Construction of an Immunostimulatory Plasmid, pUCpGs10, and Research on its Immune Adjuvant Effect

In order to overcome the instability of CpG ODN in vivo, sequence diversity, and individual differences, eleven CpG ODN fragments were meticulously selected and linked to form a Multi-CpG, which were repeatedly inserted into the cloning vector pUC19 for constructing the recombinant plasmid pUCpGs10 containing ten of Multi-CpG. Using the multi-genotype HCV E1 and multi-epitope complex HCV-T as immunogens, and plasmid pUCpGs10 as the immune adjuvant, Balb/c mice were immunized through nasal and subcutaneous immunization. Strong-specific humoral and cellular immune response were induced, which can obviously inhibit the growth of homograft expressing HCV antigen. The immune adjuvant effect of pUCpGs10 closely matched that of Freund’s complete adjuvant. The plasmid pUCpGs10 can significantly improve IgA content in serum and different mucosal extract and systematical T-cell response via intranasal immunization. In conclusions, the newly constructed immunostimulatory plasmid pUCpGs10 is able to effectively activate the humoral and cellular immune activity, and possesses activation on mucosal immune response.     

Sensitivity of Prestaining RNA with Ethidium Bromide Before Electrophoresis and Performance of Subsequent Northern Blots Using Heterologous DNA Probes

Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde–agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.     

Ecological Risk Assessment of Organochlorine Pesticides in Surface Waters of Lake Taihu,China

The residues of organochlorine pesticides (OCPs) in surface waters from Meiliang Bay, Gonghu Bay, and Xukou Bay of Lake Taihu, China, were investigated, and their ecological risks were assessed using the risk quotient method and probabilistic risk assessment. Environmental concentrations of OCPs in surface water of these bays were relatively lower compared with other rivers or lakes in China. Calculation of risk quotient associated with taxonomic groups indicates moderate ecological risks from OCPs for crustaceans and insects in these bays, while the ecological risks were low for fish and negligible for phytoplankton. The ecological risk quotients associated with individual OCPs were lower than 0.01 in these bays, suggesting a negligible risk to aquatic organisms. Ecological risk from α-HCH was relatively lower compared with DDTs, endosulfans, and γ-HCH. The combined ecological risks were evaluated using probabilistic risk assessment for only eight OCPs owing to a lack of available toxicity data for β-HCH and δ-HCH. The percentage of species with the potential to be at risk from mixture of OCPs was lower than the criteria of 5% in each bay, indicating that the combined ecological risks were acceptable.     

Mechanism for KRIT1 Release of ICAP1-Mediated Suppression of Integrin Activation

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Highlights? Cocrystal structure of KRIT1 with ICAP1 shows a bidentate interaction interface ? Cocrystal structure of ICAP1 with integrin β1 shows a PTB-peptide interaction ? KRIT1 antagonizes ICAP1-modulated integrin activation by the bidentate interaction ? KRIT1 contains a previously undescribed N-terminal Nudix domain     

Molecular characterization of methicillin-resistant Panton-valentine leukocidin positive <Emphasis Type="Italic">staphylococcus aureus</Emphasis> clones disseminating in Tunisian hospitals and in the community

Background

The spread of MRSA strains at hospitals as well as in the community are of great concern worldwide. We characterized the MRSA clones isolated at Tunisian hospitals and in the community by comparing them to those isolated in other countries.

Results

We characterized 69 MRSA strains isolated from two Tunisian university hospitals between the years 2004-2008. Twenty-two of 28 (79%) community-associated MRSA (CA-MRSA) strains and 21 of 41 (51%) healthcare-associated MRSA (HA-MRSA) strains were PVL-positive. The PVL-positive strains belonged to predicted founder group (FG) 80 in MLST and carried either type IVc SCCmec or nontypeable SCCmec that harbours the class B mec gene complex. In contrast, very diverse clones were identified in PVL-negative strains: three FGs (5, 15, and 22) for HA-MRSA strains and four FGs (5, 15, 45, and 80) for CA-MRSA strains; and these strains carried the SCCmec element of either type I, III, IVc or was nontypeable. The nucleotide sequencing of phi7401PVL lysogenized in a CA-MRSA strain JCSC7401, revealed that the phage was highly homologous to phiSA2mw, with nucleotide identities of more than 95%. Furthermore, all PVL positive strains were found to carry the same PVL phage, since these strains were positive in two PCR studies, identifying gene linkage between lukS and mtp (major tail protein) and the lysogeny region, both of which are in common with phi7401PVL and phiSa2mw.

Conclusions

Our experiments suggest that FG80 S. aureus strains have changed to be more virulent by acquiring phi7401PVL, and to be resistant to β-lactams by acquiring SCCmec elements. These novel clones might have disseminated in the Tunisian community as well as at the Tunisian hospitals by taking over existing MRSA clones.

     

Cyr61 is involved in neutrophil infiltration in joints by inducing IL-8 production by fibroblast-like synoviocytes in rheumatoid arthritis

Introduction

It is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients.

Methods

RA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay.

Results

We found that Cyr61 induced IL-8 production by RA FLS in an IL-1β and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPβ and NF-κB signaling pathways.

Conclusions

Our results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.