Hirudin (HV) is known as the most potent and specific inhibitor of thrombin. Although hirudin has many advantages , it has the bleeding side effect and this is the great shortage of hiudin for clinical application. In order to alleviate bleeding side effect of hirudin, fusion protein, named as FHV (fusion hirudin linked with FXa recognition peptide) was designed. The fusion protein gene ( fhv) was cloned into plasmid pPIC9K. FHV engineered Pichia pastoris containing high copies was chosen for fermentation and purification at 30 L fermentor scale, finally, FHV with purity of above 97% was obtained. To investigate the function of FHV in vivo, mouse tail thrombosis model was used. In the mice thrombus tail model induced by carrageenan, FHV decreased the length of tail thrombus significantly, similar to that of HV control, and had no obvious effects on the TT, PT and APTT. In conclusion, FHV is constructed and expressed in yeast. FHV fusion proteins is obtained by fermentation and purification. FHV has antithrombotic effects not influencing IT, PT and APTT after administration immediately in animal models. Therefore, FHV is a promising anticoagulant and antithrombotic drug.

OBJECTIVETo find out the regular pattern of pain reaction by pressing along the Governor Vessel at the levels of T3 to L4 on the back in patients with digestive system disease, so as to provide references for diagnosis of the disease.

METHODSThirty patients diagnosed with digestive system disease by gastroscopy and colonoscopy as well as 16 patients with digestive symptoms without accurate diagnosis by endoscopy checks were observed. Pressing was applied from Zhiyang (GV 9) to Yaoyangguan (GV 3), including non-acupoints along the Governor Vessel. Positive reaction was recognized since pain, soreness or distension sensation appeared in the space between the spinous process.

RESULTSThe positive ratios of 30 patients with endoscopy checks and 16 without the check accounted for 100.0%. The tender spots, usually 5 to 9 in number, were mainly focused on the relative acupoints and non-acupoints along the Governor Vessel from T6 to T10.

CONCLUSIONThe pain reaction from Lingtai (GV 10) to Zhongshu (GV 7), which are at the level of T6 to T10, along the Governor Vessel is closely related with digestive system disease. And it can be regarded as function criterion of the digestive system and important reference of auxiliary diagnosis.

Acupuncture Points ; Adult ; Aged ; Back ; physiopathology ; Diagnosis, Differential ; Digestive System Diseases ; diagnosis ; physiopathology ; Female ; Humans ; Male ; Meridians ; Middle Aged ; Pain ; Pain Measurement ; Sensation

OBJECTIVESTo detect the levels of reactive oxygen species (ROS), superoxide dismutase(SOD) and interleukin 8(IL-8) in seminal plasma of infertile patients, and evaluate the possible relationship between those levels.

METHODSSemen was collected from normal donors (15 cases), infertile men without infection (16 cases), and infertile men with infection (leukocytospermia, 11 cases). The routine analysis of semen was accomplished, and then the levels of IL-8, malondialdehyde (MDA), SOD, and white blood cell (WBC) were examined. The correlative analysis between the level of ROS and other parameters in these populations was made.

RESULTSIn leukocytospermic group, the levels of MDA, WBC, and IL-8 were higher than those in the other two groups (P < 0.001). Significantly positive correlation was observed between IL-8 and MDA (r = 0.852, P < 0.001) and between the levels of IL-8 and WBC.

CONCLUSIONSThese findings suggest that increased oxidative stress in patients with leukocytospermia may cause the increase of IL-8(r = 0.818, P < 0.01). The increased oxidative stress may be due to defect in ROS scavenging system.

Adult ; Cytokines ; blood ; Humans ; Infertility, Male ; blood ; complications ; Male ; Male Urogenital Diseases ; blood ; complications ; Reactive Oxygen Species ; blood ; Semen

OBJECTIVETo summarize the experience in one-stage urethroplasty with pedicled scrotal skin flap for hypospadias, and improve its therapeutic effect.

METHODSWe retrospectively analyzed the clinical data of 310 cases of hypospadias (except coronal hypospadias) treated by one-stage urethroplasty with pedicled scrotal skin flap. All the patients were followed up for 6 to 24 months.

RESULTSNo postoperative complications were observed except urinary fistula, which occurred in 12.6% of the patients. Postoperative fistula formation was associated with the type of hypospadias, the length of the urethral defect and postoperative comprehensive medication, but not with the stent indwelling time after surgery. Most of the fistulae were located at the base of the penis.

CONCLUSIONOne-stage urethroplasty with pedicled scrotal skin flap is a simple and effective option for all types of hypospadias except the coronal type, and postoperative treatment is very important.

Adolescent ; Adult ; Child ; Child, Preschool ; Humans ; Hypospadias ; surgery ; Infant ; Male ; Reconstructive Surgical Procedures ; methods ; Retrospective Studies ; Scrotum ; surgery ; Skin Transplantation ; Surgical Flaps ; Urethra ; surgery ; Young Adult

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.
Animals ; Cloning, Molecular ; Electroporation ; Hirudins ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Plasminogen Activator ; biosynthesis ; genetics

OBJECTIVE: To determine the relationship between intravoxel incoherent motion (IVIM) imaging derived quantitative metrics and serum soluble CD40 ligand (sCD40L) level in an embolic canine stroke model. MATERIALS AND METHODS: A middle cerebral artery occlusion model was established in 24 beagle dogs. Experimental dogs were divided into low- and high-sCD40L group according to serum sCD40L level at 4.5 hours after establishing the model. IVIM imaging was scanned at 4.5 hours after model establishment using 10 b values ranging from 0 to 900 s/mm². Quantitative metrics diffusion coefficient (D), pseudodiffusion coefficient (D*), and perfusion fraction (f) of ischemic lesions were calculated. Quantitative metrics of ischemic lesions were normalized by contralateral hemisphere using the following formula: normalized D = D(stroke) / D(contralateral). Differences in IVIM metrics between the low- and high-sCD40L groups were compared using t test. Pearson's correlation analyses were performed to determine the relationship between IVIM metrics and serum sCD40L level. RESULTS: The high-sCD40L group showed significantly lower f and normalized f values than the low-sCD40L group (f, p < 0.001; normalized f, p < 0.001). There was no significant difference in D*, normalized D*, D, or normalized D value between the two groups (All p > 0.05). Both f and normalized f values were negatively correlated with serum sCD40L level (f, r = −0.789, p < 0.001; normalized f, r = −0.823, p < 0.001). However, serum sCD40L level had no significant correlation with D*, normalized D*, D, or normalized D (All p > 0.05). CONCLUSION: The f value derived from IVIM imaging was negatively correlated with serum sCD40L level. f value might serve as a potential imaging biomarker to assess the formation of microvascular thrombosis in hyperacute period of ischemic stroke.
Animals ; CD40 Ligand* ; Diffusion ; Dogs ; Infarction, Middle Cerebral Artery ; Magnetic Resonance Imaging* ; Perfusion ; Stroke* ; Thrombosis

AIMTo investigate the mechanism of protective effect of SOD (superoxide dismutase) on damage of RBCs stored at 4 degrees C, the studies of erythrocyte glucose and energy metabolism were performed.

METHODSwhole blood collected from healthy donors and stored at 4 degrees C in ACD, GMA and SOD solutions. Before and post storage, some parameters were assayed. Standard methods were used for the in vitro tests. The 24-hour in vivo recoveries were measured by FTTC (Fluorescein 5-isothiocyanate) from SIGMA Company.

RESULTSAll parameters of red blood cell glucolysis rate without oxygen condition, ATP, PK (pyruvic kinase) and 24 h recoveries level were 86.2%, 56.4%, 64.3% and 86.2% of normal respectively stored in SOD solution at 4 degrees C for 75 days, distinctly more than in ACD and GMA groups at 75 days stored. The 24 h recovery at 75d in group SOD was near the recovery at 42d in group GMA.

CONCLUSIONWhole blood in SOD solution can be stored satisfactorily for 75 days at 4 degrees C, and furnished theoretical evidence for RBCs survival.

Animals ; Blood Preservation ; methods ; Erythrocytes ; cytology ; metabolism ; Humans ; Rabbits ; Refrigeration ; Superoxide Dismutase ; pharmacology

OBJECTIVE: To study the efficacy and safety of caffeine used in the early (≤72 hours after birth) and late (>72 hours after birth) stage in preterm infants with a gestational age of ≤31 weeks. METHODS: A retrospective analysis was performed for 640 preterm infants (with a gestational age of ≤31 weeks) who were admitted to the neonatal intensive care unit of eight hospitals in Jiangsu Province, China. Of the 640 preterm infants, 510 were given caffeine in the early stage (≤72 hours after birth; early use group) and 130 were given caffeine in the late stage (>72 hours after birth; late use group). The clinical data were compared between the two groups. RESULTS: There were no significant differences in birth weight, Apgar score, sex, gestational age, and age on admission between the two groups (P>0.05). Compared with the late use group, the early use group had a significantly younger age at the beginning and withdrawal of caffeine treatment (P<0.05) and a significantly shorter duration of caffeine treatment (P<0.05). There was no significant difference in respiratory support on admission between the two groups (P>0.05). Compared with the late use group, the early use group had significantly lower incidence rate of apnea (P<0.05) and significantly shorter oxygen supply time and length of hospital stay (P<0.05). There were no significant differences between the two groups in the incidence rates of neonatal intracranial hemorrhage, periventricular leukomalacia, necrotizing enterocolitis, retinopathy of prematurity, and patent ductus arteriosus at discharge and NBNA score at the corrected gestational age of 40 weeks (P>0.05). However, significant differences were found in the incidence of bronchopulmonary dysplasia and the rate of home oxygen therapy, but there was no significant difference in the mortality rate between the two groups (P>0.05). CONCLUSIONS Early use of caffeine can shorten the duration of caffeine treatment, oxygen supply time, and length of hospital stay, with little adverse effect, in preterm infants with a gestational age of ≤31 weeks.

OBJECTIVETo evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study.

METHODSGroups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization.

RESULTSThe immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively.

CONCLUSIONBALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.

Animals ; Antibodies, Bacterial ; blood ; Dose-Response Relationship, Immunologic ; Female ; Guinea Pigs ; Immunization ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plague ; prevention & control ; Plague Vaccine ; immunology ; Rabbits ; Vaccines, Subunit ; immunology

OBJECTIVETo evaluate the efficacy and safety of long pulse 1064 nm Nd:YAG laser therapy in the treatment of onychomycosis of the toenails.

METHODSA total of 104 patients with onychomycosis (461 toenails) were divided by age into ≥60 years group and <60 years group, and each group was further divided into subgroups according to Scoring Clinical Index of Onychomycosis (SCIO) scoring and the location of the compromised toenails. All the toenails were treated with 10 to12 sessions of long pulse 1064 nm Nd:YAG laser therapy at the interval of 1 week. All the patients were followed up for 48 weeks after the initial treatment to assess the clinical efficacy and adverse reactions.

RESULTSThe overall clinical response rate in these patients was 72.5% by the end of the 48-week follow-up. In patients aged <60 years, the clinical response rate and mycological cure rate were significantly higher than the rates in patients aged ≥60 years (P<0.05). No significant differences were observed in the response rates between different SCIO subgroups (P>0.05); the 2nd to 4th toenails showed better outcomes after the therapy than the 1st and 5th toenails (P<0.05). No adverse reactions related with the therapy were recorded in these patients.

CONCLUSIONLong pulse 1064 nm Nd:YAG laser is an effective and safe approach for treatment of onychomycosis of the toenails.

Humans ; Lasers, Solid-State ; Middle Aged ; Nails ; microbiology ; Onychomycosis ; therapy ; Treatment Outcome