To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.
Animals ; Biological Transport ; Cimetidine ; pharmacology ; DNA, Complementary ; Dogs ; Drug Interactions ; Humans ; Madin Darby Canine Kidney Cells ; Metformin ; pharmacology ; Organic Cation Transport Proteins ; genetics ; metabolism ; Transfection

Objective To evaluate the effect of curcumin pretreatment on brain injury induced by intestinal ischemia-reperfusion (I/R) in mice.Methods Forty-eight male C57BL/6 mice,aged 8 weeks,weighing 20-24 g,were divided into 3 groups (n =16 each) using a random number table:sham operation group (Sham group),intestinal I/R group (I/R group) and curcumin pretreatment group (CUR group).Mice were subjected to 75 min superior mesenteric artery occlusion followed by 24 h reperfusion to establish the model of brain injury induced by intestinal I/R in mice.Curcumin 200 mg/kg (in 30 ml of 10％ dimethyl sulfoxide) was intraperitoneally injected at 30 min before ischemia in CUR group,while 10％ dinethyl sulfoxide 30 ml was given instead of curcumin in Sham group and I/R group.Two percent Evans blue (EB) in 4 ml/kg of normal saline was injected via the caudal vein at 23 h of reperfusion,1 h later mice were sacrificed,and hippocampi were removed for determination of EB content.Mice were sacrificed at 24 h of reperfusion,hippocampal tissues were isolated for determination of wet to dry weight ratio (W/D ratio) and cell apoptosis (by TUNEL) and for examination of the pathological changes (with a light microscope),and brain tissues were isolated for determination of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) contents (by enzyme-linked immunosorbent assay),malondialdehyde (MDA) content (by thiobarbituric acid method),superoxide dismutase (SOD) activity (by xanthine oxidase method) and expression of caspase-3 (by Western blot).Apoptosis rate was calculated.Results Compared with Sham group,the W/D ratio of hippocampal tissues,EB content and apoptosis rate were significant increased,the contents of MDA,TNF-α and IL-6 in brain tissues were increased,SOD activity was decreased,and the expression of caspase-3 was up-regulated in I/R and CUR groups (P＜0.05).Compared with I/R group,the W/D ratio of hippocampal tissues,EB content and apoptosis rate were significant decreased,the contents of MDA,TNF-α and IL-6 in brain tissues were decreased,SOD activity was increased,and the expression of caspase-3 was down-regulated (P＜0.05),and the pathological changes of hippocampal tissues were significantly reduced in CUR group.Conclusion Curcumin pretreatment can reduce brain injury induced by intestinal I/R in mice,and the mechanism may be related to inhibiting inflammatory responses,lipid peroxidation and cell apoptosis.

OBJECTIVETo compare the effects of different contrast media on the renal function in children, and to investigate the prophylactic efficacy of hydration.

METHODSSixty patients on whom either intravenous pyelography (IVP) or enhanced CT scan was required were divided into high osmolality contrast media (HOCM) group (n = 27) and low osmolality contrast media (LOCM) group (n = 33), and each group was randomly subdivided into hydration group (HG) and non-hydration group (NHG). In HOCM group, HG had 14 cases and NHG had 13 cases; while in LOCM group, HG had 18 cases and NHG had 15 cases. A 1/5-tonic solution at a dose of 20 ml/kg was intravenously given immediately after the exposure to a contrast medium within 3 hours in the HG, while the NHG cases were not given any infusion.

RESULTSThere were no significant difference between HG and NHG in baseline serum creatinin (SCr) and creatinin clearance (Ccr). After exposure, in HOCM group, SCr of NHG (59.71 +/- 12.49) micromol/L significantly increased as compared with baseline (49.91 +/- 6.09) micromol/L (P < 0.05), while Ccr (97.81 +/- 15.10)ml/(min x 1.73 m(2)) decreased compared with baseline (71.33 +/- 7.51) ml/(min x 1.73 m(2)) (P < 0.05). No significant changes of SCr and Ccr were observed in the HG before (48.37 +/- 7.11) micromol/L, (99.81 +/- 15.41) ml/(min x 1.73 m(2)) and after (49.63 +/- 6.84) micromol/L, (88.29 +/- 12.75) ml/(min x 1.73 m(2)) (P > 0.05) the exposure to contrast medium. Contrast medium-associated nephropathy (CAN) was found in 3 cases in NHG (23.1%, 3/13) but none in HG (P > 0.05). In the LOCM group, there was no significant difference in SCr and Ccr before and after the exposure to the contrast media. The incidence of CAN was 6.7% (1/15) in the NHG and 11.1% (2/18) in the HG (P > 0.05). The average increase of SCr in HOCM group was significantly higher than that in LOCM group (Z = -2.42, P < 0.05). The average decrease of Ccr in HOCM group was significantly higher than that in LOCM group (Z = -2.83, P < 0.05). The SCr and Ccr of the 6 CAN cases in both HOCM and LOCM groups returned to baseline level within 2 weeks.

CONCLUSIONS(1) Children can develop reversible CAN after the exposure to high or low osmolality contrast medium. (2) The high osmolality contrast medium seemed to have more serious toxicity in renal function than low osmolality contrast medium. (3) The prophylactic use of hydration can effectively prevent CAN in patients who will expose to high osmolality contrast medium. (4) Children can develop reversible CAN after the exposure to low osmolality contrast medium even after hydration.

Child ; Contrast Media ; adverse effects ; Creatinine ; blood ; Fluid Therapy ; Humans ; Infusions, Intravenous ; Kidney Diseases ; chemically induced ; prevention & control ; Kidney Function Tests ; Osmolar Concentration

OBJECTIVETo study aberrant expression of cell cycle control genes in patients with myelodysplastic syndromes (MDS).

METHODSReverse transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of cell cycle control genes (cyclin D2, cyclin D3, cyclin A1, cyclin E, CDK2, CDK4, CDK6, p21, p27, p57, Rb and E2F1) in bone marrow mononuclear cells (BMMNCs) from 29 normal control, 27 MDS and 19 de novo acute myeloid leukemia (AML).

RESULTSThe expression levels of cyclin D3 (0.65 +/- 0.17, P < 0.05) and cyclin A1 (0.48 +/- 0.04, P < 0.05) in MDS were higher than those in normal control and significantly lower than those in AML. The expression rates and levels of cyclin D2 (40.7% and 0.78 +/- 0.21) and cyclin E (51.9% and 0.52 +/- 0.10) in MDS were statistically higher than those in normal control and AML. The expression level of CDK2 in MDS (0.66 +/- 0.19, P < 0.01) was higher than that in normal control (0.42 +/- 0.04) and the expression rate of CDK6 in MDS (25.9%) higher than in normal control (3.4%, P < 0.05). There was no significant difference of the expression rates and levels of CDK4 in MDS, AML and normal control. The expression rates and levels of p21 (77.8% and 1.18 +/- 0.21) and p27 (48.1% and 1.14 +/- 0.40) in MDS were statistically higher than those in normal control and AML. The expression level of p57 in MDS (0.69 +/- 0.06) was higher than that (0.53 +/- 0.05, P < 0.01) in normal control but lower than in AML (0.96 +/- 0.16, P < 0.01). The expression rate (55.6%) and level (0.85 +/- 0.17) of Rb in MDS were significantly higher than those in normal control and AML. The expression rate (7.4%) and level (0.39 +/- 0.04) of E2F1 in MDS were comparable to those in normal control but lower than those in AML.

CONCLUSIONMDS clones have aberrant mechanism of cell cycle control: high expressions of cyclin family members, CDK2 and CDK6 may lead to high proliferation; high expression of p21 and p27 may cause the G1 phase arrest.

Adolescent ; Adult ; Cell Cycle Proteins ; genetics ; Child ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; Cyclin-Dependent Kinases ; genetics ; E2F1 Transcription Factor ; genetics ; Female ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; pathology ; Retinoblastoma Protein ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

OBJECTIVETo evaluate the quantitative and functional changes of T helper (Th) cell subsets in the bone marrow of severe aplastic anemia (SAA) patients and the relationship between these changes and the patients hematopoietic function.

METHODSBy FACS, the quantity and ratio of Th1 and Th2 cells, the percentage of CD3(+)CD8(+) cells in the bone marrow were detected in 24 patients with SAA at active phase, 15 patients with SAA at recovery phase, and 16 normal controls. By radioimmunoassay, the serum levels of TNF-alpha, or IL-4 in 20 SAA patients at active phase, 12 at recovery phase and 16 normal controls were measured. The relationships between CD3(+)CD8(+) cells, TNF-alpha and Ret, ANC; and between Th1 cells and CD3(+)CD8(+) cells, TNF-alpha or Ret, ANC; between IL-4, balance of Th1/Th2 and Ret, ANC were evaluated.

RESULTSThe percentages of Th1 and Th2 cells, and ratio of Th1/Th2 in bone marrow of SAA patients at active phase were (4.87 +/- 2.64)%, (0.41 +/- 0.26)% and 21.22 +/- 5.07, respectively, being higher than those of normal controls [(0.42 +/- 0.30)% (P < 0.01), (0.24 +/- 0.17)% (P < 0.05) and (1.57 +/- 0.93) (P < 0.01), respectively] and all of them reduced to normal levels of SAA at recovery phase (P > 0.05). The percentage of CD3(+)CD8(+) cells significantly decreased from (32.32 +/- 8.69)% at active phase to (13.76 +/- 2.96)% at recovery phase (P < 0.01). The serum levels of TNF-alpha and IL-4 at active phase was (4.29 +/- 3.15) microg/L and (1.24 +/- 0.73) microg/L, respectively, being higher than those of normal controls (1.21 +/- 1.16) microg/L, (1.18 +/- 0.97) microg/L, but only the difference of TNF-alpha was statistically significant (P < 0.01). In recovery SAA patients, the serum levels of TNF-alpha significantly decreased to (1.46 +/- 1.41) microg/L (P < 0.01), and the levels of IL-4 increased markedly to (3.05 +/- 1.94) microg/L. The CD3(+)CD8(+) cells and TNF-alpha of patients negatively correlated with Ret (P < 0.05; P < 0.05) and ANC (P < 0.05; P < 0.05), Th1 cells correlated with CD3(+)CD8(+) cells and TNF-alpha positively (P < 0.01; P < 0.05), the Ret and ANC negatively (P < 0.01; P < 0.01), IL-4 and the balance of Th1/Th2 positively correlated with Ret and ANC (P < 0.05, P < 0.01; P < 0.01, P < 0.01).

CONCLUSIONThe bone marrow failure in SAA might be caused not only by the increase of Th1 cells, Th1 type effector cells and cytokines, but also by insufficient compensation of Th2 cells and Th2 type cytokines, which shifted the balance of Th1/Th2 favorable to Th1.

Adolescent ; Adult ; Anemia, Aplastic ; blood ; pathology ; physiopathology ; Bone Marrow ; metabolism ; pathology ; CD3 Complex ; blood ; CD8 Antigens ; blood ; Child ; Female ; Hematopoietic System ; metabolism ; pathology ; physiopathology ; Humans ; Interleukin-4 ; blood ; Male ; Middle Aged ; Radioimmunoassay ; T-Lymphocytes, Helper-Inducer ; metabolism ; pathology ; Th1 Cells ; metabolism ; pathology ; Th2 Cells ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; blood ; Young Adult

OBJECTIVETo measure the subsets of dendritic cells 1 (DC1) in the bone marrow of severe aplastic anemia (SAA) patients and evaluate the relationships between the CD11c+CD83+ cells and Th1 cells, CD3+CD8+ cells or hematopoietic function and explore the role of DC1 in the pathogenesis of SAA.

METHODSBy FACS, the quantities and ratios of CD11c+CD1a+ cells, CD11c+CD83+ cells, Th1 cells, and CD3+CD8+ cells in the bone marrow of SAA patients and normal controls were detected respectively. The relationships between CD3+CD8+ cells and reticulocyte absolute value (Ret) or neutrophil absolute value (ANC), between Th1 cells and CD3+CD8+ cells, Ret or ANC, between CD11c+CD83+ cells, and Th1 cells, CD3+CD8+ cells, Ret or ANC were evaluated.

RESULTSIn normal controls' bone marrow, the percentages of Th1 cells, CD11c+CD1a+ cells, CD11c+CD83+ cells and the ratio of CD11c+CD83+/CD11c+CD1a+ were (0.42 +/- 0.30)%, (0.38 +/- 0.29)%, (0.37 +/- 0.32)% and 1.07 +/- 0.10, respectively. In untreated SAA patients, they were (4.87 +/- 0.54)%, (1.73 +/- 0.24)%, (3.38 +/- 0.56)% and 2.21 +/- 0.32 respectively, which were higher than that in normal controls (P < 0.01). In recovering SAA patients, the percentages of Th1 cells, CD11c+CD1a+ cells and CD11c+CD83+ cells decreased significantly to (0.53 +/- 0.22)%, (0.61 +/- 0.23)%, (0.65 +/- 0.22)%, respectively (P < 0.01). The ratio of CD11c+CD83+/CD11c+ CD1a+ in recovering SAA patients decreased to 1.37 +/- 0.25, which was similar to that in normal controls (P > 0.05). The percentage of CD3+CD8+ cells in untreated SAA patients was (32.32 +/- 10.22)%, and in recovering SAA patients decreased to (13.67 +/- 5.24)% (P < 0.01). The percentage of CD3+CD8+ cells in SAA patients was negatively correlated with their Ret and ANC (P < 0.05), while their Th1 cell percentages were positively correlated with their CD3+CD8+ cells (P < 0.01), and negatively correlated with their Ret and ANC (P < 0.01). SAA patient's CD11c+CD83+ cell percentages were positively correlated with their Th1 cell and CD3+CD8 cells (P < 0.01, P < 0.05), but negatively with their Ret and ANC (P < 0.01).

CONCLUSIONBoth immature DC1 and activated DC1 increased in the bone marrow of SAA patients, and the balance of DC1 subsets shifted from stable form to active one, which might promote Th0 cells to polarize to Th1 cells, and cause the over-function of T lymphocytes and hematopoiesis failure in SAA.

Adolescent ; Adult ; Anemia, Aplastic ; immunology ; Antigens, CD ; immunology ; Antigens, CD1 ; immunology ; Bone Marrow ; immunology ; CD11c Antigen ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Child ; Dendritic Cells ; immunology ; Female ; Humans ; Immunoglobulins ; immunology ; Male ; Membrane Glycoproteins ; immunology ; Th1 Cells ; immunology ; Young Adult

OBJECTIVETo investigate the quantity, subset of dendritic cells (DC) and their costimulatory molecule expression in peripheral blood (PB) of the patients with myelodysplastic syndromes (MDS).

METHODSTotal DC (Lin1(+) HLA-DR(+)), myeloid DC (mDC) (Lin1(-) HLA-DR(+) CD11c(+)) and plasma DC (pDC) (Lin1(-) HLA-DR(+) CD123(+)) in fresh PB samples of 38 MDS patients and 19 normal controls were assayed by flow cytometry with the monoclonal antibodies. The expressions of costimulatory molecules CD80, CD86 and CD40 on these DCs were also assayed in the same way.

RESULTSThe number of total DC in PB of low-risk and high-risk MDS patients was significantly higher than that in normal controls [(33.7 +/- 7.0) x 10(6)/L, (56.3 +/- 29.0) x 10(6)/L vs (12.1 +/- 1.4) x 10(6)/L, respectively] (P < 0.05), that of mDC in PB of low-risk and high-risk MDS patients was higher than that of normal controls too [(16.7 +/- 6.3) x 10(6)/L, (28.7 +/- 17.6) x 10(6)/L vs (5.5 +/- 0.9) x 10(6)/L] (P < 0.05), but pDC in low-risk and high-risk MDS patients was not significantly higher than that in normal controls (P > 0.05). The percentage of total DC in PB mononuclear cells (PBMNC) of low-risk and high-risk MDS patients [(2.37 +/- 0.53)% and (3.58 +/- 1.39)% respectively and that of mDC (0.90 +/- 0.35)%, (1.51 +/- 0.70)% respectively] were higher than that of normal controls [(0.68 +/- 0.08)%, and (0.32 +/- 0.05)% respectively] (P < 0.05), but that of pDC in MDS cases was not higher than that of normal controls (P > 0.05). The expressions of CD80 and CD86 between MDS patients and normal controls had significant difference (P < 0.05).

CONCLUSIONSTotal DC and mDC were increased significantly in MDS, but pDC did not. The costimulatory molecules (CD80 and CD86) except CD40 expressed higher on the DC of MDS patients. It suggested that the inflammatory injury related APC increased in MDS, but the antitumour immunity related APC did not . What found here might be one of the mechanisms involved in the pathogenesis of MDS.

Adolescent ; Adult ; Aged ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Dendritic Cells ; immunology ; metabolism ; pathology ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; immunology ; metabolism ; pathology

OBJECTIVETo study the clinical characteristics of autoimmune hemolytic anemia (AIHA) with both warm and cold autoantibodies.

METHODSClinical and laboratory characteristics of 23 cases of AIHA with both warm and cold autoantibodies admitted to our hospital between January 1994 and April 2004 were analyzed retrospectively.

RESULTSIn comparison with the AIHA patients with both warm and cold autoantibodies in the 1980s, the present patients showed the following features: The proportion of this kind AIHA in all AIHA patients increased from 17.6% to 22.1%. There were more females, more primary cases (73.9%), more mixed subtypes of autoantibodies and more of IgM (56.5%). The hemolysis was related with thermal amplitude of autoantibodies and quantity of complement. The response to cortisone and other immunosuppressive drugs was good. The relapse rate was 77.8% in a median follow-up time of 4 months.

CONCLUSIONSAIHA with both warm and cold autoantibodies is related with the type and thermal amplitude of the autoantibody and the activation of complement. It can be treated effectively with combined immunosuppressive therapy, but the relapse rate is high.

Adolescent ; Adult ; Aged ; Anemia, Hemolytic, Autoimmune ; drug therapy ; immunology ; Autoantibodies ; immunology ; Female ; Follow-Up Studies ; Humans ; Immunoglobulin M ; immunology ; Male ; Middle Aged ; Treatment Outcome

OBJECTIVETo explore dysfunction mechanism of rat alveolar type II (AT-II) injured by bleomycin (BLM).

METHODSSD rats were injected with a single intratracheal dose of bleomycin or control saline. On day 7, 14, and 28 following intratracheal bleomycin or saline instillation, animals were killed under overdose of 1.5% sodium pentobarbital (0.25 ml/100 g, i.p.) and bronchoalveolar lavage fluid (BALF) from the lung was tested for the activity of pulmonary surfactant (PS) by the Whihelmy Film Balance. Several concentrations of bleomycin stimulated the culture of rat AT-II cells, and surfactant protein (SP) A, B, and aquaporin-1 (AQP) mRNA were analyzed by fluorescent quantitative polymerase chain reaction (FQ-PCR).

RESULTSThe activity of PS and hypoxemia significantly decreased on day 7 and improved on day 14 and completely recovered to normal status on day 28. SP-A, B, and AQP-1 mRNA expression in BLM-stimulated group were significantly lower than those in the control group (P<0.001).

CONCLUSIONBLM-injured AT-II cells decrease the levels of SP-A, B, and AQP-1 mRNA and cause PS dysfunction, resulting in hypoxemia and pneumonedema.

Animals ; Aquaporin 1 ; biosynthesis ; genetics ; Bleomycin ; administration & dosage ; toxicity ; Cells, Cultured ; Dose-Response Relationship, Drug ; Epithelial Cells ; drug effects ; metabolism ; Hypoxia ; chemically induced ; metabolism ; pathology ; Male ; Pulmonary Alveoli ; cytology ; drug effects ; Pulmonary Surfactant-Associated Protein A ; biosynthesis ; genetics ; Pulmonary Surfactant-Associated Protein B ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo evaluate the value of (18)F-FDG PET/CT in detecting residual disease and predicting relapse following first-line treatment in patients with diffuse large B cell lymphoma (DLBCL).

METHODSThe clinical data of 39 patients with DLBCL, who underwent PET/CT scan after first-line treatment, were analyzed retrospectively. Kaplan-Meier method was used to analyze the survival of patients.

RESULTSPET/CT findings were interpreted as negative, mild metabolism and positive. Seventeen patients' PET/CT findings were judged as negative, none of them relapsed with a median follow-up of 24.1 months, 13 were judged as mild metabolism, 2 of them relapsed with a median follow-up of 17.1 months. Of the rest 9 findings were judged as positive with a median follow-up of 16.3 months, 4 patients were considered as disease progression according to clinical manifestations and other radiographic results, 2 patients relapsed at the time points of 13.5 and 6.8 months after PET/CT scan respectively, the other 3 patients were diagnosed as negative by biopsy, none of them relapsed at the time points of 5.9, 9.6 and 20.0 months after PET/CT scan respectively. One-year progression-free-survival (PFS) for negative, mild metabolism and positive groups was 100%, 83% and 56%, respectively. Two-year PFS was 100%, 83% and 42%, respectively. Overall survival (OS) at 1 year for negative, mild metabolism and positive groups was 100%, 100% and 89%, respectively. Two-year OS was 100%, 100% and 63%, respectively (P = 0.004).

CONCLUSIONDLBCL patients with negative and mild metabolism PET/CT following first-line treatment had good prognosis, who needed no additional therapy. While patients with positive PET/CT had poor prognosis, those patients should receive biopsy before adjusting treatment regimen because of the high false-positive rate.

Aged ; Female ; Fluorodeoxyglucose F18 ; Humans ; Lymphoma, Large B-Cell, Diffuse ; diagnostic imaging ; therapy ; Male ; Middle Aged ; Positron-Emission Tomography ; methods ; Prognosis ; Retrospective Studies ; Treatment Outcome