they tend to show efficient gene delivery, nonviral vectors offer several advantages over viral vectors for gene therapies. They are inexpensive to produce and suitable to adopt particularly with respect to little or no specific immune responses; they are simple to use; they entail easier large-scale production; and they have a high degree quality control. hMSCs and rat marrow stromal cells were transfected with the plasmid pEGFP-N1 that encoded a green fluorescent protein component by using two nonviral methods: nucleofection and electroporation. Nucleofection provided a much better rate of transfer than electroporation particularly in hMSCs.

Implication of prostaglandin E(2) in TNF-alpha-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs.

OBJECTIVE: Calpains are known as Ca(2+)-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined. METHODS: Human chondrocytic HCS-2/8 cells were stimulated by TNF-alpha in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined. RESULTS: TNF-alpha (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E(2) (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha. CONCLUSIONS: These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process.