Serial expression of the truncated fragments of the nucleocapsid protein of CCHFV and identification of the epitope region.

The Crimean-congo hemorrhagic fever virus (CCHFV) is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections. In this study, expression vectors carrying series truncated fragments of the NP (nucleocapsid protein) gene from the S fragment of CCHFV strain YL04057 were constructed. The recombinant proteins were expressed in E.coli and purified for detection. The antigenic of the truncated fragments of NP was detected with a polyclonal serum (rabbit) and 2 monoclonal (mAbs) (14B7 and 43E5) against CCHFV by Western-blot analyses. The results showed that the three expressed constructs, which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum. The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.

Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection.

BACKGROUND: Enterovirus 71 (EV71) is a viral pathogen that belongs to the Picornaviridae family, EV71-infected children can develop severe neurological complications leading to rapid clinical deterioration and death. RESULTS: In this study, several monoclonal antibodies (MAbs) were produced by immunizing mice with the inactived EV71 Henan (Hn2) virus strain. The isolated MAbs were characterised by in vitro neutralizing analysis and peptide ELISA. ELISA assay showed that the neutralizing monoclonal antibody 4E8 specifically reacted with synthetic peptides which contain amino acid 240-250 and 250-260 of EV71 VP1. The in vivo protection assay showed that 4E8 can protect two-day-old BALB/c mice against the lethal challenge of EV71 virus. CONCLUSION: The MAb 4E8 could be a promising candidate to be humanized and used for treatment of EV71 infection.

Sequence analysis of six enterovirus 71 strains with different virulences in humans.

Enterovirus 71 (EV71) infection is the main cause of hand, foot and mouth disease (HFMD) and has been associated with severe neurological diseases resulting in high mortalities. In this study, six EV71 strains isolated from patients with different clinical symptoms were sequenced and analyzed in a mouse model of EV71 infection. In a phylogenetic tree, based on the complete VP1 gene sequence, all six strains grouped into the C4 genotype. The sequence analysis revealed that there are nucleotide changes clustered in the internal ribosome entry site (IRES) element of the 5'-nontranslated region (5'-NTR), as well as amino acid differences clustered in the non-structural proteins. Importantly, we identified a unique amino acid difference (Val(1994)-Ile(1994)) that distinguished the more virulent strains, Anhui1 (Ah1), Henan1 (Hn1) and Henan2 (Hn2) from the less virulent strains, Chongqing1 (Cq1), Chongqing2 (Cq2) and Chongqing3 (Cq3). This amino acid difference is located in the finger domain of the viral RNA-dependent RNA polymerase 3D (3D(pol)). Furthermore, two-day-old Balb/c mice were inoculated with the Ah1, Hn1, Hn2, Cq1, Cq2 and Cq3 isolates by the intracerebral or intraperitoneal routes. All of the mice inoculated with Ah1, Hn1 and Hn2 isolates developed hind-leg paralysis and subsequently died. Mice inoculated with the Cq1, Cq2 or Cq3 isolates survived throughout the 21-day observation period. These results show that clinical isolates of EV71 associated with disease of different severity in humans have characteristic sequence differences and cause different mortality rates when inoculated into mice. These data also provide a rational basis to investigate the molecular determinants of EV71 pathogenesis using a reverse genetic approach.

Effect of ginkgo biloba extract on livers in aged rats.

AIM: To investigate the protective effect of ginkgo biloba extract (GBE) on livers of aged rats and the associated mechanisms. METHODS: Two-mo- and 20-mo-old rats were treated with GBE/saline for 3 mo. Liver tissue samples from 5-mo-old rats treated with saline (group Y) and 23-mo-old rats treated with GBE (group E) or saline (group N) were used for histopathological examinations (hematoxylin-eosin and Masson staining, Lipofuscin staining-Schmorl staining) and determination of expression of tissue inhibitor-1 of metalloproteinase (TIMP-1) and the level of malondialdehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD). Blood samples were collected for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin. RESULTS: Microscopic studies with Masson staining revealed mild liver fibrosis in aged rats (group N), while the livers of aged rats receiving GBE (group E) showed amelioration in fibrosis (2.2+/-0.1 vs 2.8+/-0.1, P<0.01) and deposition of lipofuscin (33.7+/-5.3 vs 62.8+/-5.7, P<0.01). The expression of TIMP-1 and the level of liver MDA (1.0+/-0.1 vs 1.2+/-0.2, P<0.05) also decreased but the activity of GPx (97.1+/-15.3 vs 61.8+/-14.5, P<0.01) increased in group E. Compared with group Y, the level of liver MDA (0.8+/-0.1 vs 1.2+/-0.2, P<0.01), lipofuscin (32.4+/-6.0 vs 62.8+/-5.7, P<0.01) and TIMP-1 expression were increased, while the activity of GPx (103.2+/-17.6 vs 61.8+/-14.5, P<0.01) and SOD (16.7+/-4.4 vs 11.8+/-3.9, P<0.05) was decreased in group N. There was no difference in liver function among these three groups. CONCLUSION: GBE has protective effects on aging liver. The possible mechanisms might be its antioxidant activity and inhibition of TIMP-1 expression.

Ginkgo biloba extract reverses CCl4-induced liver fibrosis in rats.

AIM: To study the reversing effect of Ginkgo biloba extract (GbE) on established liver fibrosis in rats. METHODS: Following confirmation of CCl(4)-induced liver fibrosis, GbE or saline was administrated to the rats for 4 weeks. The remaining rats received neither CCl(4) nor GbE as normal control. The four groups were compared in terms of serum enzymes, tissue damage, expression of alphaSMA and tissue inhibitor-1 of metalloproteinase (TIMP-1) and metalloproteinase-1 (MMP-1). RESULTS: Compared with saline-treated group, liver fibrosis rats treated with GbE had decreased serum total bilirubin (P<0.01) and aminotransferase levels (P<0.01) and increased levels of serum albumin (P<0.01). Microscopic studies revealed that the livers of rats receiving GbE showed alleviation in fibrosis (P<0.05) as well as expression of alphaSMA (P<0.01). The liver collagen and reticulum contents were lower in rats treated with GbE than saline-treated group (P<0.01). RT-PCR revealed that the level of TIMP-1 decreased while the level of MMP-1 increased in GbE group. CONCLUSION: Administration of GbE improved CCl(4)-induced liver fibrosis. It is possibly attributed to its effect of inhibiting the expression of TIMP-1 and promoting the apoptosis of hepatic stellate cells.

[Potential molecular mechanism of multiple organ dysfunction syndrome induced by hemorrhage and endotoxin].

OBJECTIVE: To investigate the possible molecular mechanism of multiple organ dysfunction syndrome (MODS) associated with hemorrhagic shock followed by resuscitation and endotoxin. METHODS: A rabbit model of MODS after hemorrhagic shock followed by resuscitation and endotoxin was used in this study. The expression of I-kappaB kinase-beta (IKK-beta), the activity of nuclear factor-kappaB (NF-kappaB) in macrophage (PAM) and Kupffer cell (KC), the concentration of tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant were measured by in situ hybridization (ISH), electrophoretic mobility shift assay (EMSA) and enzyme linked immunoadsorbent assay (ELISA), respectively. Then the blood gas, biochemistrical and pathological changes in lungs, liver and intestines were examined in each groups. RESULTS: In the MODS group, the expression of IKK-beta mRNA (0.15+/-0.03, 0.17+/-0.04), the activity of NF-kappaB (1.49+/-0.30, 1.72+/-0.36) and the levels of TNF-alpha[(279.74+/-25.91)ng/L, (300.05+/-30.86)ng/L] in PAM and KCs were significantly higher than those of normal controls[IKK-beta mRNA 0.03+/-0.01 and 0.02+/-0.01; NF-kappaB 0.25+/-0.06 and 0.31+/-0.10, TNF-alpha (137.33+/-6.09)ng/L and (134.85+/-12.09)ng/L, respectively, all P<0.01]. Also, the contents of blood urea nitrogen (BUN), alanine aminotransferase (ALT) in plasma significantly increased, the arterial oxygen partial pressure decreased, and the severity of organ damages in lungs, liver as well as intestines increased following MODS. CONCLUSION: The IKK-beta expression, NF-kappaB activation and cytokine release may play important roles in the pathogenesis of acute lung injury and MODS.

[Effects of polymixin B on I-kappaB kinase mRNA expression in lipopolysaccharide- induced pulmonary alveolar macrophages].

OBJECTIVE: To investigate effects of polymixin B (PMB) on I-kappaB kinase(IKK-beta), inhibitor protein(IkappaB-alpha) and nuclear factor-kappa B(NF-kappaB) in lipopolysaccharide(LPS)-induced pulmonary alveolar macrophages (PAM), and explore the anti-inflammatory mechanism of PMB. METHODS: PAM from rats collected by bronchoalveolar lavage was cultured and divided into three groups. In the control group, PAM was not stimulated with LPS and not treated with PMB. In the LPS stimulated group, PAM was stimulated with LPS. In the PMB treated group, PAM was pretreated with PMB half an hour prior to LPS stimulation. The expression of IKK-beta mRNA, level of IkappaB-alpha and the activity of NF-kappaB in PAM were measured by in situ hybridization(ISH), enzyme linked immunoadsorbent assay (ELISA) and electrophoretic mobility shift assay(EMSA), respectively. RESULTS: In the LPS stimulated group, the expression of IKK-beta mRNA (0.147+/-0.015) and activity of NF-kappaB (0.828+/-0.019) in PAM significantly increased, whereas levels of IkappaB-alpha (0.228+/-0.021) decreased (all P<0.01) in PMB treated groups. The expression of IKK-beta mRNA (0.112+/-0.022) and activity of NF-kappaB (0.358+/-0.011) were down-regulated while level of IkappaB-alpha (0.477+/-0.016) was up-regulated(P<0.01). CONCLUSION: LPS might induce expression of IKK-beta mRNA, degradation of IkappaB-alpha and activation of NF-kappaB PMB could inhibit the expression of IKK-beta, degradation of IkappaB-alpha and activation of NF-kappaB, showing marked anti-inflammatory property.