[Differential expression of oxidative reductases in different subsets of mouse hematopoietic cells].

Hematopoietic stem cells (HSC) are the source of all blood cells, which can differentiate into various hematopoietic hierarchy cells. Physiological level of reactive oxygen species (ROS) plays an important role in regulating functions of HSC as excessive ROS is harmful to HSC. Oxidative reductases and antioxidants can eliminate cellular ROS to maintain ROS homeostasis and thus avoid excessive ROS-caused damages. There are several types of oxidative reductases in cells such as catalase, manganese superoxide dismutase (MnSOD), glutathione peroxidase 1 (GPX1), thioredoxin reductase 1 (Txrnd1) and Nqo1 [NAD(P)H dehydrogenase quinone 1]. However, the functional roles of various oxidative reductases in regulating ROS level in hematopoietic cells remain unclear. This study was to investigate the expression patterns of these oxidative reductases in mouse hematopoietic cells that were sorted out via flow cytometry and to find out important oxidative reductases involving in HSC ROS regulation. The expression of various oxidative reductases was detected by semi-quantitative real-time PCR. The results showed that the expression level of catalase in T cell population was 0.14 times that in LT-HSC population (P < 0.05). The expression levels of MnSOD in CLP population and myeloid cells were 0.56 and 0.47 times that in LT-HSC population respectively (P < 0.05). The expression levels of GPX1 in ST-HSC, GMP, Myeloid cells, MEP, T lymphocytes and B lymphocytes were 1.79, 2.96, 2.07, 0.58, 0.10, 0.6 times that in LT-HSC population respectively (P < 0.05). The expression levels of Txrnd1 in ST-HSC, MPP, CMP, GMP, Myeloid cells, T lymphocytes and B lymphocytes were 3.36, 3.18, 4.19, 6.39, 4.27, 0.016, 0.56 time that in LT-HSC population, respectively (P < 0.05). The expression levels of Nqo1 in ST-HSC, MPP, CMP, GMP, CLP and B cell were 0.30, 0.17, 0.25, 0.10, 0.04, 0.01 times that in LT-HSC population, respectively (P < 0.05). It is concluded that the expression levels of oxidative reductases (catalase, MnSOD, GPX1, Txrnd1 and Nqo1) in hematopoietic hierarchy cells are cell-type specific. It suggests that reductases may play divergent roles in various hematopoietic cell populations. More importantly, the expression level of Nqo1 in LT-HSC population significantly increased as compared with other cell populations, thereby suggesting its unique regulatory role in HSC.

Hypomethylation of TNF-alpha gene promoter in the patients with acute-on-chronic hepatitis B liver failure / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The present study was designed to investigate the possible epigenetic alteration in the promoter of TNF-alpha in the patients with acute-on-chronic hepatitis B liver failure (ACHBLF).</p><p><b>METHODS</b>The methylation of TNF-alpha promoter in peripheral blood mononuclear cells (PBMCs) was measured by methylation specific PCR (MSP). The level of serum TNF-alpha was determined by enzyme-linked immunosorbent assay (ELISA). Model for End-stage Liver Disease (MELD) was performed for the evaluation of liver failure.</p><p><b>RESULTS</b>The serum level of TNF-alpha in patients with ACHBLF(44.9260 +/- 26.48523) was higher than that in CHB (18.92505 +/- 9.04461) and healthy controls (11.9172 +/- 5.04612) (P < 0.05). Moreover, the serum TNF-alpha level was significantly decreased in methylation group as compared to unmethylaiton group in patients with ACHBLF (P < 0.05). MELD was not significantly different between methylated and unmethylated group of ACHBLF patients (P > 0.05). In addition, the serum level of TNF-alpha was found to be positively correlated with serum total bilirubin (r = 0.891, P < 0.01) and MELD score (r = 0.792, P < 0.01), but to be negatively correlated with prothrombin activity (r = - 0.511, P < 0.05) in patients with ACHBLF.</p><p><b>CONCLUSION</b>The TNF-alpha methylation patten is stable for the liver failure, suggesting the effect of environment on methylation.</p>

Study of oxidative stress in chronic hepatitis B patients with elevated serum total bilirubin / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate oxidative stress in chronic hepatitis B (CHB) patients with elevated serum total bilirubin (TBIL).</p><p><b>METHODS</b>75 CHB patients with elevated serum TBIL were enrolled in the present study. A, B, C, D and E group were defined. Serum Malondialdehyde (MDA), Xanthine Oxidase (XOD), Vitamin C (V(C)) and Vitamin E (V(E)) were determined. The control group contained 11 healthy donors and the carrier group contained 16 Hepatitis B surface antigen (HBsAg) carriers.</p><p><b>RESULTS</b>The concentrations of MDA and XOD were significantly higher in each group of patients than in the control (P < 0.05), while V(C) and V(E) were significantly lower (P < 0.05). The concentration of XOD was significantly higher in the carrier group than in the control (P < 0.05), while MDA, V(C) and V(E) were not significantly different (P > 0.05). The concentrations of MDA and XOD were significantly positively correlated with TBIL (r = 0.670, P < 0.01; r = 0.737, P < 0.01, respectively) in the patients, while V(C) and V(E) were significantly negatively correlated with TBIL (r = -0.463, P < 0.01; r = -0.247, P < 0.05, respectively). The concentration of MDA was significantly different among all the groups in the patients except the comparison between group A and group B. The concentration of XOD was significantly different between group A, B, C and group D, E (P < 0.05). The concentration of V(C) was significantly different between group A and group D, E and between group B, C, D and group E (P < 0.05). The concentration of V(E) was significantly different between group A, B and group E (P < 0.05).</p><p><b>CONCLUSION</b>There was a disturbance between oxidative stress and anti-oxidative ability in CHB patients with elevated serum TBIL. Oxidative stress became more serious along with the increasing of serum TBIL. In HBsAg carriers, oxidative stress level was low. The results suggest antioxidant treatment for CHB patients with elevated serum TBIL may help to improve the effect of therapy.</p>

[Intra-bone marrow infusion of human cord blood hematopoietic stem/progenitor cells improves hematopoietic reconstitution in NOD-SCID mice].

The purpose of this study was to explore the effect of intra-bone marrow infusion (iBMI) of cord blood (CB)-derived hematopoietic stem/progenitor cells (HS/PCs) on human hematopoietic reconstitution in xenotransplanted NOD-SCID mouse model. Aliquots containing 5 x 10(5) CB CD34(+) cells were transplanted into sublethally irradiated NOD-SCID mouse via intravenous infusion (iVI) or iBMI routes. 64 female NOD-SCID mice were divided randomly into 3 groups: iBMI group, iVI group and negative control group. The engraftment levels of human hematopoietic cells at 3, 5 and 8 weeks after xenotransplantation were detected by fluorescence-activated cell sorter (FACS), polymerase chain reaction (PCR), immunohistochemistry and HPC colony formation assay, and long-term hematopoietic reconstitution capacity of HSC was tested by secondary transplantation. The results showed that the percentages of human CD45(+) cells in bone marrow, peripheral blood and spleen of recipient in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group (p < 0.05). HS/PCs given through both iVI and iBMI methods had the ability of multilineage differentiation, the percentages of CD45(+)CD19(+) cells, CD45(+)CD33(+) cells, CD45(+)CD56(+) cells and CD45(+)CD34(+) cells of recipient bone marrow in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group (p < 0.05), while other lineages in iBMI group were also higher than that in iVI group (p > 0.05). alpha-satellite-specific fragment of human chromosome 17 could be detected by PCR in liver, spleen, lung, peripheral blood and bone marrow cells of long-term survival recipients in both iVI and iBMI groups. Human CD45 antigen could be detected by immunohistochemical method in spleen, liver and lung of recipients in iBMI group at 8 weeks after xenotransplantation. Total colony count in iBMI group at 8 weeks after xenotransplantation was significantly higher than that in iVI group (p < 0.05). alpha-satellite-specific fragment of human chromosome 17 could be detected in all above organs of both group recipients at 6 weeks after secondary xenotransplantation. It is concluded that iBMI of CB CD34(+) cells improves hematopoietic reconstitution in xenotransplanted NOD-SCID mouse model.

[The observation of engraftment of human umbilical cord blood-derived hematopoietic stem/progenitor cells in xenotransplanted NOD/SCID mouse model by intra-bone marrow injection].

OBJECTIVE: To explore whether intra-bone marrow injection strategy could promote the engraftment of human umbilical cord blood derived hematopoietic stem/progenitor cells (HS/PC) in xenotransplanted NOD/SCID mouse model. METHODS: Aliquots containing 1 x 10(3), 1 x 10(4), 0.5 x 10(5), 1 x 10(5) and 5 x 10(5) human umbilical cord blood (hUCB) CD34+ cells were transplanted into sublethally irradiated NOD/SCID mice via intra-venous (i.v.) and intra-bone marrow (iBM) injection. The homing and long-term engraftment capabilities of hUCB CD34+ cells from right tibia, right femur, left tibia, left femur and spleen were detected by PCR 24h after xenotransplantation and by FACS 8-week after xenotransplantation. RESULTS: Tissues of liver, spleen, lungs, or cells from peripheral blood, right tibia, right femur, left tibia and left femur 24 hours after xenotransplantation in iBM injecting 5 x 10(5) CD34+ cells recipients expressed human chromosome 17 specific alpha-satellite fragment. 8-week engraftment of human cells was observed and engraftment level indicated dose-dependent effect in injected bone (right tibia) as well as non-injected bones (including right femur, left tibia and left femur), spleen and peripheral blood in all iBM recipients. 8-week engraftment levels of human cells were (44.063 +/- 20.095)% and (45.881 +/- 22.316)% for i.v. and iBM groups respectively, when transplanted with 1 x 10(5) hUCB CD34+ cells, being no statistical difference (P >0.05). More superior 8-week engraftment levels of human cells were observed in iBM recipients [(54.019 +/- 31.338)%] than in i.v. recipients [(12.197 +/- 10.350)%] when transplanted with 1.0 x 10(4) CD34 cells (P<0.01). Human cell engraftment was observed in iBM but not in i.v. recipients when transplanted with 1.0 x 10(3) CD34+ cells, and was usually observed in non-injected bones. CONCLUSION: Intra-bone marrow strategy can efficiently increase the engraftment of umbilical cord blood derived hematopoietic stem/progenitor cells in xenotransplanted NOD/SCID mouse model.

[Study on the genetic stability of immortalized cell line of lymphocyte cell transformed by EB virus after long subculture process].

OBJECTIVE: To study the genetic stability of an immortalized cell line transformed by Epstein-Barr virus (EBV) after long subculture process. METHODS: In the present study, the genetic stability including chromosome diploidy, karyotypes and microsatellite DNA were evaluated with chromosome banding techniques and microsatellite DNA detection. The telomerase activity of the immortalized cell line was detected by using the telomerase assay kit. RESULTS: From passage 1 to 30, there were no change of the diploidy, karyotypes of chromosome and microsatellite DNA, and the telomerase activity is negative. CONCLUSION: This study indicates that the immortalized cell line remains stable genetically within limited passages.

Increased expression of inducible nitric oxide synthase in chronic hepatitis B patients is correlated with histopathological grading and staging / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the intrahepatic expression of inducible nitric oxide synthase (iNOS) in patients with chronic hepatitis B (CHB) and its relation to liver histopathology.</p><p><b>METHODS</b>The intensity and distribution of the immunohistochemical staining of intrahepatic iNOS were studied in the liver biopsy specimens obtained from 74 patients with CHB and statistical analyses were performed between intrahepatic iNOS and ALT, HbeAg, HBV DNA grading of liver inflammation and staging of fibrosis. Seven histologically normal liver sections were used as a control group.</p><p><b>RESULTS</b>Compared with the control group, the intrahepatic iNOS immunoexpression was significantly higher in patients with CHB (P < 0.05), iNOS immunoreactivity was observed mainly in hepatocytes showing a predominant cytoplasmic staining, with the positive liver cells distributed diffusely throughout the hepatic lobule. Immunopositive staining could also be detected in Kupffer cells, sinusoidal lining cells and vascular endothelial cells. Compared with patients with normal ALT, the hepatocellular iNOS immunoexpression was significantly higher in patients with elevated ALT (P < 0.05) and the iNOS immunoexpression was significantly correlated with the serum level of ALT (r=0.601, P=0.000). Statistical analysis also showed that the intrahepatic iNOS immunoexpression was positively correlated with the grading of liver inflammation and the staging of liver fibrosis (r=0.660, P=0.000; r=0.507, P=0.000). No significant correlation between iNOS and HBeAg and HBV DNA was detected. CONCLUSION The intrahepatic expression of iNOS is elevated in chronic hepatitis B patients and correlated well with the severity of the disease, which indicated that inducible nitric oxide synthase may have a critical role in the pathogenesis of chronic viral hepatitis B.</p>

Hemangiopoietin supports animal survival and accelerates hematopoietic recovery of chemotherapy-suppressed mice.

OBJECTIVE: To determine if recombinant human hemangiopoietin (HAPO), a novel growth factor for primitive cells of hematopoietic and endothelial cell lineages, accelerates hematopoietic reconstitution after high-dose chemotherapy in vivo in mice. METHODS: Male Balb/c mice after treatment of 5-fluorouracil were subcutaneously injected with HAPO or its dilution for consecutive 10 d. Their survival and body weight together with peripheral blood were routinely tested. At day 7 and 14, the numbers of bone marrow (BM) cells as well as colony-forming units (CFU) after in vitro colony culture were counted. The peripheral blood CFU and the percentage of CD34+ CD117+ cells in BM were analyzed. Transwell chamber was used for cell migratory assay. RESULTS: HAPO at different doses significantly increased the survival rate and body weight, with an optimal effect in the HAPO 10 microg/d group. The number of BM cells and the percentage of CD34+ CD117+ cells were also increased after HAPO administration. The number of granulocyte/macrophage CFU and granulocyte, erythroid, macrophage and megakaryocyte CFU in BM after HAPO treatment was greater than that from the HAPO dilution group. More circulating CFU could be observed after injection of HAPO. In addition, this novel cytokine had a chemotactic effect on the hematopoietic stem/progenitor cells. CONCLUSION: HAPO improves animal survival and accelerates hematopoietic reconstitution of mice after high-dose chemotherapy.

[Differential study on the in vivo homing potential of human hematopoietic stem/progenitor cells from different sources in xenotransplanted NOD/SCID mouse model].

OBJECTIVE: To compare the in vivo homing potential of human hematopoietic stem/progenitor cells (HS/PCs) derived from fresh umbilical cord blood (UCB), cryopreserved UCB, mobilized peripheral blood (mPB) and bone marrow (BM) in xenotransplanted NOD/SCID mouse model, and to explore the relationship between the homing potential of HS/PCs and their expression levels of membrane receptor CXCR4. METHODS: The expression levels of membrane CXCR4 on HS/PCs were assessed by flow cytometric analysis (FACS). CFSE-labeled human HS/PCs from different sources were transplanted into irradiated NOD/SCID mice. Human CD34 cells home in bone marrow and spleen of recipient mice were determined 20 hours after xenotransplantation by FACS and the homing efficiencies were calculated. Tissue sections of the recipient mice femurs were made and the distribution of CFSE-labeled human CD34 cells were observed under fluorescence microscope. RESULTS: The expression levels of membrane CXCR4 on CD34+ cells from fresh UCB, cryopreserved UCB, mPB and BM were (49.52 +/- 1.12)%, (46.12 +/- 2.29)%, (48.50 +/- 2.48)% and (65.39 +/- 1.27)%, respectively. The homing efficiencies of CD34+ cells from fresh UCB, cryopreserved UCB, mPB and BM in recipient mice BM were (3.00 +/- 0.44)%, (2.84 +/- 0.46)%, (4.06 +/- 0.70)% and (5.76 +/- 0.52)% , respectively. Human CD34+ cells mainly located within endosteal region of recipient mice femurs. CONCLUSION: CD34+ cells from UCB express lower levels of membrane CXCR4 than those from mPB and BM. The level of membrane CXCR4 on UCB CD34+ cells is down-regulated after freezing and thawing procedures. The homing efficiency of human CD34 cells from UCB in recipient mice is lower than that of mPB and BM.

Oxidative stress in patients with chronic hepatitis B before and after interferon alpha-2b treatment / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the impacts of interferon alpha-2b (IFN alpha-2b) on the oxidative stress states in the treatment of chronic hepatitis B (CHB) with different genotypes.</p><p><b>METHODS</b>Thirty-five patients with chronic hepatitis B and 18 healthy volunteers as a control were enrolled in this present study. In control and patients group, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), serum malondialdehyde (MDA) levels, serum total antioxidative stress capacity (TAC) were measured spectrophotometrically. After the therapy with interferon alpha-2b at the dose of 300 million units via intramuscular injection thrice a week for 12 weeks, these parameters were measured again in the patient group. The genotypes of hepatitis B virus were detected by polymerase chain reaction and hybridization. The effective group was defined as the patients with complete response and partial response.</p><p><b>RESULTS</b>The elevated concentrations of MDA and impaired levels of TAC in the patients with CHB were observed as compared to the healthy controls (P < 0.05 for both). There were no significant differences in serum levels of MDA and TAC in CHB patients with various genotypes (P > 0.05). The serum levels of MDA after the treatment with IFN alpha-2b were significantly lower than the pretreatment levels (P < 0.05), which even returned to the normal concentration (P > 0.05) in the effective group. There were significant increases in the TAC after the IFN alpha-2b therapy in the effective group. However, the significant differences in the TAC levels before and after the INFalpha-2b treatment were not observed in the non-responsive group.</p><p><b>CONCLUSION</b>The oxidative stress could be improved with IFN alpha-2b treatment of chronic hepatitis B patients. The results suggest that antioxidant treatment for chronic hepatitis B patients may help improve the effect of anti-virus therapy.</p>

[High expression of mirobial transglutaminase gene from Streptoverticillium mobaraense in Escherichia coli].

The microbial transglutamunase (MTG) gene was amplified from the genomic DNA of Streptoverticillium mobaraensea by using PCR and inserted into pET vector to construct the expression plasmid called pET-MTG. The pET-MTG was transfected into E. coli (Rosetta DE3) and the MTG protein was found to be highly expressed as inclusion bodies. The inclusion bodies were isolated and subjected to denaturation and re-naturation, followed by strong cation ion-exchange chromatography to purify the expressed MTG. The specific activity of purified MTG was close to that of native MTG. Taken together, this study might provide a base for the industrial production of microbial transglutaminase.

[Expression of alternatively spliced isoforms of platelet endothelial cell adhesion molecule-1 of embryonic stem cells during vasculogenesis and angiogenesis].

OBJECTIVE: To study the expression of alternatively spliced isoforms of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) of embryonic stem cells (ES cells) during vasculogenesis and angiogenesis. METHODS: Mouse ES cells of the line J1 were cultured. Another ES cells were cultured in differentiation medium to induce the formation of embryonic bodies (EBs). Then the ES cells with PECAM-1 and EBs were inoculated with methylcellulose into Petri dish, containing cell growth factor, VEGF, bFGF, EPO, and IL-6 and the ES cells cultured for 11 days were inoculated in the Petri dish with collagen for 72 hours so as to induce sprouting angiogenesis. Immunofluorescence analysis, RT-PCR, and flow cytometry were used to detect the expression of PECAM-1, Oct-4, and stage-specific embryonic antigen (SSEA)-1 in the undifferentiated ES cells, EBs, and EB sprouting. In order to delineate the alternatively spliced cytoplasmic domain isoforms of PECAM-1 specific primers were designed to span the exon-exon junctions in the regions of alternative splicing. In order to amplify the cytoplasmic domains of all possible PECAM-1 isoforms a sense primer spanning the border of exons 9 and 10 within the cytoplasmic domain and an antisense primer spanning the border of exon 16 and 3'-untranslated region were used. Then the PCR products of the cytoplasmic domain underwent subsequent sequencing to analyze the expression of the 8 known alternatively splice isoforms of PECAM-1. RESULTS: The ES cells expressed high level PECAM-1 that was mainly located at the cell-cell junctions. The SSEA-1 and Oct-4 levels rapidly decreased along with the differentiation of the ES cells. All 8 known alternatively splice isoforms of PECAM-1 were expressed in the ES cells and the EB sprouting, the expression of Delta14%15 and Delta12&14&15 being the highest. The expression level of Delta12&14&15 increased markedly and the expression of Delta15 decreased along with the differentiation of ES cells. CONCLUSION: PECAM-1 is a constitutive feature of undifferentiated ES cells. Its changes in splice form mark the differentiation and may participate in vasculogenesis and angiogenesis.

Production of a monoclonal antibody against SARS-CoV spike protein with single intrasplenic immunization of plasmid DNA.

Severe acute respiratory syndrome (SARS) is a highly infectious disease caused by a novel coronavirus (SARS-CoV). Specific monoclonal antibodies (mAbs) against the SARS-CoV are vital for early diagnosis and pathological studies of SARS. Direct intrasplenic inoculation of plasmid DNA encoding antigen is an effective and fast approach to generate specific mAb when the protein antigen is difficult to prepare or dangerous in use. In this study, we selected one fragment of SARS-CoV spike protein (S1-(3)) as antigenic determinant by immunoinformatics. Single intrasplenic immunization of plasmid DNA encoding S1-(3) induced anti-spike protein antibodies. We established one hybridoma cell line secreting specific mAb and evaluated this mAb with murine leukemia virus pseudotyped with SARS-CoV spike protein (MLV/SARS-CoV). The mAb could recognize the spike protein on the MLV/SARS-CoV-infected Vero E6 cells albeit with no neutralizing effect on the infectivity of the pseudotype virus. Our results show that a single-shot intrasplenic DNA immunization is efficient for the production of specific mAb against SARS spike protein, and a linear epitope of the spike protein is recognized in this study.

Nitric oxide and nitric oxide synthase in patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate nitric oxide (NO) and nitric oxide synthase (NOS) in patients with chronic hepatitis B.</p><p><b>METHODS</b>Nitric oxide and nitric oxide synthase, including inducible NOS (iNOS) and constitutive NOS (cNOS), were measured in patients and control groups, then were statistically analyzed.</p><p><b>RESULTS</b>NO and iNOS were significantly higher in patients with hepatitis B than in the controls (P < 0.05). NO and iNOS were significantly higher in patients with increased ALT than in the controls and in patients with normal ALT (P < 0.05). NO was significantly higher in patients with normal ALT than in the controls (P < 0.05). cNOS were not significant different among these groups. NO and iNOS significantly correlated with ALT in patients with hepatitis B (r=0.367 and r=0.474). No significant relationship was found among NO, NOS and HBV DNA. Among different genotype groups, NO and NOS had no significant difference.</p><p><b>CONCLUSION</b>NO and NOS were higher in patents with chronic hepatitis B. In patients with increased ALT, NO's damage was severe. In patients with normal ALT, there was no significant damage caused by NO. NO should be detected in patients with hepatitis B in addition to HBV markers.</p>

Hemorheologic changes in patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the variation of the hemorheology in patients with chronic hepatitis B and study its relations with HBV DNA, liver function and oxidative stress markers.</p><p><b>METHODS</b>Indices for hemorheology, oxidative stress markers, liver function, and HBV DNA were measured in 55 patients with chronic hepatitis B and correlative analysis was made.</p><p><b>RESULTS</b>The low-shear whole blood viscosity (BV), RBC aggregation index were significantly higher in hepatitis B group than those in the control group (P < 0.05), Hematocrit (HCT), the low-shear BV, RBC aggregation index were significantly higher in the patients whose ALT was higher than the patients whose ALT was normal and the controls. No significant difference was found in HBV DNA and indices of hemorheology (P > 0.05), nor in indices of hemorheology and oxidative stress markers (P > 0.05).</p><p><b>CONCLUSION</b>There is disturbance of microcirculation and oxidative stress in the body of patients with chronic hepatitis B. The hemorheology and oxidative stress markers should be regarded as useful indexes in patients with chronic hepatitis B in addition to HBV markers.</p>

[Potent neutralization antibody elicited in mice by SARS-associated coronavirus spike protein S1 domain].

OBJECTIVE: To study the antigenicity of SARS associated coronavirus (CoV) spike S1 (12-672Aa) domain. METHODS: BALB/c mice were immunized with a plasmid bearing codon-optimized SARS-CoV (Tor2 strain) S1 domain and then boosted with purified S1 protein; the SARS-CoV specific IgG antibody was tested by ELISA and neutralization antibody was determined by in vitro microneutralization assay. RESULTS: S1 domain of SARS-CoV spike, which has been demonstrated harboring the receptor binding domain, successfully elicited SARS-CoV specific IgG antibody in mouse after combined immunization with DNA and purified S1 protein; the antibody elicited solely by S1 could potently neutralize SARS-CoV (HKU-39849) in vitro, 50% of 1 000 TCID50 SARS-CoV challenged cells were protected from viral infection by a 1:1499.68 dilution of mice sera immunized with S1 protein, but negative control sera showed no protection. CONCLUSION: S1 domain of SARS-CoV spike protein, which is responsible for receptor binding, can efficiently and sufficiently induce highly potent neutralizing antibody in mice. This result suggested that S1 domain could be an effective subunit vaccines against SARS-CoV.

[Inhibition of tumor angiogenesis in nude mice by adenovirus-mediated PF4 p17-70 cDNA transfection].

OBJECTIVE: To investigate the in vivo effect of modified platelet factor 4 (PF4)-p17-70 cDNA on tumor angiogenesis in nude mice. METHODS: The p17-70 cDNA was cloned into the AdEasy system to transfect packing cell line 293 and produce viral particles encoding p17-70cDNA (Ad p17-70). The integration of p17-70 cDNA was confirmed by RT-PCR and the P17-40 peptide Western blot. The biological activity of purified recombinant adenovirus was determined by umbilical veinal endothelial cell proliferation assay in vitro and in vivo tumor angiogenesis suppression of nude mice bearing human head and neck carcinoma. RESULTS: p17-70 significantly inhibited in vitro proliferation of endothelial cells being 58% lower than that of empty vector and reduced tumor volume in vivo. The tumor mass was (0.086 +/- 0.054) g, (0.171 +/- 0.076) g and (0.195 +/- 0.067) g, the tumor volume was (16.7 +/- 5.2) mm(3), (36.5 +/- 23.7) mm(3) and (41.5 +/- 12.2) mm(3) in p17-70 cDNA transfected group, empty vector group and PBS group, respectively. Immunohistochemical staining demonstrated a decreased number of blood vessels in the tumors. CONCLUSION: P17-70 peptide mediated by adenoviral vector could inhibit the endothelial proliferation in vitro and the tumor growth in vivo.

Adenovirus-mediated gene therapy with an antiangiogenic fragment of thrombospondin-1 inhibits human leukemia xenograft growth in nude mice.

Recent investigations support the idea that angiogenesis is involved in the pathophysiology of leukemia. Within a given microenvironment, the angiogenic response is regulated by a delicate balance of angiogenesis inducers and inhibitors. Thrombospondin-1 (TSP-1) is a multifunctional extracellular glycoprotein showing angiostatic properties in multiple in vitro and in vivo assays. Interestingly, there is also proangiogenic domain in this complex molecule. Development of TSP-1 as an antiangiogenic drug has been hindered by multiplicity of its functional effects, difficulties in its production and its poor pharmacokinetics. The aim of the present study was to establish a recombinant adenovirus (ADV.TSP-1(f)) expressing antiangiogenic fragment of TSP-1 (TSP-1(f)), and to determine the feasibility for use of the adenovirally expressed TSP-1(f) in leukemia gene therapy. The results of this investigation showed that TSP-1(f) was expressed efficiently in adenovirus-transduced human myelogenous leukemia K562 cells. Compared to the controls, although there was almost no effect on proliferation of K562 cells in vitro, adenovirus-mediated TSP-1(f) transduction inhibited the growth of K562 xenografts dramatically. Furthermore, the microvessel density (MVD) was much lower in the ADV.TSP-1(f)-treated tumors compared to the controls. These data support the use of in vivo gene delivery approach to produce antiangiogenic fragment of TSP-1 for leukemia therapy.