Asian Pacific Journal of Tropical Medicine

933-939

Authors: B Senthilkumar, K Anbarasu, D Senbagam, M Rajasekarapandian

Objective To develop attenuated strains of Salmonella enterica serovar Typhi (S. typhi) for the candidate vaccine by osmolar stress. Methods S. typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Namakkal, Tamil Nadu, India. Both strains were grown in LB (Luria Bertani) medium supplemented with various concentration of NaCl (0.1–0.7M) respectively. The effect of osmolar stress was determined at molecular level by PCR using MGR 06 and MGR 07 primers corresponding to ompR with chromosomal DNA of S. typhi SS3 and SS5 strains. Attenuation by osmolar stress results in deletion mutation of the S. typhi strains was determined by agglutination assays, precipitation method, SDS PAGE analysis and by animal models. Results The 799 bp amplified ompR gene product from wild type S. typhi SS3 and SS5 illustrate the presence of virulent gene. Interestingly, there was only a 282 bp amplified product from S. typhi SS3 and SS5 grown in the presence of 0.5, 0.6 and 0.7 M NaCl. This illustrates the occurrence of deletion mutation in ompR gene at high concentration of NaCl. Furthermore, both the wild-type and mutant S. typhi outer membrane SDS-PAGE profile reveals the differences in the expression of ompF, ompC and ompA proteins. In mice, wild type and mutant strains lethal dose (LD50) were determined. The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU.mL−1. When the mice were injected with the mutants in same dosage, no clinical symptoms were observed; whereas the serum antibody titre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response. These results suggest that S. typhi SS3 and SS5 may be used as good candidate strains for the development of live attenuated vaccine against salmonellosis. Conclusions This study demonstrates that the S. typhi strains were attenuated and could be good vaccine candidates in future.

946-951

Authors: Pritom Chowdhury, Siraj Ahmed Khan, Rashmee Topno, Prafulla Dutta, R.N.S. Yadav, Jagadish Mahanta

Objective To assess the best suitable condition for virus inactivation, and to study the immunogenic potential and protective efficacy of a circulating West Nile virus (WNV) strain in Assam. Methods Bulk preparation of circulating WNV: WNIRGC07 (GeneBank ID: HQ246154), was undertaken in a bioreactor using cytodex-1. Virus Inactivation was done in three different conditions; 22 °C, 4 °C and room temperature. The virus preparations were evaluated for antigenicity by ELISA and toxicity by cell proliferation kit. Virus efficacy was done in-vivo on swiss albino mice against standard Indian WNV and Japanese encephalitis virus (JEV) strain. Humoral and cell mediated immune response was evaluated in mice sera by ELISA and neutralization assay. Results Inactivation at 22 °C was found to be more suitable in terms of less toxicity and high antigenicity. The same was selected to study the immune response and efficacy in mice. It induced neutralizing antibody titre of 1: 625 and high IgG response. In vivo experiment showed 100% protective efficacy against WNV and 20.8% cross protective efficacy against JEV. Further assessment of cellular immunity through immunized mice revealed augmentation of high levels of pro-inflammatory cytokines and moderate levels of anti-cytokines indicating a mixed balance of Th1 and Th2 response. Conclusions Findings suggest that formalin inactivated Indian WNV strain has a good immunogenic potential. This is the first study on assessment of immunogenic potential of a lineage 5 strain of WNV. Our study reveals that it would be a promising and effective candidate for vaccine studies which warrants further evaluation.

956-961

Authors: Abbas Khani, Monireh Heydarian

Objective To test fumigant and repellent properties of sesquiterpene-rich essential oil from Teucrium polium subsp. capitatum (L.). Methods The fumigant toxicity test was performed at (27±1)°C, (65±5)% relative humidity, and under darkness condition and 24 h exposure time. The chemical composition of the isolated oils was examined by gas chromatography-mass spectrometry. Results The major compounds were α-cadinol (46.2%), caryophyllene oxide (25.9%), α muurolol epi (8.1%), cadalene (3.7%) and longiverbenone (2.9%). In all cases, considerable differences in mortality of insect to essential oil vapor were observed in different concentrations and exposure times. Callosobruchus maculatus (C. maculates) (LC50=148.9 μL/L air) was more susceptible to the tested plant product than Teucrium castaneum (T. castaneum) (LC50=360.2 μL/L air) based on LC50 values. In the present investigation, the concentration of 3 μL /mL acetone showed 60% and 52% repellency against T. casteneum and C. maculatus adults, respectively. Conclusions The results suggests that sesquiterpene-rich essential oils from the tested plant could be used as a potential control agent for stored-product insects.

968-976

Authors: Thirunavukkarasu Santhoshkumar, Abdul Abdul Rahuman, Chidambaram Jayaseelan, Govindasamy Rajakumar, Sampath Marimuthu, Arivarasan Vishnu Kirthi, Kanayairam Velayutham, John Thomas, Jayachandran Venkatesan, Se-Kwon Kim

Objective To determine the efficacies of antibacterial and antioxidant activities of aqueous leaf extract of Psidium guajava mediated biosynthesis of titanium dioxide nanoparticles (TiO2 NPs). Methods Synthesized TiO2 NPs were tested by disc diffusion method against against human pathogenic bacteria. The total antioxidant activity and phenolic content (Folin-Ciocalteau method) of synthesized TiO2 NPs and aqueous plant extract were determined. The scavenging radicals were estimated by DPPH method. The synthesized TiO2 NPs were characterized by XRD, FTIR, FESEM and EDX. Results FTIR spectra of synthesized TiO2 NPs exhibited prominent peaks at 3 410 cm−1 (alkynes), 1 578 cm−1, 1 451 cm−1 (alkanes), and 1 123 cm−1(C-O absorption). The morphological characterization of synthesized TiO2 NPs was analysed by FESEM which showed spherical shape and clusters with an average size of 32.58 nm. The maximum zone of inhibition was observed in the synthesized TiO2 NPs (20 μg/mL) against Staphylococcus aureus (25 mm) and Escherichia coli (23 mm). The synthesized TiO2 NPs showed more antibacterial activity than the standard antibiotic disk, tetracycline which drastically reduces the chances for the development of antibiotics resistance of bacterial species. The plant aqueous extract and synthesized TiO2 NPs were found to possess maximum antioxidant activity when compared with ascorbic acid. The content of phenolic compounds (mg/g) in leaf aqueous extract and synthesized TiO2 NPs were found to be 85.4 and 18.3 mgTA/g, respectively. Conclusions Green synthesized TiO2 NPs provides a promising approach can satisfy the requirement of large-scale industrial production bearing the advantage of low-cost, eco-friendly and reproducible.

985-990

Authors: Bao-Qin Liu, Xin Gong, Zhe Jin

Objective To investigate the effects of traditional Chinese medicine, Danzhi decoction, on the expression angiogenesis factors in human endometrial cells during the sequelae of pelvic inflammatory disease (SPID) and explore the role of Danzhi decction in improving the blood stasis microenvironment of SPID. Methods A three-dimensional (3D) co-culture system including human vascular endothelial cells (VECs), endometrial stromal cells and glandular epithelial cells was established in vitro and treated with Danzhi decoction, sterilized water and aspirin respectively. A Milliplex multifunctional liquid chip technique was used to measure the expression levels of vascular endothelial growth factor (VEGF)-A/C/D, fibroblast growth factor −1/2, angiopoietin-2, epidermal growth factor (EGF), HB-EGF, bone morphogenetic protein-9, endoglin, endothelin-1, granulocyte colony stimulating factor, hepatocyte growth factor, interleukin-8, follistatin, placenta growth factor and leptin. The location of angiogenesis factors was monitored by immunofluorescence labeling and confocal laser scanning microscope 3D reconstruction. Results Endometrial stromal cells and glandular epithelial cells were isolated and primary cultured for establishing a 3D co-culture system. The levels of VEGF-A/C/D in Danzhi decoction group and aspirin group were significantly lower than those in mock group (P<0.05), while there was no significant difference between Danzhi decoction group and aspirin group (P>0.05). Furthermore, the alterative location of VEGF-A/C/D was observed in the cytoplasm of endometrial glandular epithelial cells. Conclusions Danzhi decoction may inhibit the expression of VEGF in the blood stasis microenvironment of SPID by targeting the cytoplasm of endometrial glandular epithelial cell.

996-999

Authors: Hong-Mei Liu, Kai-Jie Wang

Objective To investigate the therapeutic effect of the intervention treatment with different doses of Captopril on TNF-αcontents in serum of rheumatoid arthritis (RA) rats, and to provide the theoretical proofs for clinical application of Captopril in treatments of rheumatoid diseases. Methods Fifty Wistar rats were randomly divided into 5 groups, namely, Group A, Group B, Group C, Group D, Group E with 10 rats in each group. Injection of Freund's complete adjuvant was employed to establish adjuvant-induced arthritis model in rats. Group A was model group; after model establishment, rats were treated with 20 mL normal saline as placebo (ip.). Rats in Group B were treated with 8 mg/kg cyclophosphamide (ip.). Rats in Group C, D and E were intraperitoneally injected with 30 mg/kg, 100 mg/kg and 300 mg/kg Captopril respectively. Rats in each group were subjected to continuous treatment for 3 weeks, and then sacrificed. Eyeballs of rats were excised and blood was collected. TNF-αcontent in serum were detected using ELISA; each group rats were compared for the hind legs arthrocele. Right ankle tissues of rats were collected to prepare section, and microscopic observation of pathological changes was performed. Results TNF-αcontent in serum of Group A rats was significantly higher than that of rats in other 4 groups (P<0.05). TNF-αcontent in serum of Group B rats was significantly lower compared with that of rats in Groups C, D and E. The highest TNF-αcontent in serum of rats treated with Captopril was found in Group C, followed by Groups D and E (P<0.05). Right ankle arthrocele of rats in Groups B, C, D and E in early stage showed no statistical difference compared with that of Group A rats (P>0.05). From Day 8, ankle arthrocele of rats in Groups B, C, D and E was obviously relieved compared with that of Group A rats; the anti-inflammatory effects were gradually enhanced with the extension of medication time. Treatments of Groups C, D and E showed significant activities against tardive arthrocele; the degree of ankle arthrocele in rats of these three groups was lower than that of Group A rats (P<0.01). Histological observation showed that large amount of inflammatory cells and plasmocyte infiltration was found in ankle synovial tissues of Group A rats. Relief of hyperaemia and edema of right ankle synovial tissues as well as significant decrease in synoviocyte layer hyperplasia, intra-articular inflammatory cells infiltration and cartilago articularis damage degree etc. were observed in Groups B, C, D and E. Conclusions Intervention treatment with Captopril can effectively reduce the TNF-αcontent in serum of rheumatoid arthritis rats and inhibit the generation of inflammatory factors, so as to achieve the therapeutic effect.

1005-1008

Authors: Tie-Gang Jia, Jian-Qing Zhao, Jian-Hua Liu

Objective To explore the serum levels of IL-32, MMP-9,PCT and CRP in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Methods A total of 50 patients with AECOPD and 45 cases with acute asthma attack admitted from October 2013 to August 2014 were selected, and the serum levels of IL-32, MMP-9, PCT and CRP were determined and compaed by using Double antibody sandwich Enzyme linked immunosorbent assay, immunofluorescence double antibody sandwich assay and immunoturbidimetry assay. Results Serum levels of IL-32, MMP-9, PCT and CRP were significantly higher in AECOPD group than acute asthma attack group (P<0.05). IL-32 and MMP-9 were negatively correlated with lung function. MMP-9 in AECOPD patients was increased more significantly. Conclusions Serum levels of IL-32 and MMP-9 were negatively correlated with lung function, and the worse the lung function is, the more significant the increase is.