Clinical related factors of portal vein tumor thrombosis in patients with hepatocellular carcinoma: a logistic regression analysis / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the related factors of portal vein tumor thrombosis (PVTT) in patients with HCC.</p><p><b>METHODS</b>A total number of 234 patients with hepatocellular carcinoma (HCC) were included in this retrospective study. Uni-variate and multi-variate logistic regression analysis were employed to analyze the association between PVTT and 18 routine clinical parameters.</p><p><b>RESULTS</b>Among the 234 patients with HCC, 15% of patients (35/235) had PVTT. Univariate logistic regression analysis revealed significant association of age (P = 0.016), gamma glutamyl transferase (GGT, P = 0.003), number of segmental invasion (P = 0.007), microvascular invasion (P < 0.01), segment location of S2 (P = 0.001), S3 (P = 0.000), S4 (P = 0.004) and S6 (P = 0.016). Multivariate analysis shows potential significant predictors of PVTT in HCC were age (RR: 0.373; 95% CI: 0.146-0.954; P = 0.040), the tumor location of S3 (RR: 4.625; 95% CI: 1.916-11. 165;P = 0.001), GGT (RR: 4.091; 95% CI: 1.448-11.553; P = 0.008) and microvascular invasion (RR: 20.912; 95% CI: 4.745-92.172; P < 0.01).</p><p><b>CONCLUSIONS</b>PVTT occurred more commonly in the younger (< 50 years old), and those with high level of GGT, segment location of S3 and microvascular invasion.</p>

Involvement of JNK signal pathway in hypoxia related upregulation of calcium/calmodulin-dependent serine protein kinase in endothelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the expression of calcium/calmodulin-dependent serine protein kinase (CASK) induced by short-term hypoxia, and to explore the role of JNK pathway in this signal event.</p><p><b>METHODS</b>EA. hy926 cells were cultured in normoxic condition for 0, 12, 24, 48, 72 h after being exposed to hypoxic condition for 3 h, then the cellular lysates were extracted. CASK promoter luciferase reporter recombinant was constructed and transfected into EA. hy926 cells for 48h. Cellular lysates were extracted 1, 3, 6, 12 h after hypoxia treatment and were used to detect firefly luciferase activity and rinella luciferase activity with luminometer. EA. hy926 cells were cultured under hypoxic condition for 1, 3, 6, 12 h or under normoxic condition, then the cell lysates were extracted and used to detect phospho-JNK with Western blot. EA. hy926 cells were pretreated with different concentrations of JNK specific inhibitor SP 600125 (0, 10, 100 nmol/L and 1,10 micromol/L) 1h before hypoxic treatment of various duration, and the cell lysates were extracted to detect CASK expression with Western blot.</p><p><b>RESULTS</b>CASK expression was obviously elevated by hypoxia, and the high expression sustained for 72 h when the hypoxic cells were cultured in normal conditions, and it was significantly higher than that of normal controls. Dual luciferase reporter assay showed that CASK promoter activity was significantly increased after hypoxia (0.010 +/- 0.003, P < 0.01), and it reached the peak 12 hrs after hypoxia (0.192 +/- 0.023, P < 0.01). The phosphorylation of JNK was enhanced with the prolongation of hypoxic time. CASK protein expression was suppressed by JNK specific inhibitor SP600125 in a dose dependent manner, and it decreased to the lowest level with 10 micromol/L SP600125 pretreatment.</p><p><b>CONCLUSION</b>JNK signal pathway is involved in short-term hypoxia related CASK upregulation.</p>

Modification,Expression and Purification of Human Endotoxin Binding Peptide Gene / 中国生物工程杂志

Objective: To modify EBP(endotoxin binding peptide), clone and express the mutate of EBP gene and gain purified mEBP.Method: mEBPgene was cloned by PCR site-directed mutagenesis. PinpointXa-3/mEBP expression vector was designed to express human mEBP as a fusion protein in BL21 (DE3) pLysS. Digested engineering bacteria by lysozyme and collected inclusion bodies.Fusion protein was purified by Pinpoint TM Xa purification system and cleaved by factorXa,mEBP was purified by RP-HPLC. Results: Mutations at residues 5 and 18(Gln→Lys) was obtained by PCR site-directed mutagenesis, expressed and purified mEBP successfully.Conclusions: Obtaining of purified mEBP lay a foundation for its biological activity research.

Study on the expression, purification and the anti-endotoxin activity of human endotoxin binding peptide and its mutant / 中华烧伤杂志

<p><b>OBJECTIVE</b>To express endotoxin binding peptide and its mutant in E coli DH5alpha and detect the antiendotoxin activity of the purified peptides.</p><p><b>METHODS</b>(1 ) E coli DH5at containing pinpointXa3-EBP and pinpointXa3-mEBP was activated by IPTG to express biotin fusion protein. The fusion proteins were purified, and then digested by factor Xa for isolation of EBP and mEBP. The target peptide was purified with affinity chromatography and reversed-phase HPLC. Sequences of 10 amino acids at N-terminal were used for identification of mEBP. (2) PBMCs were isolated from blood of normal people, and they were stimulated with 5 mg/L FITC-LPS plus 2.0,5. 0 and 12. 5 mg/L EBP or mEBP. Then the mean fluorescent intensity was detected. PBMC was also stimulated with 1 mg/L LPS plus 2.0, 5.0 and 12.5 mg/L EBP or mEBP for 5 hours for the detection of the TNF-alpha and IL-6 level in the supernatant. (3) Thirty-five Kunming mice were randomized into normal control ( n = 5, with intraperitoneal injection of 0. 2 ml isotonic saline) , model group(n = 5, with intraperitoneal injection of LPS and 20% TBSA full-thickness burns), and treatment group (n = 15, with intraperitoneal injection of 5 mg/kg PMB or 10 mg/kg EBP or mEBP after burns). The serum contents of TNF-a and IL-6, and TNF-alpha mRNA level in hepatic tissue in each group were determined 6 hours after treatment.</p><p><b>RESULTS</b>( 1 ) EBP and mEBP were obtained after Xa digestion of biotin fusion protein, with purity reaching above 98% . The sequence of 10 amino acid at N-terminal was in accord with what expected. (2) The fluorescent intensity was decreased followed by an increase in mEBP or EBP concentration. Compared with normal PMBC, IL-6 and TNF-alpha level in the supernatant were obviously lowered in 1 mg/L LPS + 12.5 mg/L EBP group and I mg/L LPS +2. O0 , 5. 0, 12. 5 mg/L mEBP groups ( P < 0.01). (3) The serum level of IL-6 and TNF-ca in the therapeutic groups were obviously lower than that in model group ( P < 0.01 ) , and the levels of these cytokines were significantly lower in 10 mg/kg mEBP group than that in 10 mg/kg EBP group ( P <0. 01) , but they were similar to that in 5 mg/kg PMB treatment group ( P >0.05). (4) Relative optical density of TNF-alpha. mRNA in control, model, 10 mg/kg mEBP, 10 mg/kg EBP and 5 mg/kg PMB groups was 0.25, 0.93, 0.51 , 0.77 and 0.43, respectively.</p><p><b>CONCLUSION</b>Endotoxin binding peptides can be obtained by procaryon expression. Both EBP and mEBP have anti-LPS activity, but mEBP is more effective.</p>

Pathologic diagnosis of 1123 post-transplant liver biopsies from 665 liver transplant patients / 中华病理学杂志

<p><b>OBJECTIVE</b>To summarize the Chinese experience in pathologic diagnosis of liver biopsies after orthotopic liver transplantation (OLTx).</p><p><b>METHODS</b>1123 post-transplant liver biopsies from 665 OLTx patients from the Shanghai Eastern Hepatobiliary Surgery Hospital, Tianjin First Central Hospital, Guangzhou Sun Yat-sen University and Chongqing Southwest Hospital were retrospectively analyzed. All liver biopsies were stained with hematoxylin and eosin. Immunohistochemical studies for cytomegalovirus, HBsAg, CK19, CD4 and CD8 were also performed in selected examples.</p><p><b>RESULTS</b>In the involved hospitals, 4 to 12 types of complications were encountered after OLTx. The number of liver biopsies performed for each patient ranged from 1 to 9 (mean = 2.2). The timing of these biopsies varied from the second to the 2877 th post-transplant day. The 5 most common complications were acute cellular rejection (35.6%), ischemic-reperfusion injury (13.4%), biliary stricture (5.6%), drug complication (5.0%) and chronic rejection (4.7%). The 5 earliest complications after OLTx were primary non-function (occurring at day 4.7 +/- 2.1), ischemic-reperfusion injury (occurring at day 14.0 +/- 4.0), acute cellular rejection (occurring at day 32.1 +/- 62.9), hepatic artery thrombosis / stricture (occurring at day 62.9 +/- 74.2) and cytomegalovirus infection (occurring at day 107.7 +/- 93.0).</p><p><b>CONCLUSIONS</b>This study has evaluated the types, incidence and timing of major complications occurring after OLTx. The most important issue is the distinction between rejection and non-rejection pathology. Thorough understanding of atypical pathologic features of these complications is necessary. The Banff Schema (rejection activity index) for grading liver allograft rejection is useful for monitoring anti-rejection therapy and should be used routinely.</p>

Study on the protective effect of recombinant human N-terminal lipopolysaccharide binding protein in mice challenged with LPS / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of recombinant human N-terminal lipopolysaccharide binding protein in mice challenged with LPS.</p><p><b>METHODS</b>Seventy male Kunming mice were randomly divided into 3 groups, i.e. LPS challenge (Injection of LPS into abdominal cavity, n = 21); tLBP protection (Injection of LPS and tLBP into abdominal cavity, n = 21) and control (Injection of normal saline into abdominal cavity, n = 8) groups. The blood samples and tissue samples of the liver and lungs were harvested on 15 and 30 minutes and 1, 3, 6, 12 and 24 hours after the injection. The serum contents of ALT and TNF-alpha were determined by biochemical velocity analysis and RIA method, respectively. The pathomorphological changes in the liver and pulmonary tissue were examined under light microscope (LM). The mortality rate of ten mice each was observed within 24 hours after the injection of tLBP + 400 ng LPS or 400ng LPS.</p><p><b>RESULTS</b>The ALT content of tLBP group reached the peak level at 12 post-injection hour (PIH) (41.00 +/- 4.58), but it was significantly lower than that in LPS group in which it peaked at 6PIH (99.50 +/- 62.63) (P < 0.01). The TNF-alpha content in tLBP and LPS group was lower than that in LPS group, and both reached the peak level at 3 PIH (35.96 +/- 7.33). Compared with those in LPS, injury to hepatocytes in tLBP group was obviously milder without scattered necrosis. The pulmonary congestion in tLBP group was abated, and the inflammatory exudation in the alveoli was evidently less than that in LPS group. There were 9 out of 10 mice died in the LPS challenge group, while only 3 out of 10 mice died during 24 hours after LPS injection in tLBP protection group.</p><p><b>CONCLUSION</b>Preliminary results indicated that recombinant human tLBP might possess biological activity with a potential protection effect in LPS challenged mice.</p>

The purification and isolation of a new type of rabbit-origin lipopolysaccharide binding protein and the study of its biological function in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To isolate and purify a new type of lipopolysaccharide binding protein (LBP) from burn rabbit serum, and to investigate its biological functions.</p><p><b>METHODS</b>Rabbits subjected to burn injury and endotoxemia were employed. The serum from the rabbits was purified by two-steps of ion-exchange chromatography (Bio-Rex 70 Resin, Mono-Q) and gel chromatography. Furthermore, the serum was identified by flow cytometry analysis, agglomeration test with sheep erythrocyte, and amino end amino acid residue sequencing. The obtained protein was applied to cultured human monocytes (U937), and the cytokine secretion such as TNFalpha from the U937 was observed.</p><p><b>RESULTS</b>The molecular weight of the harvested protein was 48 kDa, and the 10 amino acid sequence at N end was arranged as GSQGTFTSEE, which was different to the amino acid sequence in NCBI protein bank and was so named P48. P48 possessed similar function to that of LBP and could promote the binding of LPS in a very low concentration with peripheral blood monocytes (PBMC), and also promote the TNFalpha secretion from U937.</p><p><b>CONCLUSION</b>P48, a new type of LBP, could be isolated and purified from the burn rabbit serum. P48 possessed similar biological activities to that of LBP and could promote the process of inflammatory reaction.</p>

An experimental study on the role of PGE2 and cAMP on the postburn change of the granulopoiesis in bone marrow in burned mice with endotoxemia / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the role of PGE(2) and cAMP in the postburn change in granulopoiesis in bone marrow in burned mice with endotoxemia.</p><p><b>METHODS</b>One hundred and seventy eight mice were randomly divided into burn with LPS administration, simple burn, simple LPS administration and control (injection of normal saline) groups. The COX-2 expression and the contents of PGE(2) and cAMP in myeloid cells in injured mice in all groups were determined by RIA (radioimmuno-assay) within 1 postburn week and immunohistochemistry methods. At the same time the change in granulopoiesis was dynamically observed.</p><p><b>RESULTS</b>The granulopoiesis was enhanced slightly at the early stage of burn and with endotoxin challenge, followed by suppression. The COX-2 expression in myeloid cells the contents of PGE(2) on supernatant of marrow cells and intracellular cAMP in the myeloid cells was increased at 12 postburn hour (PBH) up to 5 postburn day (PBD). Furthermore, the change in the cAMP was evidently and positively correlated with that of PGE(2) (r = 0.978, P < 0.01), but was negatively correlated with that of CFU-GM (r = -0.971, P < 0.01)</p><p><b>CONCLUSION</b>PGE(2) might play pivotal roles in the postburn granulopoiesis suppression in bone marrow during endotoxemia. This effect might be accomplished by its ligating to its special receptor and to activate adenylate cyclase so as to increase the intracellular content of cAMP in bone marrows.</p>

Molecular mechanism of the sensitivity of myocardial cell injury after severe hypoxia / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanism of the sensitivity of myocardial cell injury after severe hypoxia.</p><p><b>METHODS</b>Differential expression of the genes in rat myocardium and skeletal muscles after severe hypoxia for 6 hours was determined by DNA chip containing 4096 mice genes.</p><p><b>RESULTS</b>There were high expressions of 125 genes in skeletal muscles and 111 genes in myocardial cells after 6 hours of severe hypoxia. Among them, high expression genes in myocardial cells included those encoding apoptosis activator Mtd, complement C3, vascular cell adhesion molecule-1, integrin beta subunit and lipopolysaccharide binding proteins (LBP).</p><p><b>CONCLUSION</b>Activation of apoptosis of myocardial cells and inflammatory mediators played important roles in myocardial cell injury after 6-hour severe hypoxia.</p>