Preparation and epitope analysis of monoclonal antibodies against African swine fever virus DP96R protein.

BACKGROUND: Many proteins of African swine fever virus (ASFV, such as p72, p54, p30, CD2v, K205R) have been successfully expressed and characterized. However, there are few reports on the DP96R protein of ASFV, which is the virulence protein of ASFV and plays an important role in the process of host infection and invasion of ASFV. RESULTS: Firstly, the prokaryotic expression vector of DP96R gene was constructed, the prokaryotic system was used to induce the expression of DP96R protein, and monoclonal antibody was prepared by immunizing mice. Four monoclonal cells of DP96R protein were obtained by three ELISA screening and two sub-cloning; the titer of ascites antibody was up to 1:500,000, and the monoclonal antibody could specifically recognize DP96R protein. Finally, the subtypes of the four strains of monoclonal antibodies were identified and the minimum epitopes recognized by them were determined. CONCLUSION: Monoclonal antibody against ASFV DP96R protein was successfully prepared and identified, which lays a foundation for further exploration of the structure and function of DP96R protein and ASFV diagnostic technology.

Rational Design of an Air-Stable, High-Spin Diradical with Diazapyrene.

Stable carbon-based polyradicals exhibiting strong spin-spin coupling and slow depolarization processes are particularly attractive functional materials. A new molecular motif synthesized by a convenient method that allows the integration of stable, high-spin radicals to (hetero)aromatic polycycles has been developed, as illustrated by a non-Kekulé diradical showing a triplet ground state with long persistency (τ1/2 ≈31 h) in air. Compared to the widely used 1,3-phenylene, the newly designed (diaza)pyrene-4,10-diyl moiety is for the first time demonstrated to confer ferromagnetic (FM) spin coupling, allowing delocalized non-disjoint SOMOs. With the X-ray crystallography unambiguously proving the diradical structure, the triplet ground state was thoroughly characterized. A large ΔES-T of 1.1 kcal/mol, proving the strong FM coupling effect, was revealed consistently by superconducting quantum interference device (SQUID) measurements and variable-temperature electron paramagnetic resonance (EPR) spectroscopy, while the zero-field splitting and triplet nutation characters were examined by continuous-wave and pulsed EPR spectroscopy. A millisecond spin-lattice relaxation time was also detected. The current study not only offers a new molecular motif enabling FM coupling between carbon-based spins, but more importantly presents a general method for installing stable polyradicals into functional π-systems.

[Treadmill exercise alleviates neuropathic pain by regulating mitophagy of the anterior cingulate cortex in rats].

This study aimed to investigate the effect of treadmill exercise on neuropathic pain and to determine whether mitophagy of the anterior cingulate cortex (ACC) contributes to exercise-mediated amelioration of neuropathic pain. Chronic constriction injury of the sciatic nerve (CCI) was used to establish a neuropathic pain model in Sprague-Dawley (SD) rats. Von-Frey filaments were used to assess the mechanical paw withdrawal threshold (PWT), and a thermal radiation meter was used to assess the thermal paw withdrawal latency (PWL) in rats. qPCR was used to evaluate the mRNA levels of Pink1, Parkin, Fundc1, and Bnip3. Western blot was used to evaluate the protein levels of PINK1 and PARKIN. To determine the impact of the mitophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP) on pain behaviors in CCI rats, 24 SD rats were randomly divided into CCI drug control group (CCI+Veh group), CCI+CCCP low-dose group (CCI+CCCP0.25), CCI+CCCP medium-dose group (CCI+CCCP2.5), and CCI+CCCP high-dose group (CCI+CCCP5). Pain behaviors were assessed on 0, 1, 3, 5, and 7 days after modeling. To explore whether exercise regulates pain through mitophagy, 24 SD rats were divided into sham, CCI, and CCI+Exercise (CCI+Exe) groups. The rats in the CCI+Exe group underwent 4-week low-moderate treadmill training one week after modeling. The mechanical pain and thermal pain behaviors of the rats in each group were assessed on 0, 7, 14, 21, and 35 days after modeling. Western blot was used to detect the levels of the mitophagy-related proteins PINK1, PARKIN, LC3 II/LC3 I, and P62 in ACC tissues. Transmission electron microscopy was used to observe the ultrastructure of mitochondrial morphology in the ACC. The results showed that: (1) Compared with the sham group, the pain thresholds of the ipsilateral side of the CCI group decreased significantly (P < 0.001). Meanwhile, the mRNA and protein levels of Pink1 were significantly higher, and those of Parkin were lower in the CCI group (P < 0.05). (2) Compared with the CCI+Veh group, each CCCP-dose group showed higher mechanical and thermal pain thresholds, and the levels of PINK1 and LC3 II/LC3 I were elevated significantly (P < 0.05, P < 0.01). (3) The pain thresholds of the CCI+Exe group increased significantly compared with those of the CCI group after treadmill intervention (P < 0.001, P < 0.01). Compared with the CCI group, the protein levels of PINK1 and P62 were decreased (P < 0.001, P < 0.01), and the protein levels of PARKIN and LC3 II/LC3 I were increased in the CCI+Exe group (P < 0.01, P < 0.05). Rod-shaped mitochondria were observed in the ACC of CCI+Exe group, and there were little mitochondrial fragmentation, swelling, or vacuoles. The results suggest that the mitochondrial PINK1/PARKIN autophagy pathway is blocked in the ACC of neuropathic pain model rats. Treadmill exercise could restore mitochondrial homeostasis and relieve neuropathic pain via the PINK1/PARKIN pathway.

Highly Effective Photocatalytic Radical Reactions Triggered by a Photoactive Metal-Organic Framework.

On account of their inherent reactive properties, radical reactions play an important role in organic syntheses. The booming photochemistry provides a feasible approach to trigger the generation of radical intermediates in organic reaction processes. Thus, developing effective photocatalysts becomes the key step in radical reactions. In this work, the triphenylamine moiety with photoactivity is successfully embedded in a highly porous and stable metal-organic framework (MOF), and the obtained MOF, namely, Zr-TCA, naturally displays a photoactive property derived from the triphenylamine-based ligand. In photocatalytic studies, the triphenylamine-based Zr-TCA not only exhibits a high catalytic activity on the aerobic oxidation of sulfides via the generation of the superoxide radical anion (O2•-) under light irradiation but also shows good efficiency in the trifluoromethylation of arenes and heteroarenes by the formation of the trifluoromethyl radical (CF3•) as an intermediate. Moreover, the high performance of Zr-TCA can be well maintained over a wide range of substrates in these radical reactions, and the recycled Zr-TCA still retains its excellent photocatalytic activity. The high recyclability and catalytic efficiency to various substrates make the constructed triphenylamine-based Zr-TCA a promising photocatalyst in diverse radical reactions.

Serum level of interleukin-35 as a potential prognostic factor for gastric cancer.

AIMS: Interleukin-35 (IL-35), a novel anti-inflammatory cytokine, has recently been implicated in tumor development, progression, and survival. However, the relationship between serum IL-35 levels and gastric cancer (GC) is inconclusive. Here, we performed this study to clarify the role of serum level of IL-35 in GC patients. METHODS: We enrolled 180 GC patients and 170 healthy controls and used enzyme-linked immunosorbent assay to detect serum IL-35 levels. The clinical relevance between IL-35 and clinical pathology parameters was assessed. Univariate and multivariate logistic regressions were used to determine the feasibility of IL-35 as a clinical biomarker. RESULTS: We observed that serum IL-35 levels were significantly higher in GC patients (17.559 ± 13.266 pg/mL) than in healthy controls (8.077 ± 3.801 pg/mL, P < .001). High serum IL-35 levels were significantly associated with clinical stage (P = .048) and Helicobacter pylori (HP) infection (P < .001). The Kaplan-Meier survival analysis indicated that patients in the high-IL-35 group had poor overall survival (OS) and progression-free survival (PFS) (median OS: 26.0 vs 36.0 months, P < .001; median PFS: 18.0 vs.26.0 months, P = .044). Multivariate analyses demonstrated that serum IL-35 was an independent prognostic factor for GC (OS: hazard ratio [HR] = 1.031 [95% CI, 1.017-1.045], P < .001; PFS: HR = 1.029 [95% CI, 1.015-1.043], P < .001). CONCLUSIONS: High serum IL-35 levels are associated with poor disease prognosis in GC patients, and it may be become a new and promising biomarker for prognosis of gastric cancer.

Chang'an II Decoction ( II )-Containing Serum Ameliorates Tumor Necrosis Factor-α-Induced Intestinal Epithelial Barrier Dysfunction via MLCK-MLC Signaling Pathway in Rats.

OBJECTIVE: To investigate the effect of Chang'an II Decoction ( II ))-containing serum on intestinal epithelial barrier dysfunction in rats. METHODS: Tumor necrosis factor (TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium. Caco-2 monolayers were treated with blank serum and Chang'an II Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang'an II Decoction intragastrically at doses of 0.49, 0.98, 1.96 g/(kg·d) for 1 week, respectively. After preparation of containing serum, cells were divided into the normal group, the model group, the Chang'an II-H, M, and L groups (treated with 30 ng/mL TNF-α and medium plus 10% high, middle-, and low-doses Chang'an II serum, respectively). Epithelial barrier function was assessed by transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate (FITC)-labeled dextran. Transmission electron microscopy was used to observe the ultrastructure of tight junctions (TJs). Immunofluorescence of zonula occludens-1 (ZO-1), claudin-1 and nuclear transcription factor-kappa p65 (NF-κ Bp65) were measured to determine the protein distribution. The mRNA expression of myosin light chain kinase (MLCK) was measured by real-time polymerase chain reaction. The expression levels of MLCK, myosin light chain (MLC) and p-MLC were determined by Western blot. RESULTS: Chang'an II Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α. It alleviated TNF-α-induced morphological alterations in TJ proteins. The increases in MLCK mRNA and MLCK, MLC and p-MLC protein expressions induced by TNF-α were significantly inhibited in the Chang'an II-H group. Additionally, Chang'an II Decoction significantly attenuated translocation of NF-κ Bp65 into the nucleus. CONCLUSION: High-dose Chang'an II-containing serum attenuates TNF-α-induced intestinal barrier dysfunction. The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κ Bp65.

Elevated serum levels of interleukin-37 correlate with poor prognosis in gastric cancer.

BACKGROUND: interleukin-37 (IL-37) is as a natural suppressor of the innate inflammatory and immune responses. It has also been reported to be involved in carcinogenesis and metastasis. The present case-control study was designed to investigate the role of serum levels of IL-37 in patients with gastric cancer. METHODS: serum IL-37 levels were determined using the enzyme-linked immunosorbent assay in 180 patients diagnosed with gastric cancer and 100 healthy controls. The association between IL-37 levels and clinical factors was assessed. Univariate and multivariate analyses were performed to investigate the prognostic significance of these parameters in gastric cancer. RESULTS: serum IL-37 levels in gastric cancer patients (5.606 ± 0.837 pg/ml) were significantly higher than those in healthy controls (2.364 ± 0.210 pg/ml, p < 0.001). High serum IL-37 levels were related to a poorly differentiated histologic type (p = 0.046) and advanced T stage (p = 0.003). The Kaplan-Meier survival analysis indicated that the high-IL-37 group had a poorer overall survival and progression-free survival (overall survival [OS]: 39.0 months vs 13.0 months, p < 0.001, progression-free survival [PFS]: 25.0 months vs 10.0 months, p < 0.001). Multivariate analyses showed serum IL-37 to be an independent prognostic factor for gastric cancer patients (OS: hazard ratios [HR] = 1.842, 95% CI: 1.190-2.854, p = 0.006; PFS: HR = 1.547, 95% CI: 1.014-2.359, p = 0.043). CONCLUSIONS: in conclusion, serum IL-37 levels were associated with poor overall survival and progression-free survival in gastric cancer patients. IL-37 may be a potential predictor of prognosis in gastric cancer.

Effects of Xingnaojing Injection on Adenosinergic Transmission and Orexin Signaling in Lateral Hypothalamus of Ethanol-Induced Coma Rats.

Acute alcohol exposure induces unconscious condition such as coma whose main physical manifestation is the loss of righting reflex (LORR). Xingnaojing Injection (XNJI), which came from Chinese classic formula An Gong Niu Huang Pill, is widely used for consciousness disorders in China, such as coma. Although XNJI efficiently shortened the duration of LORR induced by acute ethanol, it remains unknown how XNJI acts on ethanol-induced coma (EIC). We performed experiments to examine the effects of XNJI on orexin and adenosine (AD) signaling in the lateral hypothalamic area (LHA) in EIC rats. Results showed that XNJI reduced the duration of LORR, which implied that XNJI promotes recovery form coma. Microdialysis data indicated that acute ethanol significantly increased AD release in the LHA but had no effect on orexin A levels. The qPCR results displayed a significant reduction in the Orexin-1 receptors (OX1R) expression with a concomitant increase in the A1 receptor (A1R) and equilibrative nucleoside transporter type 1 (ENT1) expression in EIC rats. In contrast, XNJI reduced the extracellular AD levels but orexin A levels remained unaffected. XNJI also counteracted the downregulation of the OX1R expression and upregulation of A1R and ENT1 expression caused by EIC. As for ADK expression, XNJI but not ethanol, displayed an upregulation in the LHA in EIC rats. Based on these results, we suggest that XNJI promotes arousal by inhibiting adenosine neurotransmission via reducing AD level and the expression of A1R and ENT1.

Mebendazole elicits potent antimyeloma activity by inhibiting the USP5/c-Maf axis.

c-Maf is a critical oncogenic transcription factor that contributes to myelomagenesis. Our previous studies demonstrated that the deubiquitinase USP5 stabilizes c-Maf and promotes myeloma cell proliferation and survival; therefore, the USP5/c-Maf axis could be a potential target for myeloma therapy. As a concept of principle, the present study established a USP5/c-Maf-based luciferase system that was used to screen an FDA-approved drug library. It was found that mebendazole, a typical anthelmintic drug, preferentially induced apoptosis in c-Maf-expressing myeloma cells. Moreover, oral administration of mebendazole delayed the growth of human myeloma xenografts in nude mice but did not show overt toxicity. Further studies showed that the selective antimyeloma activity of mebendazole was associated with the inhibition of the USP5/c-Maf axis. Mebendazole downregulated USP5 expression and disrupted the interaction between USP5 and c-Maf, thus leading to increased levels of c-Maf ubiquitination and subsequent c-Maf degradation. Mebendazole inhibited c-Maf transcriptional activity, as confirmed by both luciferase assays and expression measurements of c-Maf downstream genes. In summary, this study identified mebendazole as a USP5/c-Maf inhibitor that could be developed as a novel antimyeloma agent.

[Expert consensus statement on Xiyanping Injection for respiratory system infectious diseases in clinical practice(adults)].

Xiyanping Injection is widely used in the treatment of respiratory infectious diseases. However,its package insert is still less instructive for physicians in understanding the specific clinical application of Xiyanping Injection. To discover potential clinical advantages of Xiyanping Injection,the team invited clinical experts of traditional Chinese medicine and western medicine from the field of respiratory diseases,pharmacists and methodologists of evidence-based medicine to compile the consensus. The consensus was based on a combination of clinical research evidence and expert experience,involving recommendations for clinical problems supported by clini-cal evidence as well as consensus suggestions for clinical problems with no clinical evidence. The consensus recommended the indication,timing of intervention,usage,dosage,course of treatment,combined medication of Xiyanping Injection used for adults,and introduced the safety and precautions of its clinical application. It is suitable for guiding clinical medical workers to rationally use Xiyanping Injection in the treatment of adult's respiratory infectious diseases.

Herbal prescription Chang'an II repairs intestinal mucosal barrier in rats with post-inflammation irritable bowel syndrome.

AIM: The herbal prescription Chang'an II is derived from a classical TCM formula Tong-Xie-Yao-Fang for the treatment of liver-qi stagnation and spleen deficiency syndrome of irritable bowel syndrome (IBS). In this study we investigated the effects of Chang'an II on the intestinal mucosal immune barrier in a rat post-inflammation IBS (PI-IBS) model. METHODS: A rat model of PI-IBS was established using a multi-stimulation paradigm including early postnatal sibling deprivation, bondage and intrarectal administration of TNBS. Four weeks after TNBS administration, the rats were treated with Chang'an II (2.85, 5.71 and 11.42 g · kg(-1) · d(-1), ig) for 14 d. Intestinal sensitivity was assessed based on the abdominal withdrawal reflex (AWR) scores and fecal water content. Open field test and two-bottle sucrose intake test were used to evaluate the behavioral changes. CD4(+) and CD8(+) cells were counted and IL-1ß and IL-4 levels were measured in intestinal mucosa. Transmission electron microscopy was used to evaluate ultrastructural changes of the intestinal mucosal barrier. RESULTS: PI-IBS model rats showed significantly increased AWR reactivity and fecal water content, and decreased locomotor activity and sucrose intake. Chang'an II treatment not only reduced AWR reactivity and fecal water content, but also suppressed the anxiety and depressive behaviors. Ultrastructural study revealed that the gut mucosal barrier function was severely damaged in PI-IBS model rats, whereas Chang'an II treatment relieved intestinal mucosal inflammation and repaired the gut mucosal barrier. Furthermore, PI-IBS model rats showed a significantly reduced CD4(+)/CD8(+) cell ratio in lamina propria and submucosa, and increased IL-1ß and reduced IL-4 expression in intestinal mucosa, whereas Chang'an II treatment reversed PI-IBS-induced changes in CD4(+)/CD8(+) cell ratio and expression of IL-1ß and IL-4. CONCLUSION: Chang'an II treatment protects the intestinal mucosa against PI-IBS through anti-inflammatory, immunomodulatory and anti-anxiety effects.

Risk factors of community-associated infections in Chinese patients with diabetes: A meta-analysis.

OBJECTIVE: To identify the common sites and risk factors of community-associated infections in Chinese patients with diabetes. MATERIALS AND METHODS: The Wanfang, CNKI, PUBMED and VIP databases restricting to Chinese patients with diabetes were queried without restriction to time period. Randomized controlled trials (RCTs) on the risk factors of community-associated infections in Chinese patients with diabetes were considered. Polled odd ratios (OR) and 95% confidence intervals (95% CI) were used for each factor in fixed or random-effect meta-analyses. RESULTS: Twelve studies were identified that investigated seven risk factors of community-associated infections in Chinese patients with diabetes. The average infection incidence was 39.55%. The respiratory tract and urology tract were the predilection sites. Meta-analysis results are as follows: Diabetic patients with chronic complications (OR: 1.63; 95% CI 1.45-1.82), advanced age (OR: 1.30; 95% CI 1.19-1.42), longer duration (OR: 1.47; 95% CI 1.35-1.61) or ketoacidosis (OR: 1.37; 95% CI 1.13-1.66) were more prone to suffer from infections. Those with better glycemic control (OR: 0.68; 95% CI 0.61-0.76) or males (OR: 0.69; 95% CI 0.64-0.75) were less prone to suffer from infections. CONCLUSION: Chinese patients with diabetes had a high incidence of community-associated infections. We should highlight the risk factors that might provide a reference for the same.

[Study on the influence of the contents of paeonol in the cutting process of fresh Cortex Moutan].

OBJECTIVE: To explore the influence of cutting process on the quality of Cortex Moutan. METHODS: The contents of paeonol in Cortex Moutan collected by different methods were determined by HPLC. RESULTS: The cutting process of fresh Cortex Moutan was as follows: collectd the 4-years-Cortex Moutan, cleaned sliced fresh Cortex Moutan, and then dried them in the sun. CONCLUSION: This study provides a scientific basis for the cutting process of fresh Cortex Moutan.

[cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

OBJECTIVE: To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. METHODS: The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-ß-D-1-thiogalactopyranoside (IPTG). RESULTS: A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. CONCLUSION: The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

[Risk factors on chronic obstructive pulmonary disease among greenhouse workers in Liaoning province].

OBJECTIVE: To study the risk factors of chronic obstructive pulmonary disease (COPD) among greenhouse farmers in Liaoning province. METHODS: According to geographical differences in Liaoning province, stratified-cluster-random sampling method was used. A total of 5420 greenhouse farmers working in more than 800 greenhouses were investigated with unified questionnaire, physical examination, blood routine examination, blood gas analysis, chest X-ray and pulmonary function tests. Diagnosis of COPD was made according to the criteria for "chronic obstructive pulmonary disease of diagnosis and treatment guidelines" (2007 revision) by the Chinese Respiratory Disease Society. RESULTS: The total number of farmers under study was 5880, including 5420 respondants (92.18%) with effective data. Risk factors were found on age (χ2=32.530, P=0.000), time working in greenhouses every day (χ2=21.311, P=0.000), frequency of ventilation (χ2=9.791, P=0.007) and the location of the greenhouses (χ2=73.645, P=0.000). When using SPSS 17.0 for the single factor logistic regression analysis, results indicated that smoking (OR=1.976, 95%CI: 1.672 - 2.334), age (OR=4.188, 95%CI: 3.718-4.718), greenhouses for fungus (compared with vegetables, OR=1.302, 95%CI: 1.016-1.669), flowers and plants (compared with vegetables greenhouses, OR=1.503, 95%CI: 1.201-1.880) were the risk factors of COPD among greenhouse farmers while the protective factor was the years of working in the greenhouse (OR=0.684, 95%CI: 0.609-0.767). CONCLUSION: The overall morbidity of COPD among greenhouse farmers in Liaoning province was 17.47% with various risk factors.

[Effect of cyclosporine on regulatory T cells and Foxp3 in the peripheral blood of children with chronic aplastic anemia].

OBJECTIVE: To explore the expression diversification of CD4(+)CD25(+)CD127(low) regulatory T (Treg) cells and Foxp3 mRNA in the peripheral blood of children with aplastic anemia after the treatment with cyclosporine. METHODS: Fifty children with chronic aplastic anemia were enrolled, among whom 30 received cyclosporine treatment (cyclosporine group) and 20 were treated with conventional methods (conventional group). Twenty healthy children were enrolled as the control group. The expression of CD4(+)CD25(+)CD127(low) Treg cells was detected by flow cytometry. The expression of Foxp3 mRNA was detected by real-time Q-PCR. RESULTS: The expressions of Foxp3 mRNA and CD4(+)CD25(+)CD127(low)Treg cells showed no significant difference between the cyclosporine and the control groups 6 months after treatment. On the contrary, there were significantly lower expressions of both in the conventional group than in the control group (P<0.05). Meanwhile, the cyclosporine group had significantly higher expressions of Foxp3 mRNA and CD4(+)CD25(+)CD127(low) Treg cells than the conventional group (P<0.05). CONCLUSIONS: The expressions of CD4(+)CD25(+)CD127(low) Treg cells and Foxp3 mRNA in children with aplastic anemia increase after cyclosporine treatment.

[Detection and significance of CD4+ CD25int/high CD127low regulatory T cells in pediatric aplastic anemia].

OBJECTIVE: To study the expression of CD4+ CD25int/high CD127low regulatory T cells in peripheral blood (PB) and its relation to the quantity of Hb, WBC and platelet (Plt) in children with aplastic anemia (AA). METHODS: Expression of CD4+ CD25int/high CD127low in PB was detected by flow cytometry in 22 children with AA before and after treatment and in 15 healthy controls. The relationships between CD4+CD25highCD127low and the quantity of Hb, WBC and Plt were evaluated. RESULTS: Compared to controls, the percentages of CD4+ CD25+/CD4+, CD4+CD25high/CD4+, CD4+ CD25+ CD127low/CD4+ and CD4+CD25highCD127low/CD4+ in PB of AA patients decreased markedly at the active phase (P﹤0.05). By the recovery phase, the percentages of CD4+CD25+/CD4+, CD4+CD25high/CD4+, CD4+ CD25+ CD127low/CD4+ and CD4+CD25highCD127low/CD4+ increased significantly to the levels similar to the controls. There were significant positive relationships between the expression of CD4+CD25highCD127low cells and the quantity of Hb, WBC and Plt (r=0.499, 0.526, 0.540 respectively; P﹤0.05). CONCLUSIONS: The decrease of the percentage of CD4+CD25int/highCD127low regulatory T cells might be associated with the development of pediatric AA. The CD4+CD25int/highCD127low regulatory T cells can serve as a marker for the evaluation of disease severity as well as a target of further study on immune treatment of AA.

Down-regulation of CacyBP is associated with poor prognosis and the effects on COX-2 expression in breast cancer.

Calcyclin-binding protein (CacyBP) is a tumor suppressor in gastric and renal cell carcinoma, but an oncogene in pancreatic cancer. However, the function of CacyBP in breast cancer has not been well elucidated. In this study, we explored the clinical relevance of CacyBP and investigated the relationship between CacyBP and COX-2 in breast cancer. Immunohistochemical analysis in 172 cases of breast tissues showed that the positive rate of CacyBP protein expression in normal breast tissues (NBT) (89.3%) was higher than that in invasive ductal carcinoma (IDC) (56.1%) (P<0.05). RT-PCR and Western blot analysis showed that CacyBP mRNA and protein expression were significantly lower in tumor tissues as compared to those in the corresponding non-tumorous tissues (P<0.05). The expression trend of COX-2 was opposite with CacyBP in breast carcinogenesis. Moreover, the CacyBP expression was significantly negatively associated with the COX expression in the 132 breast cancer samples (correlation coefficient = 0.505, P<0.001). The clinicopathological data analysis in 132 breast cancer samples showed that CacyBP expression was positively correlated with well differentiated samples (P=0.021), low pathologic TNM stage (P=0.009), and no lymphatic metastasis (P=0.027) of patients with breast cancer. Furthermore, reduced CacyBP expression was associated with poor prognosis. Knockdown of CacyBP gene using siRNA enhanced the proliferation and invasion ability of breast cancer cells, which was dependent on COX-2 expression. In conclusion, CacyBP regulation of COX-2 expression may play an important role in human breast carcinogenesis. Restoration of CacyBP gene is a potential therapeutic target of breast cancer.

[Expression of regulatory T cells and Foxp3 gene in peripheral blood of children with aplastic anemia].

OBJECTIVE: To investigate the levels of CD4+CD25+CD127(low) regulatory T cells (Tregs) and the expression of Foxp3 gene in peripheral blood of children with aplastic anemia (AA) and to study their roles in the pathogenesis of AA. METHODS: Twenty-one children with chronic AA, 9 with acute AA and 15 healthy children were enrolled. The proportion of CD4+CD25+ CD127low Tregs in CD4+ T cells was evaluated by flow cytometric analysis. The level of Foxp3 mRNA was ascertained by RT-PCR. RESULTS: The percentage of peripheral blood CD4+T cells and CD4+CD25+ and CD4+CD25+CD127(low) Tregs in CD4+T cells in both the acute and chronic AA groups was significantly lower than that in the normal control group (P<0.05).The acute AA group had more decreased CD4+ T cells and CD4+CD25+ and CD4+CD25+CD127(low) Tregs percentage compared with the CAA group (P<0.05). The expression of Foxp3 mRNA in peripheral blood decreased obviously in the acute AA group (0.47 + or - 0.08%) compared with that in the normal control (0.71 + or - 0.12%) and the CAA groups (0.68 + or - 0.14%) (P<0.05). CONCLUSIONS: The low expression of Tregs and Foxp3 mRNA in peripheral blood may be involved in pathogenesis of AA.The more decreased Tregs and Foxp3 mRNA expression in acute AA than chronic AA suggests their possible roles in the assessment of the severity of AA.

Hepatocyte growth factor prevents advanced glycation end products-induced injury and oxidative stress through a PI3K/Akt-dependent pathway in human endothelial cells.

AIMS: Advanced glycation end products (AGEs) trigger an oxidative reaction which then accelerates endothelial cell apoptosis; this is a critical event in the process of diabetic vascular complications. We previously demonstrated that hepatocyte growth factor (HGF) protects human endothelial cells against AGE-induced injury. The present study was designed to investigate the possible involvement of MAPK and PI3K/Akt signaling in the action of HGF. MAIN METHODS: HUVECs were treated with AGEs in the presence or absence of HGF. For detection of apoptosis, the morphological Acridine Orange staining, flow cytometry, and caspase-3 activity assay were used. Generation of reactive oxygen species (ROS) and the change in mitochondrial membrane potential were measured using flow cytometry and fluorescence immune analysis. The activation of MAPK and Akt was assayed by Western blot. KEY FINDINGS: HGF exerted its prosurvival effect by inhibiting the overproduction of intracellular ROS and the depolarization of mitochondrial membrane, induced by AGEs. HGF-induced survival correlated with Akt activity and was inhibited by the specific PI3K inhibitor. ERK also was activated by HGF and rescued cells from apoptosis, although the cytoprotective effect was less marked than for PI3K/Akt. HGF-mediated survival was independent of JNK and p38MAPK pathways. Furthermore, blocking the PI3K and Akt activities with PI3K inhibitors or transfection of HUVECs with the dominant-negative p85 or Akt effectively abolished the inhibition of the intracellular ROS production and mitochondrial damage. SIGNIFICANCE: Our studies suggest that HGF, via PI3K/Akt signaling, prevents AGE-induced apoptosis and oxidative stress through the inhibition of mitochondrial damage in HUVECs.