AIM: To study the cytological characteristics and gene expression of normal cultured bEnd.3, a mouse brain microvascular endothelial cell strain. METHODS: The morphology of bEnd.3 was studied by light and electronic microscopy, its molecular markers were observed by immunocytochemistry. Cell proliferation kinetics and apoptosis were analyzed by flow cytometry and MTT assay, PGE_2 level was measured by ELISA, and expression of the genes that closely related with vascular endothelial functions was studied by gene micro-array. RESULTS: bEnd.3 had morphological characteristics of microvascular endothelial cells (MVEC) growing in a cobblestone pattern, forming tube-like structure or capillary network and having microvilli. Furthermore, bEnd.3 showed positive staining for vW factor and CD34 and secreted high level of PGE_2 (644.55?30.24 ng/L). Gene micro-array analysis showed CD31, CD36, CD105 expression, and other genes closely related to microvascular endothelial functions also expressed at relatively high level. In addition, bEnd.3 responsed sensitively to mitogen such as basic fibroblast growth factor. CONCLUSION: bEnd.3 is a kind of MVEC, and it can be utilized to study the mechanisms of some diseases such as cancers and cardio- cerebral vascular diseases.

AIM: To study the effect and the mechanism of crenulatin, an effective constituent of Chinese traditional medicine, on apoptosis of cerebral microvascular endothelial cells. METHODS: The following terminal concentrations of crenulatin were used in the study: 25 mg/L and 100 mg/L. Apoptosis of mouse cerebral microvascular endothelial cells (bEnd. 3 cell line) was evaluated by flow cytometer, immunocytochemical assay (Fas, Bcl - 2) and Western blotting (caspase - 3) after culture for 24 h. RESULTS: Compared with control group, apoptosis of bEnd. 3 cells in 25 mg/L group was significantly inhibited ( P ＜0.05), but apoptosis in the 100 mg/L group was significantly increased (P ＜ 0.05). In apoptosis inhibited group, the Fas immunocytochemical staining was weaker, the positive cells were significantly decreased ( P ＜ 0.05) and caspase - 3 expression was decreased compared with control group; however, the Bcl - 2 staining was stronger and the positive cells were significantly increased ( P ＜ 0.05). On the other hand, in apoptosis increased group ( 100 mg/L group), the changes were just opposite. CONCLUSIONS: The effect of crenulatin on apoptosis of mouse cerebral microvascular endothelial cells possesses a dual - direction change, inhibitive effect in 25 mg/L and stimulative effect in 100 mg/L group, respectively. The mechanism is related to the alterations of Fas/Bcl - 2 expression and caspase - 3 activity.

] AIM: To study the effect and the mechanism of crenulatin, an effective constituent of Chinese traditional medicine, on apoptosis of cerebral microvascular endothelial cells. METHODS: The following terminal concentrations of crenulatin were used in the study: 25 mg/L and 100 mg/L. Apoptosis of mouse cerebral microvascular endothelial cells (bEnd.3 cell line) was evaluated by flow cytometer, immunocytochemical assay (Fas, Bcl-2) and Western blotting (caspase-3) after culture for 24 h. RESULTS: Compared with control group, apoptosis of bEnd.3 cells in 25 mg/L group was significantly inhibited (P

Five new megastigmanes (1-5) were isolated from a decoction of Uncaria rhynchophylla by separation techniques of column chromatography using a combination of multiple stationary phases, including macroporous adsorbent resin, MCI resin, silica gel, Sephadex LH-20, and Toyopearl HW-40F, and reversed phase HPLC. Their structures were characterized by spectroscopic data analysis of HR-ESI-MS, NMR, and CD, in combination with Mosher's mothed as well as ECD and NMR calculations. The new compounds were named uncarphyllonone A (1), uncarphyllonols A (2) and B (3), and uncarphabscisic acids A (4) and B (5). Although the structures of 3 and 4 were previously reported, the reported NMR spectroscopic data were incorrect or do not support the assigned structures in literatures. This is also the first report of discovery of new megastigmane natural products from the Uncaria genus.

The present study was aimed to investigate the effect of pretreatment with hydrogen sulfide (H2S) on human umbilical vein endothelial cells (HUVECs) senescence and the underlying mechanism. Cultured HUVECs at twelfth and fourth passages were taken as old and young groups, respectively. Sodium hydrosulfide (NaHS, donor of H2S) group was treated with NaHS from fourth to twelfth passage. The cell senescence was determined by senescence-associated β-galactosidase (SA β-gal) staining. DAPI fluorescent dye was used to detect cellular apoptosis. Western blot was used to analyze the expression levels of xanthine oxidase (XOD), manganese-superoxide dismutase (Mn-SOD) and the subunits p67(phox) of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the HUVECs. Colorimetric method was used to detect SOD activity and cellular hydrogen peroxide (H2O2) level. The results showed that, compared with young group, the old group exhibited higher SA β-gal positive rate and cellular apoptosis, while NaHS pretreatment decreased SA β-gal positive rate and cellular apoptosis. Compared with the young group, the old group showed increased expression levels of XOD and p67(phox), as well as lower Mn-SOD expression level. With the pretreatment of NaHS, the up-regulations of XOD and p67(phox) levels and down-regulation of Mn-SOD level were inhibited. Compared with the young group, the old group showed lower SOD activity and higher H2O2 level, whereas NaHS pretreatment reversed the changes of SOD activity and H2O2 level. These results suggest that H2S delays senescence of HUVECs through lessening oxidative stress.
Apoptosis ; Cellular Senescence ; drug effects ; Down-Regulation ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Hydrogen Peroxide ; metabolism ; Hydrogen Sulfide ; pharmacology ; Oxidative Stress ; Phosphoproteins ; metabolism ; Superoxide Dismutase ; metabolism ; Xanthine Oxidase ; metabolism ; beta-Galactosidase ; metabolism

OBJECTIVETo study the mechanism and significance of peripheral blood mononuclear cell (PBMC) of neonates infected with hepatitis B virus (HBV).

METHODSEighty-four HBsAg-positive and HBeAg-negative mothers and their newborns were recruited in this study. Sixteen hepatitis B virus markers (HBVM)-negative mothers and their neonates were served as control. All these cases had no symptoms of hepatitis, serious pregnancy complications and preexisting disease. Age, gestational age and the method of delivery were matched in two groups (P > 0.05). Five ml blood samples were taken from the peripheral vein of the pregnant women before delivery and from neonates within 24 hours after birth, before inoculation of HBV vaccine (HBVac). Serum and PBMC were isolated from 2 ml and 3 ml samples respectively. The sera, PBMC and the last supernatant of PBMC washing were stored at -80 degrees C. HBVM of neonates were detected by using enzyme linked immunosorbent assay (ELISA). HBV DNA in serum, PBMC and the last supernatant of PBMC washing of mothers and neonates were detected by using a nested-polymerase chain reaction (n-PCR). Two pairs of oligonucleotide primers, the outer primer pair for first PCR and inner primer pair for second PCR, designed according to region S of HBV genome were synthesized at Shanghai Cell Biology Institute of Chinese Academy of Sciences. The neonates who were HBV DNA positive in PBMC but HBsAg and HBV DNA negative in serum were followed up for one year, HBsAb in serum and HBV DNA in PBMC were observed in the neonates.

RESULTS(1) The positive rate of HBV DNA in 84 serum and PBMC of mothers were 53.57% and 40.48%, respectively (chi(2) = 2.891, P > 0.05). All the results were weakly positive. (2) Twenty-four (28.57%) newborns in the study group were infected, including 7 who were only HBV DNA positive in serum, 11 only HBV DNA positive in PBMC and 6 in both, all the results were weakly positive. HBsAg was negative in all the newborns. None of the neonates in control group was infected with HBV. There was significant difference between the two groups (chi(2) = 4.55, P < 0.05). (3) Of all the study cases, 11 (13.10%) neonates were HBV DNA weakly positive in PBMC but HBsAg and HBV DNA negative in serum. Of their mothers, 5 were only HBV DNA positive in serum, 2 only positive in PBMC and 4 positive in both serum and PBMC. Seven of the 11 neonates were followed up for one year and at the end of follow-up, 4 were HBsAb positive and HBV DNA negative in PBMC; 3 were HBsAb negative, and among the 3 cases HBV DNA in 2 was still positive in PBMC, HBsAg and HBV DNA in serum were negative in all the 7 neonates.

CONCLUSION(1) HBV DNA positivity either in serum or in PBMC in mothers can result in infection of PBMC with HBV in their neonates. (2) PBMC infection with HBV can exist for a long time in neonates while HBsAg and HBV DNA are negative in serum, and may result in vaccination failure in neonates.

Case-Control Studies ; DNA, Viral ; blood ; Female ; Hepatitis B ; diagnosis ; immunology ; Hepatitis B Vaccines ; administration & dosage ; Hepatitis B virus ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; Leukocytes, Mononuclear ; virology ; Pregnancy

OBJECTIVETo investigate the anti-hyperglycemic effect and its mechanism of ethanol extraction from Calamintha chinensis (EJCT).

METHODFasting serum glucose (FSG) in normal mice was determined after oral administration of EJCT. Effects of EJCT on hyperglycemia mice induced by adrenaline were investigated by observing the contents of FSG and liver glucogen. Effect of EJCT on the diabetic mice induced by alloxan was investigated by observing the contents of FSG and the injured degree of pancreatic islet. The antilipid-peroxidation of EJCT on liver homogenate was measured by determination of malondiadehyde (MDA) induced by Fe2+/Cys.

RESULTEJCT showed no obvious effect on FSG in normal mice. However, EJCT 300, 600 mg x kg(-1) could remarkably decrease the contents of FSG and increase liver glucogen in hyperglycemia mice induced by adrenaline. In diabetic mice induced by alloxan, EJCT 150, 300, 600 mg x kg(-1) could remarkably decrease the contents of FSG. The damage of pancreatic islet induced by alloxan was also significantly attenuated by EJCT. Furthermore, EJCT 30, 60, 90, 120 mg x L(-1) inhibited lipid peroxidation initiated by Fe2+/Cys in liver homogenate.

CONCLUSIONThese results suggest that EJCT can significantly attenuate hyperglycemia in diabetic mice, which is probably due to decreasing the decomposition of liver glucogen, increasing the synthesis of liver glucogen, antioxidation and amelioration of damaged pancreatic islet.

Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; blood ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fasting ; Hypoglycemic Agents ; pharmacology ; therapeutic use ; Islets of Langerhans ; drug effects ; Lamiaceae ; chemistry ; Lipid Peroxidation ; drug effects ; Liver ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR

OBJECTIVETo study the role of the HBV-infected mothers' PBMC in intrauterine transmission of HBV to their fetuses.

METHODSThirty pregnant women with serum HBV DNA negative and PBMC HBV DNA positive and their newborns were used as the study group. Ten pregnant women with serum HBV negative and their infants served as the control group. HBV DNA in serum and in PBMC was detected using nested polymerase chain reaction (n-PCR). The mothers' PBMC in newborns' peripheral blood was examined using heminested-PCR.

RESULTSFour newborns were serum HBV DNA positive and 8 newborns were HBV DNA positive in PBMC in the study group. Among them, 2 newborns were HBV DNA positive in both serum and PBMC, 6 cases were positive in PBMC only, and 2 cases were positive in serum only. Five mothers had the GSTM1 gene; and it was not detected in 3 newborns. Among the 8 newborns with HBV DNA positive in PBMC, 3 did not have the GSTM1 gene, at the same time their mothers possessed the GSTM1 gene. Mothers' PBMC were detected in all of these three newborns' peripheral blood. HBV DNA in serum and in PBMC of the control group infants were all negative.

CONCLUSIONHBV-infected PBMC of the mother may serve as a vector in HBV intrauterine infection.

Adult ; DNA, Viral ; analysis ; Female ; Hepatitis B virus ; isolation & purification ; Hepatitis B, Chronic ; blood ; transmission ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; Leukocytes, Mononuclear ; virology ; Pregnancy ; Pregnancy Complications, Infectious ; virology

OBJECTIVETo understand the HBV infection rate of peripheral blood mononuclear cells (PBMCs) from fetuses of HBsAg positive mothers, associated risk factors and to explore the clinical significance of detecting HBV infected PBMCs.

METHODSSixty eight pregnant women who were delivered at the First Hospital of Xi'an Jiaotong University, China from August 1995 to February 1997, and their newborns were studied. They were divided into two groups according to their status of HBV serological markers. The study group included 50 cases who were serum HBsAg positive and 18 cases without any HBV serum markers served as control group. All these cases had no symptoms of hepatitis, high risk premature labor, premature delivery and hypertensive disorder complicating pregnancy. Age and gestational age were matched in two groups. Blood samples (5 mL) were taken from the peripheral vein of pregnant women before delivery and from newborns within 24 h after birth, before inoculation of HBV vaccine (HBVac) and injection of hepatitis B immunoglobulin (HBIG). PBMCs were isolated. The sera and PBMCs were stored at -80 degrees C. HBV-DNA in serum and PBMCs were detected with nested polymerase chain reaction (n-PCR). Two pairs of oligonucleotide primers, the outer primer pair for first PCR and inner primer pair for second PCR, designed according to region S of HBV genome were synthesized by Shanghai Cell Biology Institute of Chinese Academy of Science.

RESULTSThe detection rate of HBV-DNA in serum and PBMCs from HBsAg positive pregnant women was 60.0% (30/50) and 40.0% (20/50), respectively. The detection rate of HBV-DNA in serum and PBMCs from newborns of HBsAg positive pregnant women was 46.0% (23/50) and 30.0% (15/50), respectively. Ten newborns were HBV-DNA positive in serum only, 2 were positive in PBMCs only and 13 were positive in both serum and PBMCs. In the control group, HBV-DNA was not detected in PBMC nor in serum. The positive rate of HBV-DNA in PBMCs of newborns was significantly higher in the group of mothers who were HBV-DNA or HBeAg positive in serum (P < 0.05, P < 0.01); the positive rate was significantly higher in the group of mothers who were HBV-DNA positive in both serum and PBMC than that in the group of mothers who were serum HBV-DNA positive only (P < 0.01); and it was markedly higher in the group of mothers who were PBMC HBV-DNA positive than that in group of mothers who were HBV-DNA negative in PBMCs (P < 0.01). The positive rate of HBV-DNA in PBMCs of newborns was significantly higher in the group of newborns who were HBV-DNA positive in serum than that in the group of newborns who were HBV-DNA negative in serum (P < 0.05).

CONCLUSIONSThe positive rate of HBV-DNA in PBMCs from newborns of HBsAg positive pregnant women was 30.0% (15/30). It was related to HBV viremia level and HBV-DNA status in PBMCs of mothers and newborns. Detection of HBV-DNA in PBMCs may be an important supplementary method to determine intrauterine HBV infection, and can predict the response to HBV vaccine.

Adult ; Case-Control Studies ; DNA, Viral ; blood ; Female ; Hepatitis B Vaccines ; administration & dosage ; Hepatitis B virus ; immunology ; isolation & purification ; Humans ; Immunoglobulins ; administration & dosage ; Infant, Newborn ; blood ; Infectious Disease Transmission, Vertical ; prevention & control ; Injections, Intramuscular ; Leukocytes, Mononuclear ; virology ; Male ; Mothers ; Polymerase Chain Reaction ; Pregnancy ; blood ; Risk Factors ; Time Factors ; Treatment Outcome

Antibody-drug conjugate (ADC) is a new class of therapeutics composed of a monoclonal antibody and small cytotoxin moieties conjugated through a chemical linker. ADC molecules bind to the target antigens expressed on the tumor cell surfaces guided by the monoclonal antibody component. The binding ADC molecules can be internalized and subsequently the toxin moieties can be released within the tumor cells via chemical and/or enzymatic reactions to kill the target cells. The conjugation combines the merits of both components, i.e., the high target specificity of the monoclonal antibody and the highly potent cell killing activity of the cytotoxin moieties. However, such complexities make the pharmacokinetic and metabolic studies of ADCs highly challenging. The major challenges should include characterization of absorption, distribution, metabolism and excretion, investigation of underlying mechanisms, assessment of pharmacokinetic- pharmacodynamic relationship, and analytical method development of ADC drugs. This review will discuss common pharmacokinetic issues and considerations, as well as tools and strategies that can be utilized to characterize the pharmacokinetic and metabolic properties of ADCs.
Antibodies, Monoclonal ; pharmacokinetics ; Cytotoxins ; pharmacokinetics ; Humans ; Immunoconjugates ; pharmacokinetics ; Neoplasms ; drug therapy