Outcomes of robot-assisted versus conventional laparoscopic low anterior resection in patients with rectal cancer: propensity-matched analysis of the National Clinical Database in Japan.

BACKGROUND: Robot-assisted laparoscopic surgery has several advantages over conventional laparoscopy. However, population-based comparative studies for low anterior resection are limited. This article aimed to compare peri-operative results of robot-assisted low anterior resection (RALAR) and laparoscopy. METHODS: This retrospective cohort study used data from patients treated with RALAR or conventional laparoscopic low anterior resection (CLLAR) between October 2018 and December 2019, as recorded in the Japanese National Clinical Database, a data set registering clinical information, perioperative outcomes, and mortality. Of note, the registry does not include information on the tumour location (centimetres from the anal verge) and diverting stoma creation. Perioperative outcomes, including rate of conversion to open surgery, were compared between RALAR and CLLAR groups. Confounding factors were adjusted for using propensity score matching. RESULTS: Of 21 415 patients treated during the study interval, 20 220 were reviewed. Two homogeneous groups of 2843 patients were created by propensity score matching. The conversion rate to open surgery was significantly lower in the RALAR group than in the CLLAR group (0.7 versus 2.0 per cent; P < 0.001). The RALAR group had a longer operating time (median: 352 versus 283 min; P < 0.001), less intraoperative blood loss (15 versus 20 ml; P < 0.001), a lower in-hospital mortality rate (0.1 versus 0.5 per cent; P = 0.007), and a shorter postoperative hospital stay (median: 13 versus 14 days; P < 0.001) compared with the CLLAR group. The CLLAR group had a lower rate of readmission within 30 days (2.4 versus 3.3 per cent; P = 0.045). CONCLUSION: These data highlight the reduced conversion rate, in-hospital mortality rate, intraoperative blood loss, and length of postoperative hospital stay for rectal cancer surgery in patients treated using robot-assisted laparoscopic surgery compared with laparoscopic low anterior resection.

Effects of polypharmacy on the prevalence of adverse drug events resulting in outpatient visits and hospitalization.

A high proportion of hospitalizations is attributable to the prevalence of adverse drug events. This retrospective study included outpatients and inpatients to determine the prevalence of adverse drug events and if polypharmacy increases it. The prevalence, classification, and causality of adverse drug events were assessed based on medical records, laboratory values, and other data. Multivariate analysis (multiple logistic regression analysis) was performed with the presence or absence of adverse drug events at the time of the visit as the dependent variable and items for which the P-value was <0.25 in the univariate analysis as independent variables. The prevalence of adverse drug events was 13.0%, 10.9%, and 16.0% among all patients, the outpatient group, and the inpatient group, respectively. Multivariate analysis showed that polypharmacy (≥5 drugs) significantly increased the risk of adverse drug events in all patients. The prevalence of adverse drug events significantly increased with each additional drug used. We expect that minimizing the number of medications through moderation of the number of prescription drugs and elimination of polypharmacy will reduce the number of outpatient visits and hospitalizations due to adverse drug events.

Dexamethasone Prolongs Cardiac Allograft Survival in a Murine Model Through Myeloid-derived Suppressor Cells.

BACKGROUND: Recently, myeloid-derived suppressor cells (MDSCs) have attracted considerable attention because of their cancer-promoting and immunosuppressive effects. The glucocorticoid dexamethasone (Dex) is an important immunosuppressive agent used to treat autoimmune diseases and organ transplant rejection. However, the mechanism by which it modulates the immune system is not completely understood. MATERIAL AND METHODS: In this study, we investigated the mechanisms by which Dex modulated the immune response in mice given an allogeneic cardiac transplant. RESULTS: Dex injection significantly prolonged heart graft survival compared with phosphate-buffered saline-injected controls. Dex treatment increased the number of splenic MDSCs. Moreover, Gr-1high/CD11b+ MDSCs and CD3+/CD4+/Foxp3+ regulatory T cells (Tregs) were significantly increased in the Dex group compared with controls. Administration of anti-Gr-1 antibody (Ab) to the Dex group significantly shortened mouse heart graft survival. In addition, anti-Gr-1 Ab treatment significantly reduced Tregs in the Dex + anti-Gr-1 co-treatment group compared with the Dex group. These observations suggest that Dex treatment increased both MDSCs and Tregs, and that MDSCs regulated the incidence of Tregs in this immunosuppressive pathway. CONCLUSION: An important role of Dex in the prevention of the rejection of cardiac grafts in mice is to expand MDSCs and Tregs.

CCAT1 and CCAT2 long noncoding RNAs, located within the 8q.24.21 'gene desert', serve as important prognostic biomarkers in colorectal cancer.

BACKGROUND: 8q24.21 is a frequently amplified genomic region in colorectal cancer (CRC). This region is often referred to as a 'gene desert' due to lack of any important protein-coding genes, highlighting the potential role of noncoding RNAs, including long noncoding RNAs (lncRNAs) located around the proto-oncogene MYC. In this study, we have firstly evaluated the clinical significance of altered expression of lncRNAs mapped to this genomic locus in CRC. PATIENTS AND METHODS: A total of 300 tissues, including 280 CRC and 20 adjacent normal mucosa specimens were evaluated for the expression of 12 lncRNAs using qRT-PCR assays. We analyzed the associations between lncRNA expression and various clinicopathological features, as well as with recurrence free survival (RFS) and overall survival (OS) in two independent cohorts. RESULTS: The expression of CCAT1, CCAT1-L, CCAT2, PVT1, and CASC19 were elevated in cancer tissues (P = 0.039, <0.001, 0.018, <0.001, 0.002, respectively). Among these, high expression of CCAT1 and CCAT2 was significantly associated with poor RFS (P = 0.049 and 0.022, respectively) and OS (P = 0.028 and 0.015, respectively). These results were validated in an independent patient cohort, in which combined expression of CCAT1 and CCAT2 expression was significantly associated with a poor RFS (HR:2.60, 95% confidence interval [CI]: 1.04-6.06, P = 0.042) and a poor OS (HR:8.38, 95%CI: 2.68-37.0, P < 0.001). We established a RFS prediction model which revealed that combined expression of CCAT1, CCAT2, and carcinoembryonic antigen was a significant determinant for efficiently predicting RFS in stage II (P = 0.034) and stage III (P = 0.001) CRC patients. CONCLUSIONS: Several lncRNAs located in 8q24.21 locus are highly over-expressed in CRC. High expression of CCAT1 and CCAT2 significantly associates with poor RFS and OS. The expression of these two lncRNAs independently, or in combination, serves as important prognostic biomarkers in CRC.

Living Donor Kidney Transplantation After Brachytherapy for Prostate Cancer: Case Report.

INTRODUCTION: There is no obvious criterion about kidney transplantation for patients with pretransplant malignancy. Minimum tumor-free waiting periods differ according to type of cancer, staging, site of occurrence, response to therapy, and risk of cancer recurrence. We report a case of living donor kidney transplantation (LDKT) in a patient after brachytherapy for prostate cancer. CASE REPORT: The patient was a 65-year-old man with chronic kidney disease due to chronic glomerular nephritis. He received hemodialysis 3 times a week. His prostate-specific antigen level (PSA) was high (6.57 ng/mL), and he was diagnosed with prostate cancer (T1cN0M0, Gleason Score 3 + 4 = 7, 3/10) by needle biopsy in urology. He was treated with maximum androgen blockade (MAB) therapy and brachytherapy in May 2014. He underwent LDKT from a spousal donor at our department in December 2015, because urologists concluded that the prostate cancer was completely cured. Immunosuppression consisted of induction with basiliximab and maintenance with tacrolimus, mizoribine, and steroids. The postoperative course was uneventful. He discharged at postoperative day 29 with a serum creatinine level of 1.30 mg/dL. Three months after LDKT, his PSA level was 0.477 ng/mL, and there was no evidence of prostate cancer recurrence. CONCLUSION: This is the first case of LDKT for patients with prostate cancer after brachytherapy in combination with MAB. There is no recurrence of prostate cancer so far; however, careful follow-up including PSA is necessary and important.

Influences of Pre-formed Donor-Specific Anti-Human Leukocyte Antigen Antibodies in Living-Donor Renal Transplantation: Results With Graft Immunocomplex Capture Fluorescence Analysis.

BACKGROUND: Advances in immunosuppressants enable organ transplantation for sensitized patients. However, influences of pre-formed donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) have not been fully understood in renal transplantation (RT). On the other hand, immunocomplex capture fluorescence analysis (ICFA) is a reliable method to detect donor-specific anti-HLA antibodies and HLA antigen complexes. Graft ICFA can detect DSA in an allograft (g-DSA). METHODS: To elucidate the consequences of pre-formed DSA, 198 patients who underwent living-donor RT were enrolled for this study (observation period: 57.8 ± 34.9 months); 187 patients in the DSA- group (excluding ABO-incompatible cases) and 11 patients in the DSA+ group. Before RT, all DSA+ patients had undergone rituximab administration and plasmapheresis. For a graft ICFA, the biopsy specimen (1 × 105 cells) was dissolved, and HLA antigens were captured by anti-HLA beads. Finally, DSA-HLA complexes were detected by means of PE-conjugated anti-human IgG antibodies and analyzed by use of a Luminex system. A ratio (sample/blank beads, mean of fluorescence intensity) was calculated: ≥1.0 was determined as positive g-DSA. RESULTS: There were no significant differences in 5-year graft survival (87.9%/100% in the DSA-/DSA+ groups, respectively). In terms of antibody-mediated rejection (AMR), within 1 month after RT, pathologically determined AMR occurred 3.2% and 63.4% in the DSA- and DSA+ groups, respectively (P < .0001). However, interestingly, more than half of them (57.1%) indicated only subclinical AMR, that is, no fluctuation of S-Cr. As representative of 2 cases of subclinical AMR, g-DSA deposition could be confirmed (1.15 ± 0.04) at 1 hour after reperfusion by graft ICFA. Furthermore, g-DSA shifted to 2.20 ± 0.98 at 3 weeks after transplantation, along with a decline in s-DSA mean of fluorescence intensity (1718-506.5). CONCLUSIONS: Although pathologically determined AMR occurred more frequently in pre-formed DSA+ recipients, it can be argued that a successful de-sensitization protocol inhibits further production of DSA and graft destruction.

Effects of intravenous administration of umbilical cord blood CD34(+) cells in a mouse model of neonatal stroke.

Neonatal stroke occurs in approximately 1/4000 live births and results in life-long neurological impairments: e.g., cerebral palsy. Currently, there is no evidence-based specific treatment for neonates with stroke. Several studies have reported the benefits of umbilical cord blood (UCB) cell treatment in rodent models of neonatal brain injury. However, all of the studies examined the effects of administering either the UCB mononuclear cell fraction or UCB-derived mesenchymal stem cells in neonatal rat models. The objective of this study was to examine the effects of human UCB CD34(+) cells (hematopoietic stem cell/endothelial progenitor cells) in a mouse model of neonatal stroke, which we recently developed. On postnatal day 12, immunocompromized (SCID) mice underwent permanent occlusion of the left middle cerebral artery (MCAO). Forty-eight hours after MCAO, human UCB CD34(+) cells (1×10(5)cells) were injected intravenously into the mice. The area in which cerebral blood flow (CBF) was maintained was temporarily larger in the cell-treated group than in the phosphate-buffered saline (PBS)-treated group at 24h after treatment. With cell treatment, the percent loss of ipsilateral hemispheric volume was significantly ameliorated (21.5±1.9%) compared with the PBS group (25.6±5.1%) when assessed at 7weeks after MCAO. The cell-treated group did not exhibit significant differences from the PBS group in either rotarod (238±46s in the sham-surgery group, 175±49s in the PBS group, 203±54s in the cell-treated group) or open-field tests. The intravenous administration of human UCB CD34(+) cells modestly reduced histological ischemic brain damage after neonatal stroke in mice, with a transient augmentation of CBF in the peri-infarct area.

Functional folate receptor beta-expressing macrophages in osteoarthritis synovium and their M1/M2 expression profiles.

OBJECTIVE: The distribution of folate receptor (FR)-ß+ macrophages and their M1/M2 expression profiles were examined in osteoarthritis (OA) synovial tissues, and compared to those in rheumatoid arthritis (RA) synovial tissues and CD163+ macrophages in both OA and RA synovial tissues. METHOD: The phenotypes and fluorescein isothiocyanate (FITC)-folate uptake of FR-ß+ synovial macrophages were analysed by flow cytometry. The distribution of FR-ß+ macrophages in OA and RA synovial tissues was examined by immunofluorescent microscopy. Tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), interleukin (IL)-10, and transforming growth factor (TGF)-ß expression in FR-ß+ macrophages was detected by double-immunostaining in both OA and RA synovial tissues. RESULTS: FR-ß+ macrophages were predominantly present in the synovial lining layer in OA patients. The proportion of CD163-FR-ß+ cells in synovial mononuclear cells (MNCs) was increased in OA compared to RA synovial tissues. FR-ß(high) macrophages from OA synovial tissues represented the majority of folic acid-binding cells. Although FR-ß+ or CD163+ macrophages in the synovial tissues of OA and RA patients expressed a mixed pattern of M1 and M2 macrophage markers, there were more M2 markers expressing synovial macrophages in OA than in RA patients. CONCLUSIONS: The distribution and M1/M2 expression profiles of FR-ß+ synovial macrophages were different between OA and RA synovial tissues. Thus, the findings underscore that the M1/M2 paradigm using surface markers FR-ß and CD163 is an oversimplification of macrophage subsets. Functional FR-ß present on OA synovial macrophages provides a potential tool for the diagnosis and treatment of OA.

Glucocorticoid-induced TNF receptor-triggered T cells are key modulators for survival/death of neural stem/progenitor cells induced by ischemic stroke.

Increasing evidences show that immune response affects the reparative mechanisms in injured brain. Recently, we have demonstrated that CD4(+)T cells serve as negative modulators in neurogenesis after stroke, but the mechanistic detail remains unclear. Glucocorticoid-induced tumor necrosis factor (TNF) receptor (GITR), a multifaceted regulator of immunity belonging to the TNF receptor superfamily, is expressed on activated CD4(+)T cells. Herein, we show, by using a murine model of cortical infarction, that GITR triggering on CD4(+)T cells increases poststroke inflammation and decreases the number of neural stem/progenitor cells induced by ischemia (iNSPCs). CD4(+)GITR(+)T cells were preferentially accumulated at the postischemic cortex, and mice treated with GITR-stimulating antibody augmented poststroke inflammatory responses with enhanced apoptosis of iNSPCs. In contrast, blocking the GITR-GITR ligand (GITRL) interaction by GITR-Fc fusion protein abrogated inflammation and suppressed apoptosis of iNSPCs. Moreover, GITR-stimulated T cells caused apoptosis of the iNSPCs, and administration of GITR-stimulated T cells to poststroke severe combined immunodeficient mice significantly reduced iNSPC number compared with that of non-stimulated T cells. These observations indicate that among the CD4(+)T cells, GITR(+)CD4(+)T cells are major deteriorating modulators of poststroke neurogenesis. This suggests that blockade of the GITR-GITRL interaction may be a novel immune-based therapy in stroke.

One-year low-dose valacyclovir as prophylaxis for varicella zoster virus disease after allogeneic hematopoietic stem cell transplantation. A prospective study of the Japan Hematology and Oncology Clinical Study Group.

Varicella zoster virus (VZV) disease is a frequent complication after allogeneic hematopoietic stem cell transplantation (HSCT). We carried out a trial of 1-year low-dose valacyclovir (VCV) prophylaxis against VZV disease to evaluate its efficacy and safety. Patients received oral acyclovir (ACV) 1000mg/day until day 35 after HSCT. Oral VCV 500mg/day, 3 times a week, was started on day 36 and continued until 1 year after HSCT. The development of VZV disease was monitored until 2 years after HSCT. A total of 40 patients with a median age of 43 years were enrolled. VCV was well tolerated in all but 1 patient who discontinued it on day 224 because of thrombocytopenia of unknown cause. Seven patients developed VZV disease at a median of 479 days (range 145-651) after HSCT, with a cumulative incidence of 18.5%. Two patients developed breakthrough disease during VCV prophylaxis. The other 5 patients developed VZV disease after the discontinuation of VCV, and 3 of these had developed extensive chronic graft-versus-host disease. Visceral involvement and serious complications were completely eliminated. All patients responded to the therapeutic dose of VCV or ACV. One-year low-dose VCV can be safely and effectively administered for the prevention of VZV disease after allogeneic HSCT.

Effect of an immunotoxin to folate receptor beta on bleomycin-induced experimental pulmonary fibrosis.

It has been suggested that alveolar and interstitial macrophages play a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) by producing proinflammatory and/or fibrogenic cytokines. We showed that inflammatory macrophages expressed folate receptor beta (FRbeta) while resident macrophages in normal tissues expressed no or low levels of FRbeta. In the present study, we examined the distribution of FRbeta-expressing macrophages in the lungs of patients with usual idiopathic pulmonary fibrosis (UIP) and mice with bleomycin-induced pulmonary fibrosis (PF) and tested whether the depletion of FRbeta-expressing macrophages could suppress bleomycin-induced PF in mice. Immunostaining with anti-human or -mouse FRbeta monoclonal antibody (mAb) revealed that FRbeta-expressing macrophages were present predominantly in fibrotic areas of the lungs of patients with UIP and mice with bleomycin-induced PF. Intranasal administration of a recombinant immunotoxin, consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRbeta mAb and truncated Pseudomonas exotoxin A, increased survival significantly and reduced levels of total hydroxyproline and fibrosis in bleomycin-induced PF. In immunohistochemical analysis, decreased numbers of tumour necrosis factor-alpha-, chemokines CCL2- and CCL12-producing cells were observed in the immunotoxin-treated group. These findings suggest a pathogenic role of FRbeta-expressing macrophages in IPF. Thus, targeting FRbeta-expressing macrophages may be a promising treatment of IPF.

Relationship between tacrolimus blood concentrations and clinical outcome during the first 4 weeks after SCT in children.

The relationship between tacrolimus concentration and acute GVHD is not well known, with few published data available for lower target levels. We hypothesized that lower levels of tacrolimus would correlate with higher incidence of acute GVHD and poorer prognosis. Receiver operator characteristic curves (ROC) were used to quantify tacrolimus blood levels as predictors of grade II-IV acute GVHD. A total of 97 pediatric patients with hematological malignancies met the study criteria. On the ROC, a cutoff of 7 ng/ml provided the best balance between sensitivity and specificity (62.8 vs 68.2%, respectively). Cumulative incidence of acute GVHD was 65.9% (range 58.5-73.3%) in patients with mean tacrolimus concentration of < or =7 ng/ml and 34.8% (range 27.8-41.8%) in patients with mean tacrolimus concentration of >7 ng/ml (P=0.002). Incidence of non-relapse mortality (NRM) was higher in patients with tacrolimus of < or =7 ng/ml (42.9%; range 35.6-50.2%) than in patients with tacrolimus of >7 ng/ml (28.3%; range 17.4-39.2%; P=0.008). This translated into better EFS in patients with tacrolimus of >7 ng/ml (48.9%; range 39.8-58.0%) than in patients with tacrolimus of < or =7 ng/ml (31.8%; range 25.0-38.6%; P=0.031). Multivariate analysis showed that tacrolimus concentration was significantly associated with clinical outcomes. Mean whole-blood level of tacrolimus as continuous infusion should be maintained at > or =7 ng/ml for pediatric patients.

Oxidative stress causes alveolar bone loss in metabolic syndrome model mice with type 2 diabetes.

BACKGROUND AND OBJECTIVE: Alveolar bone loss is caused by a host response to periodontal pathogens, and its progression is often enhanced by systemic conditions such as insulin resistance. Alveolar bone dehiscence has been observed in KK-A(y) mice, which are metabolic syndrome model mice with type 2 diabetes. The aim of this study was to investigate inducements responsible for alveolar bone dehiscence in the KK-A(y) mice. MATERIAL AND METHODS: The expression of endothelial nitric oxide synthase in the mandibles of mice was detected using immunohistochemical staining and the reverse transcription-polymerase chain reaction. After administration of N-acetylcysteine, an antioxidant, to KK-A(y) mice, alveolar bone loss and the expression of endothelial nitric oxide synthase protein in gingival keratinocytes and of hydrogen peroxide concentrations in plasma, were analyzed. The effect of hydrogen peroxide on endothelial nitric oxide synthase expression in keratinocytes was examined using cultured keratinocytes. RESULTS: The expression of endothelial nitric oxide synthase was decreased in gingival keratinocytes from KK-A(y) mice compared with gingival keratinocytes from control mice. Administration of N-acetylcysteine to the mice restored endothelial nitric oxide synthase expression in the gingival keratinocytes, suppressed the alveolar bone loss and decreased the hydrogen peroxide concentrations in plasma without the improvement of obesity or diabetes. In vitro, stimulation with hydrogen peroxide decreased the expression level of endothelial nitric oxide synthase in cultured keratinocytes, which was restored by the addition of N-acetylcysteine. CONCLUSION: Reactive oxygen species, such as hydrogen peroxide, are responsible for the alveolar bone loss accompanied by decreased endothelial nitric oxide synthase expression in KK-A(y) mice. Therefore, we propose a working hypothesis that the generation of oxidative stress is an underlying systemic condition that enhances alveolar bone loss in periodontitis occurring as a complication of diabetes.

Significance of thrombomodulin release from gingival epithelial cells in periodontitis patients.

BACKGROUND AND OBJECTIVE: Thrombomodulin, a cell transmembrane glycoprotein, binds to thrombin and converts it from a procoagulant protease to an anticoagulant enzyme that activates protein C. Thrombomodulin is very important in regulating the function of thrombin. Elevated soluble thrombomodulin is present in the gingival crevicular fluid of subjects with periodontitis. The objective of the present study was to investigate the mechanisms about the elevated soluble thrombomodulin in gingival crevicular fluid. MATERIAL AND METHODS: Gingival sections from six patients with chronic periodontitis and from three periodontally healthy subjects were immunostained for thrombomodulin detection. Thrombomodulin levels were investigated in the gingival crevicular fluid of 11 subjects with chronic periodontitis. The effects of neutrophil enzymes on thrombomodulin release and on thrombomodulin in the gingival crevicular fluid were examined by an enzyme-linked immunosorbent assay or by Western blotting. RESULTS: The expression of gingival epithelial thrombomodulin was lost or decrease near infiltrating neutrophils. Thrombomodulin was rapidly released from gingival epithelial cells by neutrophil enzymes, and gingival crevicular fluid with periodontitis included the proteolytic cleavage thrombomodulin using immunoblotting analysis. The thrombomodulin release was not caused by rapid cell damage, on lactate dehydrogenase assay. There were significant differences in thrombomodulin content between gingival crevicular fluid samples from healthy and diseased sites, regardless of the degree of probing depth. CONCLUSION: Neutrophil enzymes induced rapid thrombomodulin release from the membrane surface of gingival epithelial cells. This might explain the thrombomodulin increase in gingival crevicular fluid with local diseased gingiva. Elevation of thrombomodulin in gingival crevicular fluid may be a potential marker of epithelial cell membrane injury.

Expansion of a unique macrophage subset in rheumatoid arthritis synovial lining layer.

The Z39Ig protein (complement receptor for C3b and iC3b) is expressed on resident tissue macrophages in various tissues. This study was undertaken to examine the distribution of Z39Ig+cells and their phenotypic features in rheumatoid arthritis (RA) synovium, in comparison with those of osteoarthritis (OA) and psoriatic arthritis (PsA) synovium. Monoclonal anti-Z39Ig antibody was produced by immunizing Z39Ig transfected murine pre B cells and used for the identification of Z39Ig+cells. Z39Ig+cells were further stained with antibodies to macrophages, fibroblast-like synoviocytes, complement receptors and dendritic cells by using the double immunostaining method in normal, RA, OA and PsA synovium. RA synovial mononuclear cells were double-stained using anti-Z39Ig and anti-CD11c antibodies and sorted into Z39Ig+CD11c+cells and Z39Ig+CD11c-cells. These cell populations were then analysed by electron microscopy. The expression of the Z39Ig protein was limited to intimal macrophages in normal, RA, OA and PsA synovium. The numbers of Z39Ig+CD11c+cells and the ratios of Z39Ig+CD11c+cells to Z39Ig+cells were increased in the synovial lining layer of RA as compared with those of OA and PsA. The ultrastructural analysis of Z39Ig+CD11c+cells showed the character of macrophages with many secondary lysosomes and swelling of mitochondria. Z39Ig+ cells appeared to be useful for identification of resident tissue macrophages in normal synovium and the corresponding macrophages in the synovial lining layer of inflammatory arthritis. Expansion of Z39Ig+CD11c+cells was characteristic of RA synovial lining layer.

Injectable growth/differentiation factor-5-recombinant human collagen composite induces endochondral ossification via Sry-related HMG box 9 (Sox9)expression and angiogenesis in murine calvariae.

BACKGROUND AND OBJECTIVE: The types of collagens available today as biomaterials are purified from animal tissues. A major growing concern, however, is their safety, since there are risks of viral and prion contamination and of unknown and potentially zoonotic infectious diseases. The present study aimed to assess, using immunohistochemistry, the effects of recombinant human growth/differentiation factor-5 (rhGDF-5) combined with recombinant human collagen I (rhCI) on bone formation in murine calvariae. MATERIAL AND METHODS: Composite rhGDF-5-rhCI or rhCI alone was injected subcutaneously into murine calvariae. After 3, 7 or 14 days, tissues were examined radiologically, histologically and immunohistochemically. The production of vascular endothelial growth factor (VEGF) by primary osteoblasts, periosteal cells and connective tissue fibroblasts isolated enzymatically from neonatal murine calvariae was also assessed. RESULTS: A protrusion was observed on the calvariae at the site injected with rhGDF-5/rhCI composite. Its mineral density was shown to be different from that of the existing bone by two-dimensional microcomputed tomography. Type II collagen-positive staining was restricted to newly formed tissues. Thus, the newly formed tissues seemed to be bone- and cartilage-like tissues. A number of vessels with positively stained cells for Von Willebrand factor were detected in the newly formed tissues. The rhGDF-5 enhanced VEGF production in cultured connective tissue fibroblasts. Sry-related HMG box 9 (Sox9)-positive cells were detected in the hypertrophic periosteum, and penetrated into the newly formed tissues. CONCLUSIONS: These results suggest that rhCI seems to allow the release of rhGDF-5 and that rhGDF-5-rhCI composite induces endochondral ossification via Sox9 expression and angiogenesis in murine calvariae.

The role of water channel aquaporin 3 in the mechanism of TNF-alpha-mediated proinflammatory events: Implication in periodontal inflammation.

Aquaporin 3 (AQP3) is the predominant water channel protein in human keratinocytes and acts as an inflammatory mediator in some lesions. A chronic, inflammatory process of periodontitis is related with a dramatic change of surrounding fluid homeostasis to plasma extravasation. The exact pattern of aquaporin (AQP) water channel expression and its mechanism in periodontal disease is still unknown. We describe herein an up-regulated AQP3 expression in the epithelial lesion with chronic periodontitis and its functional role. The levels of AQP3 expression in inflamed gingival epithelial tissues were significantly higher than those of healthy subjects. Consistent with these results, AQP3 expression (i.e., levels of mRNA and protein) in cultured rat primary gingival epithelial cells and the human gingival epithelial cell line Ca9-22 were strongly increased in response to TNF-alpha treatment through the 55 kDa TNF-alpha receptor (TNFR I). In this context, small interfering RNA- (siRNA)-mediated "aqp-3 gene silencing," which could reduce AQP3 expression by more than 65%, significantly attenuated selected proinflammatory events of ICAM-1 expression induced by TNF-alpha in Ca9-22. A sixfold increase in leukocyte adherence to TNF-alpha-stimulated epithelial cells was demonstrated by an adherence assay (P < 0.001) and pretreatment with AQP3 siRNA and anti-ICAM-1 antibody reduced leukocyte retention by 85% (P < 0.001). Our study indicates for the first time a novel important mode in the regulation of the inflammatory response through TNF-alpha/TNFR I ligation at the site of epithelial lesions by specialized membrane channel AQP3 and ICAM-1 protein, which is closely implicated in the development of periodontitis mechanisms.

Tumor necrosis factor-alpha stimulates gingival epithelial cells to release high mobility-group box 1.

BACKGROUND AND OBJECTIVE: High-mobility-group box 1 functions as a late-phase inflammatory mediator. It can be released extracellularly by macrophages and necrotic cells through lipopolysaccharide and tumor necrosis factor-alpha. The objective of this study was to clarify the source of high-mobility-group box 1 in chronic periodontitis tissues and tumor necrosis factor-alpha-stimulated gingival epithelial cells, and subsequently elucidate its inducible inflammatory pathway. MATERIAL AND METHODS: Chronic periodontitis and healthy gingival sections were stained for high-mobility-group box 1 by immunohistochemistry and immunofluorescence. The amounts of high-mobility-group box 1 released into the gingival crevicular fluid and supernatants from gingival epithelial cells stimulated by tumor necrosis factor-alpha were examined by western blot. The phosphorylation of mitogen-activated protein kinases (MAPKs) in gingival epithelial cells was also examined. RESULTS: High-mobility-group box 1 was detected in the cytoplasm and nucleus of gingival epithelial cells with periodontitis. Western blotting revealed a significant increase in high-mobility-group box 1 expression in the gingival crevicular fluid from periodontitis patients. High-mobility-group box 1 production in gingival epithelial cells was increased following stimulation with tumor necrosis factor-alpha. The molecular dialogue between tumor necrosis factor-alpha and gingival epithelial cells involved modulation of the activities of p38MAPK, Jun N-terminal kinase and p44/42. Interestingly, only phosphorylation of p38MAPK contributed to more than half of the signaling initiated by tumor necrosis factor-alpha-elicited high-mobility-group box 1 release. CONCLUSION: High-mobility-group box 1 is continuously released from the gingival epithelial cells modulated by tumor necrosis factor-alpha. These findings imply that high-mobility-group box 1 expression and possibly p38MAPK constitute important features in periodontitis.

Visualization of intracerebral arteries by synchrotron radiation microangiography.

BACKGROUND AND PURPOSE: Small cerebral vessels are a major site for vascular pathology leading to cerebral infarction and hemorrhage. However, such small cerebral vessels are difficult to visualize by using conventional methods. The goal of our study was the development of methodology allowing visualization of small cerebral arteries in rodents, suitable for experimental models. MATERIALS AND METHODS: Using barium sulfate as a contrast material, we obtained microangiographic images of physiologic and pathologic changes consequent to cerebral infarction in mouse brain by monochromatic synchrotron radiation (SR). To achieve high-resolution and high-contrast images, we used a new x-ray camera with a pixel size of 4.5 microm, and we set the energy level at 37.5 keV, just above the K absorption of barium. RESULTS: Small intracerebral arteries ( approximately 30 microm in diameter) were clearly visualized, as well as the cortical branches (50-70 microm in diameter) at the brain surface. The limit of detection appeared to be vessels approximately 10 microm in diameter. Compared with the noninfarcted side, the number of intracerebral arteries was dramatically decreased in the middle cerebral artery area affected by stroke. CONCLUSIONS: These results indicate the potential of SR for evaluating pathologic changes in small cerebral arteries and for monitoring the impact of pro- and antiangiogenic therapeutic strategies.