Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindII, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.

OBJECTIVETo compare the sensitivity and practicability of modified Bethesda assay and Nijmegen assay in detecting factor VIII (FVIII) inhibitor.

METHODSModified Bethesda assay and Nijmegen assay were used to screen FVIII inhibitors in 237 patients with hemophilia A. The buffer plus universal coagulation reference plasma (UCRP) was used to establish a standard curve for FVIII: C assay in modified Bethesda method, instead of Nijmegen plasma plus FVIII deficiency plasma in Nijmegen method. The cutoff value for positive FVIII inhibitors is > or = 0.6 BU/ml.

RESULTSThe positive rate of FVIII inhibitors was 5.5% (n = 13) when using modified Bethesda assay and was 8.4% (n = 20) when using Nijmegen assay (P > 0.05).

CONCLUSIONModified standard Bethesda assay is a convenient and feasible method for detecting FVIII inhibitors.

Adolescent ; Adult ; Autoantibodies ; blood ; Blood Coagulation Tests ; methods ; Child ; Child, Preschool ; Factor VIII ; immunology ; Female ; Hemophilia A ; blood ; immunology ; Humans ; Male ; Middle Aged ; Sensitivity and Specificity ; Young Adult

Objective To identify risk factors for a human orf disease outbreak in a village in Chongqing city. Methods Standardized case-definition was set and a case-finding program was conducted among all the residents of the village. All the patients were interviewed using a standardized questionnaire and collected fluids in the skin rash for laboratory testing. A retrospective cohort investigation was conducted among all the village residents who introduced the black goats to analyze the risk of orf infection, in relation to the mode and frequency of contacts to the infected goats. Results We found 18 cases (including 16 suspected cases and 2 confirmed cases) among the members of 10 families that introduced the black goats. Village residents who had ever used their legs to grip the goats were nearly five times as likely to develop orf disease as those who did not (RR=4.98, 95％CI: 1.34-75.27). Village residents who had ever washed and wiped the goats were three times as likely to develop orf disease as those who had not (RR = 3.09,95％CI: 0.98-45.38). The frequency of contacts with the infected goats was associated with the risk of developing orf disease in a dose-response fashion (x2 test for trends: P= 0.006).Frequently wearing long trousers when dealing with the goats appeared as a protective factor (RR=0.30,95％CI: 0.15-0.78). Conclusion This outbreak was caused by the introduced black goats which carried and infected by the orf virus. Direct physical contact with infected goats but without wearing protective clothing were risk factors for the development of human orf disease.

OBJECTIVETo evaluate the discrimination of serum procalcitonin (PCT) and interleukin-6 (IL-6) between patients with sepsis and non-infectious inflammatory response syndrome (SIRS) and the prediction power of clinical outcome.

METHODSA perspective study was performed in 27 patients with sepsis and 30 patients with non-infectious SIRS. The serum concentrations of PCT, IL-6, C-reactive protein (CRP), white blood cell count, percentage of neutrophil, absolute neutrophil count, and maximal body temperature were obtained less than 24 hours after clinical onset of sepsis or SIRS.

RESULTSThe serum levels of PCT and IL-6 and percentage of neutrophil were significantly higher in patients with sepsis than in those with non-infectious SIRS (PCT: 5.54 [1.20, 32.74] microg/L vs 0.77 [0.22, 3.90] microg/L, P=0.001; IL-6: 163.66 [33.60, 505.26] ng/L vs 37.72 [22.52, 110.78] ng/L, P=0.004; CRP [15.28 +/- 8.41] g/L vs [9.51 +/- 7.65] g/L, P=0.010; and percentage of neutrophil: 0.91 +/- 0.04 vs 0.88 +/- 0.04, P=0.010). Receiver operating characteristic curve showed that the power of PCT and IL-6 were the best of all above. There was significant correlation between serum PCT or IL-6 and the acute physiology and chronic health evaluation (APACHE II) or sepsis-related organ failure assessment (SOFA) scores, so was between serum PCT and the intensive care unit (ICU) length of stay.

CONCLUSIONSPCT and IL-6 are more reliable indicators to differentiate sepsis and non-infectious SIRS than the conventional inflammatory markers, and correlate with the disease severity. PCT levels are significantly correlated with ICU length of stay.

APACHE ; Adult ; Aged ; C-Reactive Protein ; metabolism ; Calcitonin ; blood ; Calcitonin Gene-Related Peptide ; Diagnosis, Differential ; Female ; Humans ; Interleukin-6 ; blood ; Length of Stay ; Male ; Middle Aged ; Prospective Studies ; Protein Precursors ; blood ; Sepsis ; blood ; diagnosis ; physiopathology ; Systemic Inflammatory Response Syndrome ; blood ; diagnosis ; physiopathology

OBJECTIVETo analyze the clinical characteristics and effects of rehabilitation treatment on hemophiliacs with iliopsoas hemorrage.

METHODSThe hemophilia patients with iliopsoas bleeding treated in Peking Union Medical College Hospital between January 2006 to December 2010 were enrolled. The clinical characteristics including symptoms, signs, complications, and rehabilitation treatment were analyzed retrospectively.

RESULTSAll of the forty-one hemophiliacs with iliopsoas bleeding were male, 20 cases wee the left bleeding, 18 the right, and 3 the bilateral. The median median age was 18 (6 - 61) years old(y). The median age of the iliopsoas bleeding for the first time was 17 (6 - 20) y. 34 patients accompanied with femoral nerve injury, 19 of them had secondary knee bleeding on the same side. 20 patents had quadriceps atrophy. Pelvic pseudotumor developed in 2 patients and permanent abnormal posture in 2 patients. The main finding of the ultrasound image was low-echo mass in iliopsoas muscles or inguinal region. 34 patients received rehabilitation therapy for 8 - 12 weeks under the support of factor replacement, complete hematomas absorption in 33 of them, with hip range of motion recovering back to baseline. 27 of 32 (84.4%) cases with femoral nerve injury got quadriceps strength above 4/5 grade, 20 cases of femoral nerve injury (62.5%) still had numbness on front of their thigh after treatment.

CONCLUSIONSIn this cohort of iliopsoas bleeding, most of the patients are adolescent. High prevalence of the femoral nerve injury and the secondary knee bleeding are found. Rehabilitation treatment under the support of factor replacement is safe and effective on hematoma absorption and neurological function recovery.

Adolescent ; Adult ; Child ; Hematoma ; etiology ; rehabilitation ; Hemophilia A ; complications ; rehabilitation ; Hemorrhage ; etiology ; rehabilitation ; Humans ; Male ; Middle Aged ; Muscular Diseases ; diagnosis ; etiology ; rehabilitation ; Psoas Muscles ; physiopathology ; Retrospective Studies ; Young Adult

The purpose of this study is to investigate the Sal I, Nru I and Mse I restriction fragment length polymorphisms (RFLPs) of factor IX gene in Chinese Han people. The frequencies of FIX-192 and FIX-793 for A and G, and FIX-698 for T and C were analyzed by polymerase chain reaction (PCR) in unrelated normal Chinese Han people. A sample of 214, 210 and 206 unrelated X chromosomes were analyzed for FIX-192 and FIX-793 and FIX-698, respectively. The results showed that the frequencies for FIX-192 were 0.878 for A and 0.122 for G, with a heterozygosity rate of 0.213, and the frequencies for FIX-793 were 0.552 for A and 0.448 for G, with a heterozygosity rate of 0.494, the frequencies for FIX-698 were 0.311 for T and 0.689 for C, with a heterozygosity rate of 0.429. It was concluded that the SalIand NruI and MseI RFLPs of FIX gene may be useful markers for carrier detection and prenatal diagnosis in Chinese families with hemophilia B patients.
China ; DNA ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Factor IX ; genetics ; Female ; Gene Frequency ; Heterozygote ; Humans ; Male ; Polymorphism, Restriction Fragment Length

Biomarkers ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cysteine Proteinase Inhibitors ; biosynthesis ; genetics ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Ki-67 Antigen ; analysis ; Liver Neoplasms ; metabolism ; pathology ; Male ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo verify the clinical efficacy of acupuncture on motor dysfunction in ischemic stroke of subacute stage.

METHODSThe multi-central randomized controlled trial was adopted. One hundred and twenty-six cases of ischemic stroke of subacute stage were randomized into an acupuncture group (61 cases) and a conventional treatment group (65 cases). The basic treatment of western internal medicine and rehabilitation training were applied to the patients of the two groups. In the acupuncture group, acupuncture was supplemented at the body points located on the extensor of the upper limbs and the flexor of the lower limbs. In combination, scalp acupuncture was applied to NS5, MS6 and MS6 on the affected side. The treatment was given 5 times a week and totally 8 weeks were required. The follow-up observation lasted for 3 months. The scores in Fugl-Meyer scale and NIHSS scale and Barthel index were compared between the two groups before treatment, in 4 and 8 weeks of treatment and the 3-month follow-up observation after treatment separately.

RESULTSIn 4 and 8 weeks of treatment and the follow-up observation, Fugl-Meyer scale score was improved obviously in the patients of the two groups (all P<0. 01). In 8 weeks of treatment and the follow-up observation, Fugl-Meyer scale score in the acupuncture groupwas im proved much apparently as compared with that in the conventional treatment group [68. 0 (43. 0,86. 5) vs 52. 5 (30.3, 77.0), 77.0 (49.5, 89.0) vs 63. 0 (33.0, 84.0), both P<0. 05]. Except that NIHSS scale score was not reduced apparently in 4 weeks of treatment in the conventional treatment group (P>0.05), the results of NIHSS scale at the other time points were all decreased obviously as compared with those before treatment in the patients of the two groups (all P<0. 01). In 8 weeks of treatment and the follow-up observation, the results in the acupuncture group were reduced much apparently as compared with those in the conventional treatment group [5. 0 (3.0,8.0) vs 7. 0 (3.0,13.8), 4. 0 (1.5,7.0) vs 6.0 (2.0,11.7) ,both P<0. 05]. In 8 weeks of treatment and the follow-up observation, Barthel index was improved obviously as compared with that before treatment in the patients of the two groups (all P<0. 05). The improvement in the acupuncture group was much more significant as compared with the conventional treatment group [75. 0 (60. 0,87. 5) vs 65. O (36. 3, 87. 5), P<0. 051.

CONCLUSIONBased on the conventional treatment, Acupuncture achieves the satisfactory clinical efficacy on motor dysfunction in ischemic stroke of subacute stage.

Acupuncture Therapy ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Motor Activity ; Stroke ; physiopathology ; therapy ; Treatment Outcome

OBJECTIVETo examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L-OVHM) injected into the spleen on liver metastasis in mice.

METHODSOVHM cells were inoculated into the spleen of 6 to 8 week-old female B6C3F1 (C57BL/6N x C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVHM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-gamma, TNF-alpha, IL-12, IL-4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded.

RESULTSThe mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rate in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-gamma, TNF-alpha and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (p < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05).

CONCLUSIONThe data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Th1 cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.

Animals ; CD11c Antigen ; metabolism ; CD40 Ligand ; genetics ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Liver Neoplasms ; pathology ; secondary ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Ovarian Neoplasms ; metabolism ; pathology ; Spleen ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVEScreening the intron 1 inversion of factor VIII (FVIII) in the population of severe haemophilia A(HA) in China and performing carrier detection and prenatal diagnosis.

METHODSUsing LD-PCR to detect intron 22 inversions and multiple-PCR within two tubes to intron 1 inversions in severe HA patients. Carrier detection and prenatal diagnosis were performed in affected families. Linkage analysis and DNA sequencing were used to verify these tests.

RESULTSOne hundred and eighteen patients were seven diagnosed as intron 22 inversions and 7 were intron 1 inversions out of 247 severe HA patients. The prevalence of the intron 1 inversion in Chinese severe haemophilia A patients was 2.8% (7/247). Six women from family A and 2 from family B were diagnosed as carriers. One fetus from family A was affected fetus.

CONCLUSIONIntron 1 inversion could be detected directly by multiple-PCR within two tubes. This method made the strategy more perfective in carrier and prenatal diagnosis of haemophilia A.

Adult ; Chromosome Inversion ; genetics ; DNA Mutational Analysis ; Factor VIII ; genetics ; Female ; Hemophilia A ; diagnosis ; genetics ; Humans ; Introns ; genetics ; Male ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis ; methods