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Objective:To develop a recombinant protein vaccine based on KPC-2, a drug resistance target in Klebsiella pneumoniae, and evaluate its immunogenicity, protective efficacy and mechanism in a mouse model of pneumonia. Methods:KPC-2 was expressed in Escherichia coli and purified using GST affinity chromatography. A recombinant protein vaccine was prepared with KPC-2 and used to immunize New Zealand rabbits through subcutaneous injection. Serum samples were isolated from cardiac blood and Protein G chromatography was used to purify polyclonal antibodies against KPC-2. Opsonophagocytic killing assay was used to assess the bactericidal activity of the polyclonal antibodies in vitro. Female BALB/c mice were immunized three times with the recombinant protein vaccine, and the titers of specific IgG antibodies in serum were measured by indirect ELISA. One week after the last vaccination, the mice were infected with Klebsiella pneumoniae strain SRT through tracheal intubation, and received a single intravenous dose of meropenem (0.1 mg) 1 h later. The protective efficacy of the KPC-2 recombinant protein vaccine was evaluated by comparing the survival rates, bacterial colonization and histopathological changes between vaccine group and adjuvant group as well as the survival rates between meropenem group and normal saline group. Moreover, the protective efficacy of polyclonal antibodies against KPC-2 was evaluated through passive immunization. Results:The level of specific IgG antibodies in serum was significantly higher in the vaccine group than in the adjuvant group ( t=4.325, P<0.05). The survival rate in the vaccine group was also higher than that of the adjuvant group [70% (7/10) vs 10% (1/10), P<0.05]. Furthermore, lung inflammation was less severe and bacterial burden was reduced in the vaccine group as compared with those of the control group ( t=3.127, P<0.05). Both active and passive vaccination strategies demonstrated strong protective efficacy against Klebsiella pneumoniae infection, and had a synergistic effect when used in combination with antibiotic therapy. The polyclonal antibodies against KPC-2 had bactericidal activity in vitro ( t=5.427, P<0.05). Conclusions:The prepared KPC-2 vaccine has better immunogenicity and protective efficacy. It can induce strong humoral immune responses. This study suggest that drug resistance target may be used as a candidate antigen for future vaccine development.
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AIM:To observed the protective effect of diminazene aceturate ( DIZE) , an angiotensin-converting enzyme 2 (ACE2) activator, on diabetic nephropathy (DN) rats.METHODS:Male Wistar rats (n=30) were randomly divided into normal control (NC) group, DN group and DIZE group (each group consisted of 10 rats).The rats in DN group and DIZE group were induced by intraperitoneal injection of streptozotocin at dose of 65 mg/kg.After 12 weeks, the rats in DIZE group and DN group received subcutaneous injection of DIZE (15 mg· kg -1 · d-1 ) or vehicle for 4 weeks. The samples of blood and urine were collected at week 16, and ratio of kidney weight to body weight (KW/BW), plasma glucose (GLU), 24 h urinary protein (24UP) and serum creatinine (SCr) were measured.The renal pathological changes in each group were observed by periodic acid-Schiff ( PAS) staining and immunohistochemistry .The levels of AngⅡ and Ang-(1-7) in the plasma, and TGF-β1 and VCAM-1 in the renal tissues were measured by ELISA .The mRNA and protein levels of collagen I and FN were determined by quantification real-time PCR and immunohistochemistry .The effects of DIZE on the expression of ACE2 in DN rats were determined by Western blot .RESULTS:DIZE remarkably increased the expression of ACE2 and Ang-(1-7) in DN rats.Compared with NC group , the GLU, KW/BW, 24UP, SCr, and the ex-pression of collagen I , FN, TGF-β1 and VCAM-1 in DN group and DIZE group were increased .However , after treatment of the DN rats with DIZE, these indicators were decreased except the KW/BW.The GLU showed no significant change . CONCLUSION:DIZE raised the activity of ACE2 and increased the expression of Ang-(1-7), thus alleviating fibrosis and inflammation in the kidney and having therapeutic potential for diabetic nephropathy .
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This study aimed to evaluate emerging trends of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) among 290 former blood donor HIV-1 infected patients in Hubei,China,from 2004 to 2006,all of whom had received anti-HIV-1 therapy.The presence of NRTI- and NNRTI-associated mutations were established by sequencing; genotypic and predicted phenotypic drug resistance were evaluated using HIVdb Program version 5.0.1 (http://hivdb.stanford.edu/pages/algs/HIVdb.html).Genotypic drug resistance analysis showed significant increases in percentages of patients carrying HIV-1 strains with M41L,T215Y/F,D67N,K103N,G190A/S,Y181C/F or L210W mutations.Of the variants' predicted phenotypic drug resistance,highly significant increases were detected in percentages of patients carrying HIV-1 with high resistance to zidovudine (AZT) or stavudine (D4T) in NRTIs,and to delavirdine (DLV),efavirenz (EFV) or nevirapine (NVP) in NNRTIs; intermediate resistance to abacavir (ABC),AZT,D4/T,didanosine (DDI) or tenofovir disoproxil fumarate (TDF) in NRTIs,and to etravirine (ETR) in NNRTIs; and low and potential low resistance to lamivudine (3TC),ABC,emtricitabine (FTC) or TDF in NRTIs,and to ETR in NNRTIs.