Effect of miR-96-5p targeting IRS1 on apoptosis of PC12 cells induced by aluminum maltol / 中华劳动卫生职业病杂志

Objective: To investigate the effect and mechanism of miR-96-5p on apoptosis of PC12 cells induced by maltol aluminum. Methods: In January 2021, PC12 cells at logarithmic growth phase were divided into blank control group and low, medium and high dose group. Cells in each group were treated with 0, 100, 200 and 400 μmol/L maltol aluminum for 24 hours respectively. Cells were collected and cell apoptosis rates were detected by flow cytometry, miR-96-5p and insulin receptor substrate 1 (IRS1) mRNA expressions were detected by qRT-PCR, and the protein expression levels of cysteine protease 3 (Caspase3) 、activated cysteine protease 3 (Cleaved-caspase3) 、IRS1、phosphorylated protein kinase B (p-AKT) and phosphorylated glucose synthesis kinase 3β (p-GSK3β) were detected by western blotting. The target binding relationship between miR-96-5p and IRS1 was detected by double luciferase reporter gene experiment. The miR-96-5p inhibitor cells and negative control cells were constructed after transfecting PC12 cells with miR-96-5p inhibitor for 24 hours. The cells were divided into blank control group, negative control group, aluminum exposure group, aluminum exposure+negative control group, aluminum exposure+miR-96-5p inhibition group, and miR-96-5p inhibition group. After transfecting PC12 cells with miR-96-5p inhibition and IRS1 siRNA for 24 h, the cells were divided into aluminum exposure+miR-96-5p inhibition+negative control group and aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group. The control group was cultured in complete culture medium, and cells in the aluminum exposure group were treated with 200 μmol/L maltol aluminum for 24 hours. Cells in each group were collected and the apoptosis rate, miR-96-5p and IRS1 mRNA expression levels, as well as protein expression levels of Caspase3, Cleaved-caspase3, IRS1, p-AKT, and p-GSK3β were measured. Results: After 24 hours of exposure, compared with blank control group and low-dose group, the apoptosis rates, relative expressions of Caspase3 and Cleaved-caspase3 proteins, and relative expressions of miR-96-5p in the medium and high-dose groups of PC12 cells were significantly increased, while the relative expression levels of IRS1 mRNA, IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05). Targetscan prediction and double luciferase report experiment both proved that IRS1 was a direct target gene of miR-96-5p. In the transfection experiment, compared with the aluminum exposure group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins, the relative expression of miR-96-5p in the aluminum exposure+miR-96-5p inhibition group were significantly decreased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly increased (P<0.05). In the IRS1 low expression experiment, compared with the aluminum exposure+miR-96-5p inhibition+negative control group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins in the aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group were significantly increased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05) . Conclusion: The increased expression of miR-96-5p and the targeted inhibition of IRS1 may be one of the mechanisms of apoptosis of PC12 cells induced by maltol aluminum exposure.

Graphite Carbon Nitride and Its Composites for Medicine and Health Applications.

With the progress of science and technology and the improvement of people's living standards, the performance of traditional materials can no longer fully meet the needs of social development. Graphitic phase carbon nitride (g-C3 N4 ), as a new type of nanomaterial, has good properties. Its unique graphite like structure and stable thermodynamic characteristics have led an increasing number of researchers to explore its diverse functions and use this as a basis to develop related energy and products for applications in various fields. Among them, applications in the field of medicine health have become popular in recent years. Therefore, this review summarizes the synthesis methods of g-C3 N4 and its composites, as well as their applications in food, medicine, environmental monitoring and disease treatment, in the hope of providing references and basis for further expanding the applications of g-C3 N4 in large health areas.

4-Sulfonyloxy/alkoxy benzoxazolone derivatives with high anti-inflammatory activities: Synthesis, biological evaluation, and mechanims of action via p38/ERK-NF-κB/iNOS pathway.

In an effort to discover new agents with high anti-inflammatory activity, 22 new 4-sulfonyloxy/alkoxy benzoxazolone derivatives were synthesized, characterized, and evaluated for their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production and TNF-α expression in RAW 264.7 cells in vitro. Most of these compounds displayed greater inhibitory ability against NO production than the lead compound 4-o-methyl-benzenesulfonyl benzoxazolone, and the most active compound 2h exhibited the strongest inhibitory activity against NO, IL-1ß, and IL-6 production with IC50 values 17.67, 20.07, and 8.61 µΜ, respectively. The effects of 2h were comparable or stronger than those of the positive control celecoxib. Compound 2h also displayed higher activity in vivo than celecoxib in a mouse model of xylene-induced ear edema, based on their inhibitory rates of 42.69% and 30.87%, respectively. Further molecular analysis revealed that compound 2h significantly reduced the iNOS levels in cell supernatant and suppressed the protein expression of iNOS, p-p38, p-ERK, and nuclear NF-κB. The results indicated that the anti-inflammatory effect of 2h might be realized through the regulation of ERK- and p38-mediated mitogen-activated protein kinase (MAPK)-NF-κB/iNOS signaling, thereby reducing the excessive release of NO, IL-1ß, and IL-6. Our findings demonstrated that compound 2h, a new benzoxazolone derivative, could inhibit activation of the MAPK-NF-κB/iNOS pathway, supporting its potential as a novel anti-inflammatory agent.

Design and synthesis of new disubstituted benzoxazolone derivatives that act as iNOS inhibitors with potent anti-inflammatory activity against LPS-induced acute lung injury (ALI).

Acute lung injury (ALI) is a pulmonary disease that acts as a severe acute inflammatory response with no specific drugs. iNOS, a catalyst of the excessive production of NO, has been demonstrated to participate in the inflammatory process, and targeting iNOS may be a promising therapeutic pathway to alleviate ALI. In our research, eighteen new disubstituted benzoxazolone derivatives were synthesized, characterized, and evaluated for activity against NO production in an LPS-induced RAW264.7 cell. The results showed that these compounds could obviously inhibit the over-generation of NO and disubstitution at the 4, N-position of the benzoxazolone ring, presenting better potency than substitution only at the 4-position. Among the analogues generated, compounds 2c, 2d, and 3d showed NO inhibitory activity with IC50 values of 16.43, 14.72, and 13.44 µM and iNOS inhibitory activity with IC50 values of 4.605, 3.342, and 9.733 µM, respectively. Meanwhile, compounds 2c, 2d, and 3d could also inhibit the release of IL-6, IL-1ß in vitro and suppress xylene-induced ear edema in vivo to realize anti-inflammatory activity. Furthermore, compound 2d could significantly protect the LPS-induced ALI, presenting as decreased inflammatory cytokines and obvious pathological changes. Immunohistochemistry and molecular modeling demonstrated that compound 2d significantly inhibited the expression of iNOS in vivo and interacted with iNOS through two hydrogen bindings with the MET368 and ILE195 residues of the iNOS protein. These results demonstrated that compound 2d could be a promising lead structure for iNOS inhibitors, with anti-inflammatory activity to treat LPS-induced acute lung injury.

LncRNA KCNQ1OT1 affects cell proliferation, apoptosis and fibrosis through regulating miR-18b-5p/SORBS2 axis and NF-ĸB pathway in diabetic nephropathy.

BACKGROUND: It has been reported that long non-coding RNAs (lncRNAs) play vital roles in diabetic nephropathy (DN). Our study aims to research the function of lncRNA KCNQ1OT1 in DN cells and the molecular mechanism. METHODS: Human glomerular mesangial cells (HGMCs) and human renal glomerular endothelial cells (HRGECs) were cultured in high glucose (30 mM) condition as models of DN cells. KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) and miR-18b-5p levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mRNA and protein levels of Sorbin and SH3 domain-containing protein 2 (SORBS2), Type IV collagen (Col-4), fibronectin (FN), transcriptional regulatory factor-beta 1 (TGF-ß1), Twist, NF-κB and STAT3 were measured by qRT-PCR and western blot. Cell viability was detected by cell counting kit-8 (CCK-8) assay for selecting the proper concentration of glucose treatment. Additionally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry assay were employed to determine cell proliferation and apoptosis, respectively. The targets of KCNQ1OT1 was predicted by online software and confirmed by dual-luciferase reporter assay. RESULTS: KCNQ1OT1 and SORBS2 were elevated in DN. Both knockdown of KCNQ1OT1 and silencing of SORBS2 restrained proliferation and fibrosis and induced apoptosis in DN cells. Besides, Overexpression of SORBS2 restored the KCNQ1OT1 knockdown-mediate effects on proliferation, apoptosis and fibrosis in DN cells. In addition, miR-18b-5p served as a target of KCNQ1OT1 as well as targeted SORBS2. KCNQ1OT1 knockdown repressed NF-ĸB pathway. CONCLUSION: KCNQ1OT1 regulated DN cells proliferation, apoptosis and fibrosis via KCNQ1OT1/miR-18b-5p/SORBS2 axis and NF-ĸB pathway.

Anti-inflammatory effects of 4-o-methyl-benzenesulfonyl benzoxazolone (MBB) in vivo and in vitro as a novel NSAIDs lead compound.

BACKGROUND: Great attention has been paid to the development of novel anti-inflammatory drugs to overcome the adverse reactions of traditional drugs. Recently, a new compound 4-o-methyl-benzenesulfonyl benzoxazolone (MBB) we have prepared attracted our attention for its promising anti-inflammatory activity and low toxicity. The present study aimed to further investigate the anti-inflammatory effects of MBB both in vivo and in vitro in order to determine its potential as a novel NSAIDs lead compound. METHODS: The anti-inflammatory effects in vivo were evaluated using acetic acid-induced mice writhing, xylene-induced mice ear edema and collagen-induced rat arthritis. NO, TNF-α, IL-6, IL-1ß and iNOS productions by LPS-stimulated RAW264.7 cells were determined to investigate the basis of anti-inflammatory effects. Finally, the COX inhibition effect was tested in vitro using COX inhibitor screening assay kit. RESULTS: MBB could significantly decrease the writhing and ear swelling in a dose-dependent manner, and it also had a moderate anti-arthritic potential associated with an attenuation of arthritis index score, arthritis swelling, and inhibition of TNF-α and IL-1ß. MBB could inhibit the activity of NO, TNF-α, IL-6, IL-1ß and iNOS to perform its activity in vitro, but it had no effect against COX-1 and COX-2. The anti-inflammation effect may be mediated via the inhibition of iNOS to reduce the production of inflammatory mediators which should be further confirmed. CONCLUSIONS: The compound MBB displayed anti-inflammatory and anti-arthritic effect, and it could be considered as a new NSAIDs lead compound for the further structure modification to develop novel anti-inflammatory drugs.

Ultrasonic differential diagnosis of scar pregnancy and lower uterine cavity non-scar pregnancy / 中国医学影像技术

Objective To investigate the value of two-dimensional and three-dimensional ultrasonography in differential diagnosing cesarean scar pregnancy (CSP) and lower uterine cavity non-scar pregnancy.Methods Totally 67 patients with CSP (CSP group)and 29 patients with lower uterine cavity non-scar pregnancy (lower uterine cavity non-scar pregnancy group) were enrolled.Two-dimensional and three-dimensional ultrasonography were performed to observe the gestational sac implantation site,the relationship between gestational sac and cesarean section scar and the main source of blood flow,and the residual muscle thickness of cesarean section scar was measured as well.Logistic regression model was established,and the diagnostic efficacy was evaluated with ROC curve.Results Statistical differences of relationship between gestational sac and scar,the main source of blood flow and residual muscle thickness were found between CSP group and lower uterine cavity non-scar pregnancy group (all P＜0.001).The area under the ROC curve of the Logistic regression model was 0.878 (P＜0.001).Taking prediction accuracy of 0.680 as a cut-off value,the accuracy of this model in predicting was 86.46％,the sensitivity was 89.55％ and the specificity was 79.31％.Taking gestational sac implanted scars and nourish the blood flow from the lower anterior wall of the uterus as the diagnostic criteria for CSP,the Kappa value of two-dimensional and three-dimensional ultrasonograpby in diagnosis CSP and non-scar pregnancy was 0.699 and 0.711,respectively.Conclusion Comprehensive analysis of the relationship between gestational sac and scars,the main source of blood flow and residual muscle thickness with Logistic regression model can improve differential diagnostic efficacy of CSP with lower uterine cavity non-scar pregnancy.

Inhibitory effect of compound W3D on LPS-induced release of inflammatory mediators of RAW264.7 cells through TLR4-MyD88-NF-κB signaling pathway / 中国药理学通报

Aim To investigate the effect of the novel benzoxazolone derivative 4-( 5′-dimethylamino )-naph-thalenesulfonyl-2 ( 3H )-benzoxazolone ( W3D ) on TLR4-MyD88-NF-κB signaling pathway in LPS-in-duced RAW264.7 cells. Methods The cell viability was detected by MTT assay, and the contents of TNF-α, IL-6, IL-1β and COX-2 in the cell supernatant were analyzed using ELISA methods. The protein ex-pression of IL-6, TLR4, MyD88, p-IRAK4 and NF-κB were investigated by western blot analysis, and the mRNA expressions of TLR4, MyD88 and IL-6 were an-alyzed by RT-PCR. Results W3D could obviously in-hibit the production of TNF-α, IL-6 and IL-1β in LPS- induced RAW264.7 cell supernatant, but it had no effect on the release of COX-2. Compared with the model group, the expressions of TLR4, MyD88 and IL-6 were decreased significantly in a dose dependent manner. Meanwhile, the expressions of p-IRAK4 and nucleus of NF-κB were decrease in W3D treated group compared with the model group. Conclusion The no-vel compound W3D could inhibit the release of the in-flammatory mediators through the regulation of TLR4-MyD88-NF-κB signaling pathway.

Hepatotoxicity and proteomic mechanism of Di-n-butyl-di-(4-chlorobenzohydroxamato)tin(IV) (DBDCT) in vivo.

Di-n-butyl-di-(4-chlorobenzohydroxamato)tin(IV) (DBDCT) is an anti-tumour organotin(IV) compound with hepatotoxicity. To investigate the hepatotoxicity and mechanisms of DBDCT in vivo, proteomic technology 2D gel combined with MALDI-TOF-MS was used in our research. Results indicated that DBDCT increased AST, AKP and ACP activities and decreased ALT activity. Further, sporadic eosinophilic changes and nuclear pyknosis were visible in hepatic pathological observation. Proteomic analysis showed that twenty-two proteins involved in amino acid, nucleic acid, carbohydrate and lipid metabolism, stress response, multicellular organism development and cell apoptosis were differentially expressed and identified. Notably, a considerable amount of the altered proteins, such as OAT, HPPD, M2GD, GSTM2, Glud1, GSTa, HS90ß and PDIA3 participated in multi-metabolic pathways and oxidative stress reactions. Our findings indicated that the inhibition of enzyme activity and oxidative stress were the major mechanisms by which DBDCT induced hepatotoxicity, and the altered proteins could be potential drug targets for the further design of new type of organic tin with high activity and low toxicology.

The effects of salinity on nitrification using halophilic nitrifiers in a Sequencing Batch Reactor treating hypersaline wastewater.

With annual increases in the generation and use of saline wastewater, the need to avoid environmental problems such as eutrophication is critical. A previous study identified ways to start up a halophilic sludge domesticated from estuarine sediments to remove nitrogen from wastewater with a salinity of 30 g/L. This investigation expands that work to explore the impact of salinity on nitrogen removal. This study demonstrated that the mixed halophilic consortia removed nitrogen from wastewater with a salinity of 30-85 g/L. A kinetic analysis showed that halophilic nitrifiers selected based on hypersalinity were characterized by low Ks, µmax and specific ammonium oxidization rates. This explains the decrease in ammonium removal efficiency in the high salinity operational phases. Salinity inhibited ammonia oxidizing bacteria (AOB) activity, as well as the number of dominant AOB, but did not significantly affect the AOB dominant species. Three most dominant AOB lineages in the halophilic sludge were Nitrosomonas marina, Nitrosomonas europaea, and Nitrosococcus mobilis. Nitrosomonas europaea and Nitrosococcus mobilis were mainly affected by salinity, while nitrite accumulation and ammonia loading played the key role in determining the abundance of Nitrosococcus mobilis and Nitrosococcus europaea. The study contributes insights about shifts in halophilic nitrifying bacterial populations.

DW MRI at 3.0 T versus FDG PET/CT for detection of malignant pulmonary tumors.

Emerging evidence suggests that diffusion-weighted magnetic resonance imaging (DW MRI) could be useful for tumor detection with N and M staging of lung cancer in place of fluorine 18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT). DW MRI at 3.0 T and FDG PET/CT were performed before therapy in 113 patients with pulmonary nodules. Mean apparent diffusion coefficient (ADC), maximal standardized uptake value (SUVmax ) and Ki-67 scores were assessed. Quantitatively, specificity and accuracy of ADC (91.7 and 92.9%, respectively) were significantly higher than those of SUVmax (66.7 and 77.9% respectively, p < 0.05), although sensitivity was not significantly different between them (93.5 and 83.1%, p > 0.05). Qualitatively, sensitivity, specificity and accuracy of DW MRI (96.1, 83.3 and 92.0%, respectively) were also not significantly different from that of FDG PET/CT (88.3, 83.3 and 86.7%, respectively, p > 0.05). Significant negative correlation was found between Ki-67 score and ADC (r = -0.66, p < 0.05), ADC and SUVmax (r = -0.37, p < 0.05), but not between Ki-67 score and SUVmax (r = -0.11, p > 0.05). In conclusion, quantitative and qualitative assessments for detection of malignant pulmonary tumors with DW MRI at 3.0 T are superior to those with FDG PET/CT. Furthermore, ADC could predict the malignancy of lung cancer.

Der p2 recombinant bacille Calmette-Guerin priming of bone marrow-derived dendritic cells suppresses Der p2-induced T helper17 function in a mouse model of asthma.

BACKGROUND AND OBJECTIVE: Previous studies have demonstrated that our recombinant bacille Calmette-Guerin (rBCG), which expresses Der p2 in house dust mite (Der p2 rBCG) suppresses asthmatic airway inflammation by regulating the phenotype and function of dendritic cells (DC) and reprogramming T helper (Th) 0 cell differentiation into different T cell (Th1/Th2/Treg) subtypes. However, the exact role of Der p2 rBCG in reprogramming Th17 differentiation and the relevant mechanisms are not known. The aim of this study was to examine whether Der p2 rBCG-mediated inhibition of allergic airway inflammation is mediated by regulating Th17 differentiation in a murine asthma model. METHODS: Primary mouse bone marrow-derived dendritic cells (BMDC) were infected with Der p2 rBCG and adoptively transferred to Der p2-intranasally sensitized mice. The role of Der p2 rBCG-BMDC on the regulation of airway inflammation and Th17 cell differentiation was assessed. RESULTS: Adoptive transfer of Der p2 rBCG-BMDC suppressed airway inflammation and mucin secretion. Der p2 rBCG-BMDC inhibited excessive Th17 immune responses but not BCG-BMDC. Furthermore, Der p2 rBCG decreased jagged-2 and increased delta-like-4 expressions on BMDC to a greater extent than BCG. CONCLUSIONS: These findings suggest that DC plays a key role in Der p2 rBCG-induced immunoregulation. Der p2 rBCG also displayed a potent inhibitory effect on Th17 differentiation, and these findings increase our understanding of the cellular basis of Der p2 BCG-mediated inhibition of asthma.

End-binding protein 1 stimulates paclitaxel sensitivity in breast cancer by promoting its actions toward microtubule assembly and stability

Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.

Der p 2 recombinant bacille Calmette-Guérin targets dendritic cells to inhibit allergic airway inflammation in a mouse model of asthma.

BACKGROUND: Previous studies showed that a recombinant bacille Calmette-Guérin (rBCG) which expressed the Der p 2 of house dust mites (Der p 2 rBCG) could suppress asthmatic airway inflammation. There are two possible mechanisms: (1) Der p 2 rBCG elicits immune deviation from Th2 to Th1, and (2) Der p 2 rBCG induces antigen-specific regulatory T cells. However, the role of dendritic cell (DC) Der p 2 rBCG in this protective effect and in reprogramming T-cell commitment still needs to be studied. OBJECTIVES: The aim of this study was to determine whether DCs play a central role in the Der p 2 rBCG-mediated inhibition of allergic airway inflammation. METHODS: DCs were collected from Der p 2 rBCG-immunized mice (Der p 2 rBCG-DCs) and adoptively transferred to Der p 2-sensitized mice. The effects of DCs on airway inflammation and immune regulation were analyzed. RESULTS: Adoptive transfer of DCs from Der p 2 rBCG-immunized mice suppressed asthmatic responses, including airway inflammation, mucin secretion and airway responsiveness. Der p 2 rBCG-DCs could effectively inhibit excessive Th2 immune responses and induced a subtype of CD4+CD25+Foxp3+ anti-specific regulatory T cells in this asthma model. Furthermore, Der p 2 rBCG immunization recruited more plasmacytoid DCs in abdominal draining lymph nodes. CONCLUSIONS: These findings suggest that DCs played a key role in Der p 2 rBCG-induced immunoregulation. Compared with BCG, Der p 2 rBCG displayed a more potent inhibitory effect on asthma responses, which may be related to the increase in plasmacytoid DC recruitment. These results improve our understanding of the cellular basis of Der p 2 BCG-mediated inhibition of asthma.

Diagnosis and treatment of capillary leak syndrome in children / 中国小儿急救医学

Capillary leak syndrome (CLS) is a common and life-threatening disorder in children.The common pathogenies are sepsis,severe trauma,shock,asphyxia,extracorporeal circulation,ischemical reperfusion injury and so on.It is characterized by shock resulting from leakage of plasma,which is refected by accompanying hemoconcentration,hypoalbuminemia and edema,sometimes followed by multiple organ dysfunction syndrome.Clinicians should consider the diagnosis in patients with unexplained edema and hypotension.To know the pathophysiology and clinical course will be benefit to proper medicine choosen.

Risk factors for capillary leak syndrome in neonates / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the risk factors for capillary leak syndrome (CLS) in neonates.</p><p><b>METHODS</b>The clinical data of 52 neonates with CLS (case group) were retrospectively reviewed. Fifty hospitalized neonates without CLS were used as the control group. The possible factors for the development of CLS were identified by univariate analysis. The independent risk factors for CLS were determined by multivariate logistic regression analysis.</p><p><b>RESULTS</b>The univariate analysis showed that the incidences of hyperglycemia, respiratory distress syndrome (RDS), sepsis and cold injury syndrome in the case group were significantly higher than those in the control group (P<0.05). The logistic regression analysis showed that sepsis (OR=5.004, P=0.001), RDS (OR=3.880, P=0.013) and cold injury syndrome (OR=3.207, P=0.023) were the independent risk factors for the development of CLS.</p><p><b>CONCLUSIONS</b>RDS, sepsis and cold injury syndrome are independent risk factors for CLS in neonates. Hyperglycemia may be associated with the development of CLS.</p>

Suppression of allergic airway inflammation in a mouse model by Der p2 recombined BCG.

Allergic asthma is a chronic inflammatory disease mediated by T helper (Th)2 cell immune responses. Currently, immunotherapies based on both immune deviation and immune suppression, including the development of recombinant mycobacteria as immunoregulatory vaccines, are attractive treatment strategies for asthma. In our previous studies, we created a genetically recombinant form of bacille Calmette-Guerin (rBCG) that expressed Der p2 of house dust mites and established that it induced a shift from a Th2 response to a Th1 response in naive mice. However, it is unclear whether rBCG could suppress allergic airway inflammation in a mouse model. In this article we report that rBCG dramatically inhibited airway inflammation, eosinophilia, mucus production and mast cell degranulation in allergic mice. Analysis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in bronchoalveolar lavage fluid (BALF) and lung tissue revealed that the suppression was associated with a shift from a Th2 response to a Th1 response. At the same time, rBCG induced a CD4(+) CD25(+) Foxp3(+) T-cell subtype that could suppress the proliferation of Th2 effector cells in vitro in an antigen-specific manner. Moreover, suppression of CD4(+) CD25(+) T cells could be adoptively transferred. Thus, our results demonstrate that rBCG induces both generic and specific immune responses. The generic immune response is associated with a shift from a Th2 to a Th1 cytokine response, whereas the specific immune response against Der p2 appears to be related to the expansion of transforming growth factor-beta (TGF-beta)-producing CD4(+) CD25(+) Foxp3(+) regulatory T cells. rBCG can suppress asthmatic airway inflammation through both immune deviation and immune suppression and may be a feasible, efficient immunotherapy for asthma.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

OBJECTIVE: To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. METHODS: The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. RESULTS: The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. CONCLUSIONS: Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

[The effects of rBCG expressing Der p2 in the form of lipoprotein on murine immune response].

AIM: To investigate the effects of rBCG vaccination containing foreign antigen Der p2 in the form of lipoprotein on murine immune response. METHODS: 6 to 8 weeks old and newborn BALB/c mice were vaccined intraperitoneally with 10(6) CFU rBCG or BCG. At the same time, the control group was injected with saline. Six weeks later, all animals were injected with Der p2 (20 microg). After two weeks later, the concentrations of IL-4 and IFN-gamma in the serum and splenocyte culture supernatant (STLCS) were determined by ELISA, and Th subgroups were determined by double fluorescent staining and flow cytometry. RESULTS: After vaccination, the serum and STLCS from both rBCG-immunized and BCG-immunized group of adult and newborn BALB/c mice had significantly higher level of IFN-gamma and lower level of IL-4 than those from control groups. Besides, there was the larger percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined and BCG-vaccined mice than that from control group. However, the percentage of CD4 (+) IL-4 (+) cells in spleen cells from rBCG-vaccined and BCG-vaccined group was lower than that from control group. Moreover, the level of IFN-gamma in STLCS from rBCG-immunized was significantly higher, compared with that from BCG-immunized mice. At the same time, the percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined mice was larger than that from BCG-vaccined group. CONCLUSION: Both rBCG and BCG could stimulate Th1 predominant immune response, when injected intraperitoneally into adult or newborn BALB/c mice, The Der p2 expressed on the cell wall of BCG can work as the component of BCG and be recognized by the immune system of mice, therefore stimulates Der p2-specific Th1 predominant immune response. These data indicate that recombinant BCG-expressing antigens can be used as the antigen-specific vaccines against allergic diseases by regulating the balance of Th1/Th2.

[Using flow cytometry to detect peripheral blood eosinophils and their related molecules].

AIM: To set up an accurate and rapid method to detect the phenotype of peripheral blood eosinophils using flow cytometry. METHODS: 10(-4) mol/L of theophylline, 10(-4) of dexamethasone and 10(-8) mol/L of rhIL-5 were added to peripheral blood sampled from normal human subjects. Anti-CD16-PE mAb and FITC-labelled mAbs to eosinophil's molecules were used to perform double labeling staining. Simultaneously, CD16-FL2 was used to install gating so as to accurately locate eosinophils. And then their molecules were examined and analyzed. RESULTS: The eosinophils were accurately located. Theophylline and dexamethasone effectively inhibited activation of eosinophils and shedding of CD62L on eosinophils caused by IL-5. CONCLUSION: Our method can accurately detect eosinophils in peripheral blood samples and their molecules. In addition, only a tiny amount of blood sample is needed and few artificial factors are involved in the detection. Therefore, this method may be an ideal both in basic immunological research and clinical laboratory examination.