Comprehensive analysis of gene expression of isolated pancreatic islets during pretransplant culture

Background: The aim of this study was to investigate the effect of pretransplant culture on the survival of pancreatic islet grafts, and to determine the biological characteristics of isolated islets during pretransplant culture. Methods: The survival of islets from Wistar rats, transplanted to diabetic C57BL/B6 mice, was compared between fresh islets and cultured islets. A comprehensive gene expression analysis was employed to investigate biological processes during pretransplant culture, and in vitro validation studies were performed. Results: Survival of cultured xenografts was significantly prolonged as compared to that of fresh islets (fresh: 12.5 ± 1.9 days, 1-day cultured: 16.0 ± 1.3 days (p= 0.017), 3-day cultured: 17.0 ± 2.6 days (p= 0.014)). Comprehensive gene expression analysis identified significant upregulation of annotated functions associated with inflammation in cultured groups. Six proinflammatory genes, including heme oxygenase 1 (HO-1) and IL-6, were significantly upregulated during culture. Validation studies revealed significantly higher levels of IL-6 in the supernatant of cultured islets and HO-1 in the cultured islets when compared with fresh islets. Conclusion: Transplantation of cultured islets induced significant but minimal prolongation of graft survival in xenogeneic combinations. Comprehensive analysis of gene expression in cultured islets showed biological processes associated with proinflammation during culture.     

Success rates in minimal stimulation cycle IVF with clomiphene citrate only

Journal of Assisted Reproduction and Genetics - To determine age-adjusted overall success rates for patients undergoing clomiphene citrate only minimal stimulation cycle (mini) in vitro...     

Elevation of circulating neutrophil extracellular traps,interleukin (IL)-8, IL-22, and vascular endothelial growth factor in patients with venomous snake mamushi (Gloydius blomhoffii) bites

Mamushi bites cause swelling and pain that extend from the bitten site. The coagulopathic, anti-coagulopathic, and vasculopathic actions of mamushi venom result in various laboratory abnormalities, occasionally with muscular, renal, and other organ damage. We investigated the serum biomarkers that were associated with the pathogenesis of mamushi bites, focusing on markers related to tissue-damage and neutrophil activation. Twenty patients (one case of grade 2, 13 cases of grade 3, and six cases of grade 4 of severity) seen by us in one summer season were enrolled. Peripheral blood samples were taken from the patients on day 0, day 2, and day 7 after mamushi bites. In addition to routine blood examination, serum samples were subjected to enzyme-linked immunosorbent assay for citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-17A, IL-22, vascular endothelial growth factor (VEGF), high mobility group box protein 1 (HMGB1), tumor necrosis factor (TNF)-α, and IL-33. Creatinine kinase (CK) values significantly correlated with prothrombin time (PT) levels, suggesting that muscular damage is associated with exaggerated coagulation and fibrinolysis. In the vast majority of patients, HMGB1, TNF-α, and IL-33 were under detection levels. Neutrophil counts did not correlate with PT or CK, indicating that the coagulation disorder and muscular damage were virtually independent of the neutrophil activation. The neutrophil number significantly correlated with CitH3, a representative marker of neutrophil extracellular traps. Moreover, there were significant correlations between neutrophil number, CitH3, IL-8, IL-22, and VEGF. Our study suggests that there are two major cascades in mamushi bites. One is an already characterized venom effect on coagulation, vessels, and muscles. In the other novel cascade, we propose that neutrophil activation with IL-8 leads to the production of IL-22 and VEGF. This sequential event may contribute to both vascular damage and repair.     

Porphyromonas gingivalis fimbriae induce unique dendritic cell subsets via Toll-like receptor 2

Backgound and Objective:  Dendritic cells (DCs) play a critical role in the activation of T cells as well as in shaping immune responses. We have reported previously that Porphyromonas gingivalis lipopolysaccharides ( Pg LPS) induced a CD14⁺CD16⁺ DC subset with a weak immuno-stimulatory activity. In contrast, Escherichia coli LPS ( Ec LPS) induced fully matured DCs with strong immunostimulatory activities. Since Pg LPS as well as Pg fimbriae have been indicated to work as Toll-like receptor (TLR) 2 ligands, we speculate that the TLR usage of bacterial antigens may be critical for DC maturation.
Material and Methods:  We investigated the effect of Pg fimbriae on the phenotype and function of human peripheral blood DCs in comparison with a TLR2 ligand, peptidoglycan, and a TLR4 ligand, Ec LPS.
Results:  Flow cytometry revealed that Pg fimbriae and peptidoglycan but not Ec LPS induced CD14 and CD16 expression on peripheral blood DCs (CD14⁻CD16⁻). A monoclonal antibody against TLR2 abrogated this induction, but an antibody against TLR4 had no effect. Dendritic cells stimulated with Pg fimbriae had a weaker capability to induce allogenic T cell proliferation and exhibited a weaker production of interleukin-8 and regulated upon activation, normal T cell expressed and secreted (RANTES) than DCs stimulated with Ec LPS.
Conclusion:  These results indicate that different TLR usage affects mature DC phenotype and function and is thus crucial to the regulation of immunity to the pathogen.     

Effect of electrical stimulation of the internal capsule on nociceptive neurons responding to orofacial stimuli in the medullary dorsal horn of the rat

Internal capsule (IC) stimulation has been used clinically to alleviate central pain. However, the neuronal mechanisms underlying pain relief by IC stimulation are poorly understood. In order to elucidate the analgesic mechanism, the effect of IC conditioning stimulation on nociceptive neurons in the rat medullary dorsal horn was investigated in the present study. Rats were anaesthetized with N(2)O-O(2) (2:1) and 0.5% halothane and were immobilized with pancuronium bromide. Two kinds of nociceptive neurons, wide dynamic range (WDR) and nociceptive specific (NS) neurons, responding to noxious stimulations of the face and oral structures were recorded in the trigeminal caudal nucleus and the medial reticular subnuclei. A test stimulus with a single rectangular pulse (2ms in duration, 5-70V) was applied to the centre of the receptive field. Responses in 55.9% of the WDR neurons and in 60% of the NS neurons were inhibited by conditioning stimuli to the ipsilateral IC with trains of 33 pulses (100-300microA) at 330Hz. The percents of peak inhibitory effects on WDR neurons and NS neurons were 78.1+/-25.0% (n=19) and 89.0+/-13.6% (n=3), respectively. The inhibitory effect continued for conditioning-test intervals of up to 500ms. Effective sites for conditioning stimulation were concentrated in the lateral side of the IC. These findings suggest that modulation of nociceptive transmission by IC stimulation occurs at second-order neurons via a presynaptic phenomenon by corticofugal fibers in the IC.     

Rationale behind the design and comparative evaluation of an all-in-one self-etch model adhesive

Objective

To investigate and compare bonding and dentin sealing efficacy of a marketed all-in-one and an experimental model adhesive with minimum effective amounts of acidic monomer and water.

Materials and methods

Composition of model adhesive (NAD) in mass%: UDMA (45), 4-META (20), H₂O (7.5), and acetone (27.5). For characterization of a reasonable NAD application procedure shear bond strengths (SBS, n = 8) were determined on human enamel and dentin. Clearfil S³ Bond (TSB; Kuraray) served as reference. SBSs were evaluated after 10 min, 1 and 7 days, and 1 month, marginal adaptation (n = 8) was assessed in cylindrical butt-joint dentin cavities. Diffusive and convective water fluxes through 1 mm thick adhesive-coated dentin disks (n = 6) were qualitatively and quantitatively analyzed.

Results

SBSs proved that application of NAD in one coat with 20 s agitated dwell time was ≥20 MPa, enamel SBSs (24 h) were 25 MPa, p > 0.05. Dentin SBSs for TSB and NAD were not different (p > 0.05) at the four stages (means: 18.9, 23.5, 25.4, and 23.6 MPa). Five and seven of the eight bonded restorations with TSB and NAD were gap-free (p > 0.05). Dentin disks treated with EDTA from both sides or one side only were highly permeable for liquid, whereas adhesive-coated dentin disks showed no permeability at 0 and 2.5 kPa water pressure.

Conclusions

Within the limitations of this study the model adhesive tested represents a promising basic composition for all-in-one adhesives, eliminating common problems encountered with single step adhesives such as phase separation and permeability.     

Proteolysis of ICAM-1 on human oral epithelial cells by gingipains

Cysteine proteinases (gingipains) from Porphyromonas gingivalis are considered key virulence factors of severe periodontitis and host immune evasion. Since expression of intercellular adhesion molecule-1 (ICAM-1) on gingival epithelium is indispensable in polymorphonuclear leukocyte (PMN) migration at the site of periodontitis, we examined the effects of gingipains on the expression of ICAM-1 on human oral epithelial cell lines (KB and HSC-2) by flow cytometry and Western blotting. We found that three purified forms of gingipains efficiently reduced ICAM-1 expression on the cells in a time- and dose-dependent manner. Gingipains reduced the expression on fixed cells and degraded the ICAM-1 in the cell membranes, indicating that the reduction resulted from direct proteolysis. They then disturbed the ICAM-1-dependent adhesion of PMNs to the cells. These results indicate that gingipains cleave ICAM-1 on oral epithelial cells, consequently disrupting PMN-oral epithelial cell interaction, and are involved in immune evasion by the bacterium in periodontal tissues.