Inhibition of CD62L+ T-cell response in vitro via a novel sulfo-glycolipid, beta-SQAG9 liposome that binds to CD62L molecule on the cell surface

We previously reported that synthetic sulfo-glycolipid, 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG(18:0)) which was deduced from sulfonoquinovosyl-diacylglycerols of sea urchin possessed immunosuppressive effects, such as human mixed lymphocyte reaction (MLR) and skin allograft in rat, and that these effects were caused by contact inhibition between T-cells and antigen presenting cells (APCs). Here, we investigated the mechanism of these immunosuppressive effects on human MLR by beta-SQAG9 which had been newly synthesized from beta-SQDG(18:0) to improve structural stability in water solution. CD62L+ T-cells in peripheral blood predominantly respond to APCs, and beta-SQAG9 inhibited the response of CD62L+ T-cell subset in human allogeneic MLR. Surprisingly, it was demonstrated that beta-SQAG9 bound to L- and P-selectin (CD62L and P) molecule in vitro. Meanwhile, beta-SQAG9 efficiently formed liposome structure and bound to L-selectin on the cell surface of CD62L+ T-cell subset but might not be incorporated into the cells. Because the immunosuppressive effects of beta-SQAG9 disappeared when beta-SQAG9 liposome was changed to soluble form by detergent, the liposome structure of beta-SQAG9 was presumed to be essential for these effects. These findings suggested beta-SQAG9 to be a novel drug with a unique immunosuppressive action.     

Characterization of Toxin Complex Produced by a Unique Strain of Clostridium botulinum Serotype D 4947

A unique strain of Clostridium botulinum, serotype D 4947 (D-4947), produces a considerable amount of a 650 kDa toxin complex (L-TC) and a small amount of a 280 kDa M-TC, a 540 kDa TC, and a 610 kDa TC. The complexes are composed of only un-nicked components, including neurotoxin (NT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33 and HA-17). Unlike other NTs from all serotype strains, separation of D-4947 NT from L-TC, except for M-TC, during chromatography required highly alkaline conditions around pH 8.8. The separated NT and NTNHA/HAs complex can be reconstituted to L-TC that is indistinguishable from the parent L-TC with respect to toxicity, hemagglutination activity and gel filtration profile. The isoelectric points of NT and NTNHA/HAs were close together depending on the number of HA-33/17 molecules. We have established a new method to separate the unique D-4947 NT from the complex, which will yield valuable information on structure of botulinum toxin.     

Down-regulation of E-cadherin by antisense oligonucleotide enhances basement membrane invasion of pancreatic carcinoma cells

Pancreatic carcinoma shows a marked invasiveness around tissues lymph node and/or hematogenous metastases resulting in poor prognoses of the patients. We examined on whether E-cadherin is associated with these malignant behaviors of pancreatic carcinoma cells using a human pancreatic adenocarcinoma cell line, JHP-1. Immunohistochemically, E-cadherin expression of JHP-1 cells was remarkably inhibited by treatment with E-cadherin antisense oligonucleotide. By invasion-MTT assay, JHP-1 cells treated with E-cadherin antisense oligonucleotide showed a significant increase of invasiveness compared to those treated with the control oligonucleotide (P < 0.001), whereas the proliferation of JHP-1 cells was not affected by the presence of either E-cadherin antisense or control oligonucleotide. Thus, down-regulation of E-cadherin of pancreatic carcinoma cells induced the invasiveness into the basement membrane. These results suggest that the reduction in E-cadherin expression plays a key role not only in detachment of cell-cell adhesion but also in invasion and metastasis of pancreatic carcinoma cells.     

Senescence marker protein 30 protects intestinal epithelial cells against inflammation-induced cell death by enhancing Nrf2 activity

Senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases during senescence. SMP30 deficiency increases susceptibility to cytokine-induced apoptosis in the liver and to radiation-induced apoptosis in the small intestine. Furthermore, colonic epithelial cell death is associated with the severity of colitis. Therefore, in the present study, we investigated the function of SMP30 during intestinal inflammation. In SMP30 deficient mice, colitis was significantly exacerbated as demonstrated by increased mortality (p?=?0.001), body weight loss (p?=?0.0105 at day 8), rectal bleeding (p?=?0.0047 at day 8) and diarrhea (p?=?0.0030 at day 8), histological scores (ulcers, p?=?0.0002; edema, p?=?0.0125; leukocyte infiltration, p?=?0.0016) and productions of pro-inflammatory cytokines (IL-1α, p?=?0.0452; IL-6, p?=?0.0074; G-CSF, p?=?0.0036). In addition, greater proportions of apoptotic cells and lower levels of anti-apoptotic marker proteins (total PARP-1 and Bcl-2) were observed in the inflamed intestines of SMP30 deficient mice than in wild type controls. In vitro experiments on colonic epithelial cells showed that stable SMP30 expression inhibited but that SMP30 siRNA expression increased TNF-α-induced apoptosis. SMP30 inhibition decreased Nrf2 mRNA expression levels (p?<?0.0001), but SMP30 overexpression increased Nrf2 mRNA expression levels (p?=?0.0495). The underlying mechanism by which SMP30 protected cells appeared to be by inhibiting Nrf2 ubiquitination and Keap1 expression, and thus enhancing Nrf2 activity. Moreover, SMP30 deficiency increased the incidence of colitis-associated colon cancer as determined by increased mortality (p?=?0.0572) and average polyp number (p?=?0.0277). Collectively, these findings suggest that SMP30 protects intestinal epithelial cells from apoptosis and this can contribute to amelioration of colitis and colitis-associated colon cancer.     

Prehospital Lactated Ringer's Solution Treatment and Survival in Out-of-Hospital Cardiac Arrest: A Prospective Cohort Analysis

Background

No studies have evaluated whether administering intravenous lactated Ringer''s (LR) solution to patients with out-of-hospital cardiac arrest (OHCA) improves their outcomes, to our knowledge. Therefore, we examined the association between prehospital use of LR solution and patients'' return of spontaneous circulation (ROSC), 1-month survival, and neurological or physical outcomes at 1 month after the event.

Methods and Findings

We conducted a prospective, non-randomized, observational study using national data of all patients with OHCA from 2005 through 2009 in Japan. We performed a propensity analysis and examined the association between prehospital use of LR solution and short- and long-term survival. The study patients were ≥18 years of age, had an OHCA before arrival of EMS personnel, were treated by EMS personnel, and were then transported to hospitals. A total of 531,854 patients with OHCA met the inclusion criteria. Among propensity-matched patients, compared with those who did not receive pre-hospital intravenous fluids, prehospital use of LR solution was associated with an increased likelihood of ROSC before hospital arrival (odds ratio [OR] adjusted for all covariates [95% CI] = 1.239 [1.146–1.339] [p<0.001], but with a reduced likelihood of 1-month survival with minimal neurological or physical impairment (cerebral performance category 1 or 2, OR adjusted for all covariates [95% CI] = 0.764 [0.589–0.992] [p = 0.04]; and overall performance category 1 or 2, OR adjusted for all covariates [95% CI] = 0.746 [0.573–0.971] [p = 0.03]). There was no association between prehospital use of LR solution and 1-month survival (OR adjusted for all covariates [95% CI] = 0.960 [0.854–1.078]).

Conclusion

In Japanese patients experiencing OHCA, the prehospital use of LR solution was independently associated with a decreased likelihood of a good functional outcome 1 month after the event, but with an increased likelihood of ROSC before hospital arrival. Prehospital use of LR solution was not associated with 1-month survival. Further study is necessary to verify these findings. Please see later in the article for the Editors'' Summary     

Tuning Colors of Silver Nanoparticle Sheets by Multilayered Crystalline Structures on Metal Substrates

We report a new concept of tuning plasmonic colors of two-dimensional crystalline silver nanoparticle sheets with layer-by-layer structures. The multilayered crystalline sheets fabricated by the Langmuir–Schaefer method keep the localized surface plasmon resonance bands at the same position (λ _max?=?465 nm) on quartz, while they change their colors drastically on metal substrates depending on the number of layers (one to five layers). The response of the absorption spectra was absolutely nonlinear, with maximum absorption for two or three layers. The obtained results were well reproduced by the finite difference time domain simulation. The simulation confirmed that these plasmonic colors originate not only from near-field coupling of plasmon resonance but also far-field nano-optics of the multilayered silver nanoparticle sheets.     

SSCP Screening of the Dihydropyrimidine Dehydrogenase Gene Polymorphisms of the Japanese Population Using a Semi-automated Electrophoresis Unit

The single-strand conformation polymorphism (SSCP) procedure has been applied in routine testing for hereditary diseases. Temperature, running buffer, gel composition, and fragment length can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes, such as the dihydropyrimidine dehydrogenase (DPYD) gene, which codes the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). The authors have optimized the condition of SSCP with an automated system (GenePhor system, GE Healthcare UK Ltd.) to screen genetic polymorphisms in the DPYD gene. The efficiency of the method was evaluated using 21 positive controls (DNA samples with polymorphisms in the DPYD gene, previously characterized) and DNA samples from 35 Japanese. Results showed that the use of three different running buffers (pH 7.4, 8.3, and 9.0) in combination with other optimized conditions (10% polyacrylamide gel, 60-90 min at constant 900 V at 5 degrees C) resulted in a high polymorphism detection rate (95.3%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for pharmacogenetic studies on 5FU.     

Alterations of NOS, arginase, and DDAH protein expression in rabbit cavernous tissue after administration of cigarette smoke extract

Cigarette smoking is an independent risk factor for vasculogenic erectile dysfunction (ED). Nitric oxide (NO) has been demonstrated to be the principal mediator of cavernous smooth muscle relaxation and penile erection. Therefore, we examined whether or not enzyme activities and factors involved in the NO generation pathway are affected in rabbit corpus cavernosum after administration of nicotine- and tar-free cigarette smoke extract (CSE). CSE was prepared by bubbling a stream of cigarette smoke into phosphate-buffered saline. CSE was injected subcutaneously into adult male rabbits once a day for 5 wk. In the CSE group, significantly decreased cyclic GMP production as a marker of NO generation was associated with attenuated overall nitric oxide synthase (NOS) activity, enhanced arginase activity, accumulation of endogenous NOS inhibitors such as monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity as an metabolizing enzyme of endogenous NOS inhibitors. Neuronal NOS (nNOS) and DDAH I protein expression were decreased without altering endothelial NOS expression, while arginase I expression was upregulated. These results suggest that impaired NO production would result from blunted NOS activity, which is possibly brought about by the downregulation of nNOS protein, accumulation of endogenous NOS inhibitors, and enhanced arginase activity together with upregulation of arginase I protein in cavernous tissue. The impaired DDAH activity due to decreased expression of DDAH I protein would result in an accumulation of endogenous NOS inhibitors with CSE. These alterations may be relevant to induction of the erectile dysfunction following CSE.