OBJECTIVETo observe the expression of phospholipid scramblase 1 (PLSCR1) in matrine (MAT) induced differentiation of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) cells, and to explore its correlation to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal pathway.

METHODSNB4 (an APL cell line sensitive to ATRA) and NB4-R1 (a resistant strain of ATRA) were observed as subjects in this study. Effects of combined treatment of 0.1 mmol/L MAT and 1 [mol/L ATRA on the differentiation of two cell lines were detected using nitroblue tetrazolium (NBT) reduction test and flow cytometry (CD11b). Expressions of PML/RARot and PLSCR1 protein/gene were detected using Western blot and Real-time fluorescence quantitative PCR assay. Meanwhile, H89, PKA antagonist, was used to observe cell differentiation antigen and changes of aforesaid proteins and genes.

RESULTSMAT combined ATRA could significantly elevate positive rates of NBT and CD11 b in NB4-R1 cells, and significantly down-regulate the expression of PML/RARapha-fusion protein/gene (P < 0.05, P < 0.01). ATRA used alone could obviously enhance the expression of PLSCRI in NB4 cells at protein and mRNA levels (P < 0.01). But the expression of PLSCR1 was up-regulated in NB4-R1 cells, but with statistical.difference only at the protein level (P <0. 01). In combination of MAT, PLSCR1 protein expression was further elevated in the two cell lines (P < 0.01). Besides, there was statistical difference in mRNA expressions in NB4-R1 cells (P < 0.05). All these actions could be reversed by treatment of 10 micromol/L H89 (P < 0.05, P < 0.01).

CONCLUSIONMAT combined ATRA could significantly induce the differentiation of NB4-R1 cells, and inhibit the expression of PML/RARalpha fusion gene/protein, which might be associated with up-regulating PLSCR1 expression.

Alkaloids ; Antineoplastic Agents ; Cell Differentiation ; Cell Line, Tumor ; Down-Regulation ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; Phospholipid Transfer Proteins ; metabolism ; Quinolizines ; RNA, Messenger ; Signal Transduction ; Tretinoin ; Tumor Cells, Cultured ; Up-Regulation

Objectiv e:To investigate the effect of different culture conditions on the differentiation of Treg and Th17 to lay a foundation for exploring the methods to reverse the immune tolerance induced by tumor microenvironment.Methods:The IL-6 gene was cloned and stablely transferred into the tumor cell line expressing TGF-β.The conditioned mediums ( CM) were prepared by collecting the culture supernatants of tumor cell lines with or without IL-6 expression and used in the in vitro culture of peripheral blood mononuclear cells ( PBMC ) .The changes of Treg and Th17 in PBMC treated with different CM were detected with flow cytometry ( FCM) .Results:The expression of TGF-βin BEL-7402 was higher than that in HepG2.Thus the BEL-7402 was selected for preparation of cell line stablely transfected with IL -6 gene.ELISA detection confirmed the effective expression of IL -6 by the identified cell lines.It was showed that the Treg increased in PBMC treated with culture supernatants of tumor cells .However,the presence of IL-6 reversed the increase of Treg and promoted the differentiation of Th 17.Conclusion: The culture supernatants of tumor cells increases the proportion of Treg.However,the presence of IL-6 in this CM can reverse the increase of Treg and raise the proportion of Th 17.

OBJECTIVETo study the effect of dimethoate on the expression of heat shock protein 70 (HSP70) in peripheral blood lymphocytes of human beings and to explore the feasibility of HSP70 in biomonitoring among workers exposed to organophosphorous pesticides.

METHODSPeripheral blood lymphocytes were isolated from subjects, comprising 11 people of the control group and 35 workers of the exposure group exposed to dimethoate. Flow cytometry was used for detecting both the basic level and the level of the dimethoate-induced expression of HSP70. The activity of whole blood acetylcholinesterase (AChE) was examined at the same time. Then the potential influential factors to HSP70 expression and AChE activity were analyzed.

RESULTSThe basic level of HSP70 expression in the exposure group and the control group was 41.24% +/- 10.45% and 23.97% +/- 4.29% respectively. The activity of AChE in these two groups were (125.23 +/- 7.97) and (145.36 +/- 8.78) U/ml respectively. Both differences were statistically significant (P < 0.01). Among the exposure group, the basic level of HSP70 expression of the two categories comprising operators and packers, were 47.34% +/- 11.87% and 38.05% +/- 8.20% respectively (P < 0.05), while there was no significant difference (P > 0.05) in AChE activity between these two categories. The factors that had significant influence on the HSP70 basic level of the exposure group were the health condition, the environmental concentration of dimethoate and the exposure time in order, according to their significance of influence. At least 88% variance of HSP70 could be explained by these factors. The only factor that could influence AChE activity significantly was the exposure time, and it could only explain about 12% variance of AChE activity. After the treatment of dimethoate in vitro, the level of the induced expression of HSP70 in the control group was significantly higher than that of the exposure group (P < 0.01). The increasing order was the control group, the group of packers and the group of operators according to the increasing extent and there were significant difference among them (P < 0.01). The factors that could significantly influence the change ratio of HSP70 expression were the environmental concentration and the exposure time.

CONCLUSIONHSP70 is a potential index that can reflect the individual and environmental conditions of workers exposed to dimethoate comprehensively.

Acetylcholinesterase ; blood ; Adult ; Cells, Cultured ; Dimethoate ; toxicity ; Female ; HSP70 Heat-Shock Proteins ; biosynthesis ; Humans ; Insecticides ; toxicity ; Lymphocytes ; drug effects ; metabolism ; Male ; Middle Aged ; Occupational Exposure

OBJECTIVETo estimate the biological exposure limit (BEL) using benchmark dose (BMD) based on two sets of data from occupational epidemiology.

METHODSCadmium-exposed workers were selected from a cadmium smelting factory and a zinc product factory. Doctors, nurses or shop assistants living in the same area served as a control group. Urinary cadmium (UCd) was used as an exposure biomarker and urinary beta2-microgloburin (B2M), N-acetyl-13-D-glucosaminidase (NAG) and albumin (ALB) as effect biomarkers. All urine parameters were adjusted by urinary creatinine. Software of BMDS (Version 1.3.2, EPA.U.S.A) was used to calculate BMD.

RESULTSThe cut-off point (abnormal values) was determined based on the upper limit of 95% of effect biomarkers in control group. There was a significant dose response relationship between the effect biomarkers (urinary B2M, NAG; and ALB) and exposure biomarker (UCd). BEL value was 5 microg/g creatinine for UB2M as an effect biomarker, consistent with the recommendation of WHO. BEL could be estimated by using the method of BMD. BEL value was 3 microg/g creatinine for UNAG as an effect biomarker. The more sensitive the used biomarker is, the more occupational population will be protected.

CONCLUSIONBMD can be used in estimating the biological exposure limit (BEL). UNAG is a sensitive biomarker for estimating BEL after cadmium exposure.

Acetylglucosaminidase ; urine ; Albuminuria ; urine ; Biomarkers ; urine ; Cadmium ; toxicity ; urine ; Dose-Response Relationship, Drug ; Female ; Humans ; Male ; Occupational Exposure ; Spectrophotometry, Atomic ; beta 2-Microglobulin ; urine

OBJECTIVEBased on two sets of data from occupational epidemiology, Benchmark dose (BMD) was applied to estimate biological exposure limit (BEL).

METHODSCadmium exposed workers were selected from a cadmium smelting and a zinc products factory and control group was selected from doctors or nurses and staff from shops living in the same area; Urinary cadmium (UCd) was used as exposure biomarker and urinary beta(2) microglobulin (UBM), NAG (UNAG) and albumin (UALB) were as effect biomarkers. All urine parameters were adjusted by urinary creatinine. Software of BMDS (Version 1.3.2, EPA.U.S) was used to calculate BMD.

RESULTSCalculated abnormal prevalence was based on the upper limit of 95% of effect biomarkers in control group; There are significant dose response relationship between the prevalence of effect biomarkers (UBM, UNAG and UALB) and exposure biomarker (UCd); BEL was 5 microg/g creatinine for UBM as effect biomarker, It consists with the recommendation of WHO; BEL was 3 microg/g creatinine for UNAG as effect biomarker; BEL can be estimated by using the method of BMD; the more sensitive biomarker would used, the more occupational people would protected.

CONCLUSIONThe application of BMD in estimating biological exposure limit (BEL) is proper. UNAG is suggested as most sensitive biomarker to be used to estimate BEL for cadmium exposure.

Acetylglucosaminidase ; urine ; Albuminuria ; urine ; Biomarkers ; urine ; Cadmium ; adverse effects ; urine ; Dose-Response Relationship, Drug ; Female ; Humans ; Male ; Occupational Exposure ; Reference Values ; beta 2-Microglobulin ; urine

OBJECTIVETo investigate the influence of pyrrolidine dithiocarbamate (PDTC) on the expression of transforming growth factor beta(1) (TGF-beta(1)), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor-1 of metalloproteinase (TIMP-1) in rats with pulmonary damage induced by paraquat (PQ).

METHODSFifty-four healthy male SD rats were randomly assigned into the control group (normal saline), the PQ-treatment groups (4 groups) and the PDTC treatment groups (4 groups). Except the rats in the control group, the rats in the PQ group were gavaged only with 40 mg/kg PQ, and PDTC group with 40 mg/kg PQ plus immediate injection 120 mg/kg PDTC (i.p). On the 3rd, the 7th, the 14th and 28th day after treatments, one group rats of each treatments were sacrificed and lung and blood samples were collected. The level of TGF-beta(1) protein in the plasma, the mRNA expression of TGF-beta(1), MMP-2 and TIMP-1 were evaluated using RT-PCR and real-time quantitative PCR, while pathological changes of lung were examined under optical microscope and electrical microscope.

RESULTSThe TGF-beta(1) protein, TGF-beta(1) and MMP-2 mRNA expression were increased significantly in the earlier stage and then decreased after PQ administration (P < 0.05 or P < 0.01), while the mRNA level of TIMP-1 was augmented continuously (P < 0.01) throughout the study compared to the control group. In comparison with the PQ group, in the PDTC treatment group, the TGF-beta(1) mRNA expression on the 3rd and the 14th day, 0.54 +/- 0.08 and 0.72 +/- 0.04 respectively, the MMP-2 mRNA expression on the 7th and 14th day, 1.62 +/- 0.50 and 1.97 +/- 0.34 respective-ly, and the TIMP-1 mRNA on the 7th and 21st day, 1.79 +/- 0.21 and 2.00 +/- 0.34 respectively, were significantly decreased (P < 0.05 or P < 0.01).

CONCLUSIONPDTC could attenuate paraquat-induced up-regulation of TGF-beta(1) and its mRNA expression, MMP-2 and TIMP-1 mRNA levels, which indicates that PDTC may exert its protective effects on paraquat-induced pulmonary damage by alleviating the earlier inflammation damage and adjust-ing the balance between MMPs and TIMPs. However, further studies are still warranted to investigate and clarify the underlying mechanisms involved in this complicated process.

Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; Disease Models, Animal ; Lung ; metabolism ; pathology ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Paraquat ; poisoning ; Pyrrolidines ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

A dominant H-2d restricted Th epitope P34 was found to be contained in recombinant particulate hepatitis E virus (HEV) vaccine HEV 239. In this paper, the cellular immune response induced in P34 immunized BALB/c mice were studied and the priming effect of P34 was characterized. Groups of BALB/c mice were subcutaneously (s. c.) immunized with P34, splenocytes were then stimulated with P34 and HEV 239 protein, cellular immune response was assayed by IFN-gamma-ELISPOT, flow cytometry and T cell proliferation experiments. Results showed that P34 immunized BALB/c splenocytes responsed to P34 and HEV 239 protein stimulation in IFN-gamma-ELISPOT, flow cytometry and T cell proliferation experiments. After depletion of the CD4+ T cells from the immunized splenocytes by magnetic separation, the response decreased to the background level while almost no influence was observed after CD8 + T cells depletion which showed that the cells responsible for IFN-gamma secretion were mainly CD4+ T cells. Then mice were primed with P34 and boosted with its vector protein, E2, the E2 specific antibody titer were assayed. Results showed that after P34 priming, some of the 10 microg, 20 microg E2 boosted mice could develop anti-E2 antibody 1 week later and all the mice had detectable antibody 3 weeks after boosting. In the control peptide P18 priming group, even after boosting with 20 microg E2, anti-E2 antibody couldn't be detected until the end of this experiment. The results showed that priming with P34 epitope could increase the immunogenicity of its vector protein, E2, in BALB/c mice.
Animals ; Antibodies, Viral ; blood ; immunology ; Cell Proliferation ; Cell Survival ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; immunology ; Female ; Flow Cytometry ; Hepatitis E virus ; immunology ; Immunization ; methods ; Immunization, Secondary ; Interferons ; metabolism ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; immunology ; metabolism ; T-Lymphocytes ; cytology ; immunology ; metabolism ; Viral Vaccines ; administration & dosage ; immunology

OBJECTIVETo investigate the clinical features of Candida albicans sepsis in preterm infants.

METHODSRetrospective analysis was performed on the clinical data of 13 preterm infants with Candida albicans sepsis, who were born at 28 to 36 weeks of gestational age and who weighed between 1400 and 2815 g.

RESULTSThe infants were infected with Candida albicans at the age of 19±11 d, with the main clinical manifestations being apnea, poor response, poor skin perfusion, blood oxygen concentration decrease, dark skin, yellowish skin, heart rate increase in the rest state, copious phlegm and difficulty in weaning from the ventilator. The infants showed significantly decreased platelet and increased C-reactive protein (CRP), platelet distribution width (PDW), alanine transaminase (ALT), creatine kinase isoenzyme-MB (CK-MB), total bilirubin (TBIL), creatine kinase (CK), and lactate dehydrogenase (LDH). CK and LDH were significantly decreased after 2 weeks of antifungal therapy. Only 3 cases developed drug resistance to fluconazole and these showed response when treated with voriconazole instead. Of the 13 cases, 10 were cured, 2 abandoned therapy and 1 died.

CONCLUSIONSThe clinical manifestations of Candida albicans sepsis are nonspecific in preterm infants. Infectious diseases are probably caused by Candida albicans in preterm infants 2-3 weeks after birth. Preterm infants show decreased platelet and increased CRP, PDW, ALT, CK-MB, TBIL, CK, and LDH when infected with Candida albicans.

Candida albicans ; isolation & purification ; Candidemia ; complications ; diagnosis ; drug therapy ; Female ; Humans ; Infant, Newborn ; Infant, Premature ; Male

OBJECTIVETo investigate the spatial learning and exploration along with the CNS excitatory amino acid neurotransmitters profiles in adult rats subchronically exposed to the anticholinesterase organophosphorus insecticide dimethoate.

METHODSRats were gavaged daily with dimethoate (0, 5, 10 or 20 mg/kg via oral) in NS. for 90 days. Morris water maze tasks were used to test the spatial learning and memory in the rats after the dimethoate exposure. Simultaneously, rats were decapitated for the determination of brain cholinesterase AChE activities, glutamate concentrations, and the NMDA receptor NMDA-R densities and affinities in hippocampus.

RESULTSLatencies to find a hidden escape platform were significantly longer in dimethoate dosed groups than that of the control group in the place navigation tests. Subsequently, the times of crossing the location of platform which had been removed obviously decreased in the highest dose group compared with that of the control in the spatial probe tests (P < 0.05). AChE activity was significantly reduced 42% approximately 78% by all three doses of dimethoate (P < 0.05). Glutamate concentrations were increased significantly 132.9% approximately 134.5% by the two highest doses of dimethoate (P < 0.05). In addition, the NMDA receptor bindings were reduced 21.2% approximately 23.2% with the statistical significance at the same two highest doses (P < 0.05). Furthermore, the receptor affinities was reduced 33.1% by the highest dose group (P < 0.05). The lesions of spatial memory were statistically corrected with the decrease of the NMDA-R affinities (P < 0.05).

CONCLUSIONThe cholinergic lesion as well as the excitatory amino acid system alteration might attribute to the inferior ability in spatial learning and memory in dimethoate subchronically exposed rats.

Acetylcholinesterase ; metabolism ; Animals ; Chronic Disease ; Dimethoate ; toxicity ; Disease Models, Animal ; Glutamic Acid ; metabolism ; Insecticides ; toxicity ; Learning ; drug effects ; Male ; Memory ; drug effects ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Toxicity Tests, Subchronic

It has been reported that baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. We have previously constructed recombinant baculovirus BacV-CMV-EGFPA and have proven that mammalian cells could be effectively infected by the recombinant baculovirus. In this report, we studied the efficiency of baculovirus to deliver exogenous gene into twenty mammalian cells, including twelve human cell lines (WI-38, Hela, HepG2, 293, PLC/PRF/5, 143B, MCF-7, BGC-223, DMS 114, CNE, Raji, LCL-cm), seven murine cell lines (BNL 1ME A.7R.1, CHO-K1, L-929, JC, PT67, NIH3T3, P815) and one monkey cell line (CV1). Results showed that most mammalian cell lines could be transduced by the recombinant baculovirus, the transduction efficiencies of the human and monkey cell lines were markedly higher than that of murine cell lines, and the transduction efficiencies in adherent culture cell lines higher than that of suspend culture cell lines, implying that the infection efficiency of the baculovirus may be correlative with the organism used and the growth properties of the cell lines. The plasmid pcDNA3. 1-EGFP, which contains the CMV promoter and EGFP reporter gene, was next transfected by LipofectAMINE into a number of mammalian cells, especially those cells that were low in the baculovirus transfection. Results showed that the CMV promoter could effectively direct the expression of the reporter gene in these mammalian cells. Therefore the gene-expression efficiencies in different mammalian cell lines by the recombinant baculovirus which contains the same CMV promoter were dictated by the ability of the baculovirus to enter the cell lines. This study suggested that the recombinant baculovirus vector is more suitable for gene expression in primate adherent culture cells than in murine cells and suspend culture cells.
Animals ; Baculoviridae ; genetics ; Cytomegalovirus ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Promoter Regions, Genetic ; Spodoptera