AIMTo study the pharmacokinetic (PK) properties in rabbits treated with N-Ile(1)-Thr(2)-63-desulfato-r-hirudin (rH) newly developed in China by means of bioassay in order to provide preclinical experiment basis for its development as a novel anticoagulant agent.

METHODSrH plasma concentration was determined using bioassay based on ex vivo antithrombin activity of rH. Normal rabbits received iv rH 4.0, 2.0 and 1.0 mg/kg or sc rH 2.0 mg/kg, respectively. The rabbits with acute severe renal failure were given iv rH 2.0 mg/kg.

RESULTSThe bioassay described in this paper met requirements for study of PK in rabbits. The major PK parameters after iv dosing were as follows: t(1/2beta) 58.4-59 min. V(d) 0.09-0.12 L/kg, CL 0.0035-0.0040 L/(kg.min); AUC were proportional to the doses, t(1/2) and CL did not change significantly with the doses. The sc bioavailability reached 94%. The rabbits suffering from acute severe renal failure presented 11-fold longer t(1/2beta) and 13-fold greater AUC than normal healthy rabbits.

CONCLUSIONrH exhibited rapid elimination, distribution was only limited to extracellular space and good absorption from sc site. The excretion of rH by kidneys played a very important role in the elimination of rH. The PK of rH could be described by the two- and one-compartment model after iv and sc dosing, respectively, and followed linear kinetics.

Algorithms ; Animals ; Biological Assay ; methods ; Computer Simulation ; Hirudins ; blood ; pharmacokinetics ; Metabolic Clearance Rate ; Models, Biological ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity ; Thrombin Time ; methods

BACKGROUND/AIMS: Upregulated CD64 expression on neutrophils is the most useful marker for acute bacterial infections and systemic inflammation. However, it is unknown whether CD64 is involved in the pathogenesis of acute pancreatitis (AP). This study was designed to determine whether CD64 is implicated in severe acute pancreatitis (SAP), and thus, is a suitable marker for SAP. METHODS: SAP was induced in rats with an intraperitoneal injection of L-arginine. CD64 expression in the rat pancreas was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Additionally, the CD64 mRNA expression in peripheral blood leukocytes from 21 patients with mild acute pancreatitis (MAP) and 10 patients with SAP was investigated at the time of admission and during remission by qRT-PCR. RESULTS: CD64 mRNA and protein expression in the pancreas was significantly higher in rats with SAP, compared to the controls. The CD64 expression was higher in the patients with SAP than in the patients with MAP. During remission, CD64 mRNA decreased in both the MAP and SAP patients. The area under the curve of CD64 expression for the detection of SAP was superior to both the Ranson and the Acute Physiology and Chronic Health Evaluation II scores. CONCLUSIONS: The CD64 level was significantly increased in correlation with the disease severity in SAP and may act as a useful marker for predicting the development of SAP.
Acute Disease ; Adolescent ; Adult ; Aged ; Animals ; Arginine/toxicity ; Female ; History, Ancient ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Pancreatitis/*metabolism ; RNA, Messenger/metabolism ; Rats, Sprague-Dawley ; Receptors, IgG/*metabolism ; Up-Regulation ; Young Adult

OBJECTIVETo investigate the potential and early effect of hypertonic saline resuscitation on T-lymphocyte subpopulations in rats with hemorrhagic shock.

METHODSA model of rat with severe hemorrhagic shock was established in 18 Sprague-Dawley (SD) rats. The rats were randomly divided into Sham group, HTS group (hypertonic saline resuscitation group) and NS group (normal saline resuscitation group). Each group contained 6 rats. The CD4(+) and CD8(+) subpopulations of T-lymphocytes in peripheral blood were detected respectively before shock and after resuscitation by double antibody labelling and flow cytometry.

RESULTSIn the early stage after hemorrhagic shock, fluid resuscitation and emergency treatment, the CD4(+) lymphocytes of peripheral blood in HTS and NS groups markedly increased. Small volume resuscitation with HTS also induced peripheral CD8(+) lymphocytes to a certain extent, whereas NS resuscitation showed no effect in this respect. Consequently, compared with Sham and HTS groups, CD4(+)/CD8(+) ratio of peripheral blood in NS group was obviously increased, and showed statistically differences.

CONCLUSIONIn this model of rat with severe hemorrhagic shock, small volume resuscitation with HTS is more effective than NS in reducing immunologic disorders and promoting a more balanced profile of T-lymphocyte subpopulations regulating network.

Animals ; Blood Pressure ; CD4-CD8 Ratio ; Disease Models, Animal ; Isotonic Solutions ; administration & dosage ; Male ; Rats ; Rats, Sprague-Dawley ; Resuscitation ; methods ; Saline Solution, Hypertonic ; administration & dosage ; Shock, Hemorrhagic ; immunology ; physiopathology ; therapy ; T-Lymphocyte Subsets ; immunology

The baculovirus-induced actin polymerization is mainly associated with the virus nucleocapsid protein P78/83, which is homologous with WASP proteins that can activate Arp2/3 complex and induce the actin polymerization. In order to explore the role of Arp2/3 complex in the baculovirus replication, the P40 subunit of Arp2/3 complex from Sf9 (Spodoptera frugiperda 9) cell line was cloned and characterized. Immunofluorescent microscopy assay indicated that P40 was recruited to the inner-side of nuclear membrane during virus infection, which was in accordance with nuclear F-actin distribution in virus-infected cells as documented in our previous research, suggesting P40 could be used to track Arp2/3 complex subcellular distribution changes during virus infection. In addition, co-immunoprecipitation assay demonstrated that P40 interacted with P78/83 only in virus-infected cells, suggesting that actin polymerization induced by P78/83-Arp2/3 complex during baculovirus infection was regulated by some unidentified virus factors.
Actin-Related Protein 2-3 Complex ; chemistry ; genetics ; metabolism ; Amino Acid Sequence ; Animals ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Humans ; Insect Proteins ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Nucleopolyhedrovirus ; genetics ; metabolism ; Phylogeny ; Protein Binding ; Sequence Alignment ; Sf9 Cells ; Spodoptera ; chemistry ; genetics ; metabolism ; virology

We proposed a new stitching method based on sift features to obtain an enlarged view of transmission electron microscopic (TEM) images with a high resolution. The sift features were extracted from the images, which were then combined with fitted polynomial correction field to correct the images, followed by image alignment based on the sift features. The image seams at the junction were finally removed by Poisson image editing to achieve seamless stitching, which was validated on 60 local glomerular TEM images with an image alignment error of 62.5 to 187.5 nm. Compared with 3 other stitching methods, the proposed method could effectively reduce image deformation and avoid artifacts to facilitate renal biopsy pathological diagnosis.
Algorithms ; Artifacts ; Humans ; Image Processing, Computer-Assisted ; methods ; Kidney Glomerulus ; ultrastructure ; Microscopy, Electron, Transmission ; methods

The study was aimed to analyze the characteristics of LRP16 gene promoter and its activity in order to explore the possible regulation mechanism of LRP16 gene expression. A 2.6 kb genomic DNA sequence of LRP16 5'-end was obtained from NCBI by BLAST software. The 7 target sequences between 0.2 - 2.6 kb from a healthy blood donor DNA sample were amplified by PCR, then identified by DNA sequencing and semi-nest PCR. The verified sequences were analyzed on-line. The results showed that the 7 target sequences were about 400 bp different from each other. All 7 sequences were the same to these GenBank described. At last, all 7 promoter sequences were ligated with luciferase vector, and then the luciferase activity was analyzed in HeLa cells. A known gene promoter sequence can be freely obtained from NCBI database. It is concluded that LRP16 promoter is a standard type II promoter and its activity is strongest in the region from -200 to -600 bp.
Base Sequence ; Chromosomes, Human, Pair 11 ; Gene Expression ; Humans ; Luciferases ; metabolism ; Molecular Sequence Data ; Neoplasm Proteins ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA

AIM:To investigate the protective effect of mucin 2(MUC2)on intestinal mucosa of colitis model mice,and to explore the correlation between the expression of anti-CBir1 flagellin antibody and MUC2.METHODS:The mice were randomly divided into normal control group,2,4,6-trinitrobenzenesulfonic acid(TNBS)group,lipopolysaccha-ride(LPS)+ovalalbumin(OVA)+TNBS group and ketotifen+TNBS group.The expression of MUC2 in colon tissue was determined by PAS staining and immunohistochemistry, and the anti-CBir1 antibody level in the serum of mice in each group was measured by ELISA.RESULTS:The scores of disease activity index and histological index in TNBS group were higher than those in normal control group(P<0.05).The scores in LPS +OVA+TNBS group were much higher than those in TNBS group(P<0.05).However, the values in ketotifen +TNBS group were lower than those in TNBS group (P<0.05).PAS staining showed a decrease in goblet cells in TNBS group.Compared with TNBS group,the colonic mu-cosa integrity in LPS+OVA+TNBS group was destroyed, and the number of goblet cells in ketotifen +TNBS group in-creased significantly.Immunohistochemical staining showed that the expression of MUC 2 in the intestinal tract of each mo-del group was basically consistent with the results of PAS staining.The serum anti-CBir1 antibody level in TNBS group was higher than that in normal control group(P<0.05), and that in LPS+OVA+TNBS group was significantly higher than that in TNBS group(P<0.05),whereas that in ketotifen +TNBS group was decreased slightly(P<0.05).CONCLU-SION:MUC2 plays a protective role in the pathogenesis of colitis in mice,and there is a negative correlation between the expression of MUC2 and the bacterial flagellin in the intestinal mucosa of mice with colitis.

AIM:To detect the expression of CBir1 in the serum and colon tissue and mast cell degranulation in the tissue of 2,4,6-trinito-benzene-sulfonic acid ( TNBS)-induced colitis in mice with different interventions. ME-THODS:SPF male BALB/c mice were randomized into 6 groups (12 mice in each group):normal control group, normal saline group, 50% alcohol group, 50% alcohol+TNBS group, 50% alcohol+TNBS+lipopolysaccharide (LPS) +ovalbu-min (OVA) group and 50% alcohol+TNBS+ketotifen group. Corresponding treatment was given to each group, and the disease activity index (DAI) of the mice was evaluated. The mice were sacrificed on day 22 after treatment. The colon tis-sues were evaluated by histological index (HI) scoring. Serum concentrations of anti-CBir1, mast cell tryptase (MCT) and histamine were measured by ELISA. The expression of CBir1, toll-like receptor 5 (TLR5) and MCT in the colon tissues was detected by immunohistochemistry. RESULTS:Compared with the normal control group, the DAI score, HI score and CBir1, anti-CBir1, MCT, TLR5, histamine concentrations in colon tissues and serum were all significantly higher in 50% alcohol+TNBS group, 50% alcohol+TNBS+ketotifen group and 50% alcohol+TNBS+LPS+OVA group (P<0.05). The DAI score, HI score and anti-CBir1, CBir1, MCT, histamine levels in 50% alcohol+TNBS group were lower than those in 50% alcohol+TNBS+LPS+OVA group (P<0.05). The DAI score, HI score and anti-CBir1, TLR5, hista-mine, CBir1 Levels in 50% alcohol+TNBS group were higher than those in 50% alcohol+TNBS+ketotifen group ( P<0.05). Normal saline group and 50% alcohol group had no statistically significant difference in comparison with normal control group. In TNBS model group, serum concentration of anti-CBir1 was positively correlated with MCT concentration (r=0.648, P<0.01) and histamine concentration (r=0.751, P<0.01). CONCLUSION:The heavier degree of in-flammation in TNBS-induced colitis, the higher levels of the CBir1 and the degranulation of mast cells. There is a positive correlation between the expression of CBir1 and the degranulation of mast cells in TNBS-induced colitic mice.

AIM:To observe the effect of interleukin-27 (IL-27) on the pathological changes and the expres-sion and activation of NOD-like receptor protein 3 (NLRP3) inflammasome in the colonic tissues of the mice with dextran sodium sulfate (DSS)-induced experimental colitis. METHODS:Male C57BL/6 mice (n=48) were randomly divided into control group (given unrestricted diet), DSS group (drinking 3% DSS solution), IL-27 (500 ng) group and IL-27 (1 &mu;g) group (intraperitoneal injection of 500 ng and 1 &mu;g IL-27 on the basis of drinking DSS solution, respectively). After treatment for 12 d, intestinal inflammation in the mice was evaluated, the pathological changes of the colonic tissues were observed by HE staining, and the disease activity index ( DAI) score and histological index ( HI) score were calculated. The colonic tissues were collected for immunohistochemistry, qPCR and Western blot detections. The serum was prepared for ELISA. RESULTS:Compared with control group, the DAI score and HI score in model group indicated that the colo-nic inflammation was more obvious (P<0.05), the mRNA expression of NLRP3 and IL-1β was increased, the protein levels of NLRP3 and cleaved caspase-1 were elevated, and the releases of IL-1β and IL-18 in the serum were also increased (P<0.05). Compared with DSS group, the DAI score and HI score in IL-27 (1 &mu;g) group indicated that the colonic in-flammation was obviously attenuated, the mRNA expression of NLRP3 and IL-1β was decreased, the protein levels of NLRP3 and cleaved caspase-1 were suppressed, and the releases of IL-1β 和 IL-18 in the serum were also decreased (P<0.05). No difference of the above indexes between DSS group and IL-27 (500 ng) group was observed except the de-creases in the releases of IL-1β and IL-18 in the serum in IL-27 (500 ng) group. CONCLUSION:IL-27 alleviates the inflammation in DSS-induced colitis mice and inhibits the expression and activation of NLRP3 inflammasome.

Objective To study the infection status and the molecular characteristics of Vibrio parahaemolyticus isolated from diarrheal patients in Shenzhen, in 2007 to 2008 and to provide evidence for the prevention and control of diarrheal diseases caused by Vibrio parahaemolyticus. Methods More than 80 fecal specimens from four sentinel surveillance hospitals were collected and cultured each month. A total of 361 isolates of Vibrio parahaemolyticus were sero-typed and examined by real-time PCR for the presence of two major virulence genes, tdh and trh. Of 361 strains, 60 O3: K6 strains isolated from six suspected outbreaks in August, 2007 and in September, 2008 were typed by pulsed-field gel electrophoresis (PFGE). Results 4384 stool samples were detected in four sentinel surveillance hospitals and with 361 Vibrio parahaemolyticus strains isolated that belonged to 28 serotypes. Serotype O3:K6, O4:K8 and O1:KUT accounted for 67.90%, 7.50% and 6.10%, respectively. Of 361 strains, 337 strains belonged to tdh + trh- , 11 strains were tdh-trh- and 13 strains were tdh + trh +. The most prevalent serotype which caused diarrheal diseases was tdh + trh-in Shenzhen. The 60 isolates were discriminated into twenty different PFGE patterns, which belonged to three clones. Among the 60 isolates, most of the PFGE patterns of isolates from the suspected outbreak locations were identical and some strains isolated from different year were different. Conclusion Vibrio parahaemolyticus isolates in Shenzhen were dominated by O3:K6 strains. Most of these isolates carried tdh gene and few carried trh gene. Meanwhile, the identical patterns of isolates from 6 suspected outbreaks locations demonstrated that Vibrio parahaemolyticus outbreaks occurred in July 2007 and in September 2008 in Shenzhen. However, the dominated strains' PFGE patterns were different each year, indicating that the sources of Vibrio parahaemolyticus had a multiplex nature and the multiplex sources such as water, sea food and pickled products should be integrated monitored. Laboratory based surveillance of diarrheal diseases could contribute in establishing early warning system for the better prevention and control of diarrheal diseases.