Objective To evaluate the diagnosis and therapy of intestinal lymphangiectasia.Methods In this study 15 patients were admitted in our hospital during recent 7 years.Clinical manifestations included hypoalbuminemia,symmetrical edema,emaciation,diarrhea and lymphopenia.Lymphangiography,lympanscintigraphy and biopsy were performed for diagnosis.Therapy conducted included conservative therapy,low-fat and medium-chain triglycerides(MCT)diet,albumin infusions,diuretics,total parenteral nutrition and octreotide.Surgical therapy ineluded thoracic duct-vein anastomasis and segmental resection.Results In this group 8 patients receiving conservative therapy were followed-up from 1.5 to 7 years(average 2.5 years).Symptoms were alleviated in 6 patients.Seven patients underwent operative therapy,among them,4 patients received thoracic duct-exterior jugular vein anastomasis and followed-up from 1 to 5 years,with symptoms mitigated in 2 patients.3 patients underwent local intestinal resection,follow-up from 1 to 3 years found one patient was cured,one was improved,and 1 patient died 3 months afterthe operation. Conclusion Intestinal lymphangiectasia is rather rare and there was no definite and effective therapy.

OBJECTIVETo prepare anti-von Willebrand factor A3 (vWF-A3) domain monoclonal antibodies(mAbs) which block vWF-A3 binding to collagen, and characterize their biochemical properties and functions.

METHODSBALB/c mice were immunized with purified recombinant vWF-A3 protein (rvWF-A3). Murine anti-human vWF-A3 mAbs were developed by standard hybridoma technology and identified with ELISA. The recognition of the mAbs with rvWF -A3 and reduced human vWF was identified by Western-blot. The effect of mAbs on binding of purified human vWF to human placenta or calf skin collagen III was studied with collagen binding inhibition test.

RESULTSA group of 30 murine anti-human vWF-A3 mAbs was obtained, from which 2 clones were identified as inhibitory ones and designated as SZ-123 and SZ-125. SZ-123 and SZ-125 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that SZ-123 and SZ-125 could recognize a 27 x 10(3) band of rvWF-A3 and 2 reduced human vWF bands at 250 x 10(3) and 170 x 10(3). SZ-123 and SZ-125 not only inhibited the binding of purified human vWF (1.5 and 3.0 microg/ml) to human type III collagen and to calf skin collagen III in a dose dependent manner, but also inhibited the binding of plasma vWF from human, rhesus monkeys or Beagle dogs to the two collagens.

CONCLUSIONSZ-123 and SZ-125 are neutralizing mAbs against vWF-A3 domain and may have therapeutic potential as an antithrombotic agent.

Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Collagen ; immunology ; Mice ; Mice, Inbred BALB C ; von Willebrand Factor ; immunology

OBJECTIVETo explore anti-angiogenesis effect of Mer, a member of tyrosine kinase receptor family, and its mechanism.

METHODSHuman Mer full length plasmid was transfected into HMEC-1 cells through liposome. G418 was used to select positive clone. Expression of Mer at mRNA and protein level was detected by real-time PCR and Western-blot, respectively. Transwell and Matrigel were used to evaluate the effect of overexpressed Mer on migration and angiogenesis of HMEC-1 cells. Primary angiogenesis associated factor VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGFR-1, VEGFR-2 were screened by real-time PCR.

RESULTSAfter G418 selection, the Mer expression in transfected HMEC-1 cells was increased 3.61- and 2.12 fold at mRNA and protein level, respectively. Compared with negative control, the migration of Mer-HMEC-1 was decreased (21 +/- 6 vs 36 +/- 11), and angiogenesis capability on Matrigel significantly decreased. By real-time PCR, the expression of VEGF-C and VEGFR-2 was down-regulated to 44.7% and 25.6% of the negative control.

CONCLUSIONOverexpressed Mer tyrosine kinase receptor can inhibit the migration and angiogenesis of HMEC-1 cells through VEGF-C/VEGFR-2 signal pathway.

Cell Line ; Cell Movement ; Cell Proliferation ; Endothelial Cells ; metabolism ; physiology ; Endothelium, Vascular ; cytology ; Humans ; Neovascularization, Physiologic ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Transfection ; Vascular Endothelial Growth Factors ; metabolism ; c-Mer Tyrosine Kinase

OBJECTIVETo develop a monoclonal antibody (mAb) directed to FVIII C2 domain and investigate its effect on FVIII activity.

METHODSFVIII C2 protein was expressed in E. coli and purified. A murine antihuman FVIII C2 domain mAb SZ-132 was developed by standard hybridoma technology and characterized. In coagulation assays, different concentrations of SZ-132 were incubated with freshly collected pooled human plasma and the residual activity of FVIII and activated partial thromboplastin time (APTT) were determined. The effects of SZ-132 on rhFVIII binding to purified human vWF, phosphatidylserine (PS) and platelets were assessed by enzyme linked immunosorbent assays (ELISA).

RESULTSSZ-132 could inhibit FVIII procoagulant activity in a dose-dependent manner within the concentrations of 0-25 microg/ml and the FVIII activity was completely inhibited on above 25 microg/ml. It could also prevent rhFVIII from binding to vWF, PS and platelets.

CONCLUSIONSSZ-132 is a neutralizing mAb against FVIII C2 domain and can inhibit FVIII procoagulant activity by preventing FVIII from binding to vWF and PS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Factor VIII ; immunology ; metabolism ; Humans ; Male ; Mice ; Mice, Inbred BALB C

The von Willebrand factor-cleaving protease (vWF-cp) is a newly identified plasma metalloproteinase and plays an important role in the pathogenesis of thrombotic microangiopathy. In the present study, the metalloproteinase domain of vWF-cp was expressed by IPTG-induced the recombinant engineered E.coli strain harbouring pET28a (+)-vWF-cp/MD and purified using chromatography on Ni-NTA column. Then the BALB/c mice were immunized with the recombinant protein to prepare the monoclonal antibodies (McAb) against vWF-cp and the obtained McAbs were characterized. Furthermore, the expression panels of vWF-cp in human normal tissues were investigated using immunohistochemistry. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 28% of total bacteria protein. Three monoclonal antibodies against the metalloproteinase domain of vWF-cp were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belong to IgG(1). The concentration of them in ascites was 4 mg/ml, and their titers were as high as 1 x 10(-5). The data of ELISA showed that SZ-112 and SZ-113 recognized different epitopes of recombinant protein. The Western blot and immunoprecipitation data showed that the two monoclonal antibodies reacted not only with the recombinant protein, but also with a 200 kD protein in platelet lysate. Moreover, the vWF-cp was found to be present in the cytoplasm of many human tissues such as liver, prostate, ovary, etc. However, the protease could not be detected in brain tissue. In conclusion, the above-mentioned research work contributed not only to the further study of the structure and function of this protease, but also to the establishment of the method for quantifying the vWF-cp antigen in plasma.
ADAM Proteins ; biosynthesis ; genetics ; immunology ; ADAMTS13 Protein ; Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Binding Sites ; genetics ; Blotting, Western ; Epitopes ; immunology ; Escherichia coli ; genetics ; Humans ; Immunohistochemistry ; Immunoprecipitation ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology ; von Willebrand Factor ; biosynthesis ; genetics ; immunology

OBJECTIVETo observe therapeutic effect of acupuncture at Neimadian on pain after operation of four limbs.

METHODSSixty-two patients were randomly divided into an observation group and a control group, 31 cases in each group. The observation group were treated with electroacupuncture at Neimadian for 30 min, and the control group with oral administration of tramadoli hydrochloridum. Changes of pain within 24 hours were observed.

RESULTSThe analgesic effect in the observation group was better than that in the control group (P < 0.05).

CONCLUSIONAnalgesic effect of acupuncture at Neimadian on pain after operation of four limbs is superior to that of oral administration of tramadoli.

Acupuncture Analgesia ; Acupuncture Therapy ; Electroacupuncture ; Humans ; Pain Management ; Postoperative Period

OBJECTIVETo compare the effects of protein kinase C (PKC) and calcium (Ca2+) on platelet aggregation and platelet membrane surface GP I b expression in thrombin receptors activation, and investigate the role of Gq signal transduction pathway in such activation.

METHODSPeptide SFLLRN (PARI-AP) and AYPGKF (PAR4-AP) were used to stimulate platelet, and the effects of Ro-31-2220 (inhibitor of PKC) and BAPTA/AM (calcium chelator) on the platelet aggregation and GP I b were analyzed.

RESULTSEither 25 micromol/L PAR1 or 250 micromol/L PAR4 peptide could induce absolute platelet aggregation with a reversible internalization of GP I b. Platelet aggregation was inhibited by Ro-31-2220 or BAPTA while the morphological change curve still occurred upon PARs activation. In addition, Ro-31-2220 decreased GP I b centralization upon PAR1 stimulation [(87.00 +/- 0.04)% and (73.00 +/- 0.08)%, respectively at 1, 2 min, P<0.05], albeit it blocked the internalization of GP I b in PAR4 activation [(44.00 +/- 0.01)% and (46.00 +/- 0.05)%, respectively at 10, 30 min, P <0.05]. Meanwhile, GP I b internalization was blocked by BAPTA in both peptides [(94.00 +/- 0.08)% and (95.00 +/- 0.00)% at 1 min, (92.00 +/- 0.02)% and (94.00 +/- 0.01)% at 2 min, (91.00 +/- 0.02)% and (91.00 +/- 0.02)% at 5 min, (90.00 +/- 0.04)% and (87.00 +/- 0.03)% at 10 min, respectively, P <0.05].

CONCLUSIONPKC and calcium play an important role in thrombin receptor activation. Calcium is closely correlated with such activation, being similar in the two PARs signal pathways. PK C promotes GP I b centralization in PAR1 pathway and accelerates GP I b return to membrane surface in PAR4 pathway.

Adult ; Blood Platelets ; metabolism ; Calcium ; metabolism ; physiology ; Female ; Humans ; Male ; Platelet Aggregation ; physiology ; Platelet Glycoprotein GPIb-IX Complex ; metabolism ; Protein Kinase C ; metabolism ; physiology ; Receptors, Thrombin ; metabolism ; Signal Transduction

OBJECTIVETo investigate the effect of thalidomide on Annexin II (AnxA2) gene regulation in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide.

METHODSRPMI8226 and HMEC-1 cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry (FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide.

RESULTSAnxA2 mRNA level in RPMI8226 cells treated with thalidomide at 12.5 microg/ml, 25.0 microg/ml and 50.0 microg/ml was decreased compared with the control group (0.60+/-0.15, 0.33+/-0.14, 0.42+/-0.16, vs 1.07+/-0.16, respectively, P<0.05) and did so in HMEC-1 cells (0.21+/-0.20, 0.08+/-0.08, 0.17+/-0.16 vs 1.16+/-0.24, respectively, P<0.05). The AnxA2 protein level in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39+/-0.32, 2.82+/-0.28, 3.21+/-0.23 vs 5.53+/-0.32, respectively, P<0.05) and that did so in HMEC-1 cells (0.72+/-0.11, 0.64+/-0.08, 0.67+/-0.08 vs 1.40+/-0.15, respectively, P<0.05).

CONCLUSIONSThalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalidomide in multiple myeloma patients.

Annexin A2 ; genetics ; metabolism ; Cell Line ; Endothelial Cells ; metabolism ; Endothelium, Vascular ; cytology ; Humans ; Multiple Myeloma ; metabolism ; pathology ; RNA, Messenger ; genetics ; Thalidomide ; pharmacology

OBJECTIVETo study the fibrinolytic activity in patients with acute promyelocytic leukemia (APL) and its alteration in all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) treatment.

METHODSPlasma fibrinogen concentration was determined with the conventional method, and the levels of fibrin degradation products (FDP) and D-dimer were quantified with ELISA. Plasminogen was measured by chromogenic assay. Cell surface expression of Annexin II and u-PAR and their mRNA levels were measured by flow cytometry and real time-PCR, respectively.

RESULTSThe levels of FDP and D-dimer in APL were remarkably higher in APL patients than that in normal controls, while fibrinogen and plasminogen were lower. Both Annexin II and u-PAR were highly expressed on APL cells, which declined after treatment with ATRA and/or ATO, but remained higher than those on normal bone marrow mononuclear cells.

CONCLUSIONAbnormally high levels of Annexin II and u-PAR expression on APL cells may contribute to the increased production of plasmin, leading to primary hyperfibrinolysis in APL. ATRA and ATO therapy induces down-regulation of Annexin II and u-PAR expression, which may be contribute, at least in part, to the relief of the hemorrhagic complications in APL.

Adolescent ; Adult ; Aged ; Annexin A2 ; analysis ; Arsenicals ; therapeutic use ; Female ; Fibrinolysis ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; physiopathology ; Male ; Middle Aged ; Oxides ; therapeutic use ; RNA, Messenger ; genetics ; Tretinoin ; therapeutic use ; Urokinase-Type Plasminogen Activator ; analysis ; Young Adult

This study was purposed to investigate the expression of annexin II in patients with hematologic malignancies and its role in genesis and development of hematologic malignancies. The expression levels of annexin II in bone marrow cells from untreated 81 patients with acute leukemia, 6 patients with MM and 20 patients with iron deficiency anemia were detected by real-time PCR assay. The results showed that the expression of annexin II mRNA significantly increased in M(3) patients as compared with others, the expression of annexin II gene in groups M(5), MM, M(4) were higher than that of other groups except M(3) group, and there were no significant difference in expression of annexin II gene between M(1) + M(2) groups and controls. It is concluded that the expression of annexin II gene significantly increased in patients M(5), M(4), MM, who showed higher ratio of infiltration than other patients. It is inferred that the annexin II participates in invasion and infiltration of hematologic malignancies probably through enhancing the degradation of extracellular matrix by cells of hematologic malignancies.
Adolescent ; Adult ; Annexin A2 ; genetics ; Bone Marrow Cells ; metabolism ; Female ; Hematologic Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Male ; Middle Aged ; Young Adult