Asian Pacific Journal of Tropical Medicine

420-425

Authors: Chang-Wei Hsieh, Wen-Ching Ko, Wai-Jane Ho, Chao-Kai Chang, Guo-Jhou Chen, Jen-Chieh Tsai

Objective To investigate suitable condition for extraction of the active components from Ajuga nipponensis (A. nipponensis). Methods Orthogonal experimental design was used to determine the optimal extraction parameters for ecdysterones and flavonoids. Finally, the hepatoprotective abilities of A. nipponensis extracts were evaluated by CCl4-induced animal models. Results Maximum yields of flavonoids (7.87 ± 0.10) mg/g and ecdysterones (0.73 ± 0.02) mg/g could be obtained when the extraction time was 50 min, the extraction temperature was 60 °C, and the ratio of sample to 70% (v/v) ethanol was 1:20 (w/w). The antioxidant property of A. nipponensis was correlated to the concentration of its extracts. At 5 mg/mL, A. nipponensis extract scavenged 84.8% of DPPH radical and had absorbance values of 2.43 ± 0.04 reducing power. Upon CCl4-induced liver injury, glutamic oxaloacetic transaminase and glutamic pyruvic transaminase decreased significantly after the mice were treated with A. nipponensis. Histological researches also explained that A. nipponensis reduced the extent of liver lesions induced by CCl4. Conclusions A. nipponensis exhibited potent antioxidant activity in chemical experimental models and hepatoprotective effect against CCl4-induced liver damage.

432-436

Authors: Silmara Baroni, Bruno Ambrosio da Rocha, Juliana Oliveira de Melo, Jurandir Fernando Comar, Silvana Martins Caparroz-Assef, Ciomar Aparecida Bersani-Amado

Objective To evaluate the effect of hydroethanolic extract of yacon on the hyperglycemia induced by streptozotocin (STZ) in neonatal rats. Methods Wistar rats aged two days old received an intraperitoneal injection of STZ (160 mg/kg); after seven weeks, glycosuria was determined and animals with glucose levels above 250 mg/dL were included in the study. Groups of diabetic and non-diabetic rats were treated orally with yacon extract at a dose of 400 mg/kg/d for 14 d. Tests were made for phytochemical characterization, glucose tolerance and toxicity. Results The results showed that treatment with the extract reduced the glucose levels of fed diabetic rats and did not change the glucose levels of fasting diabetic and normal rats. Additionally, also it was observed that treatment with the extract reduced blood glucose levels of diabetic rats during the oral and intravenous glucose tolerance tests. There was no change in body weight, liver enzymes or mortality with yacon extract treatment. The phytochemical screening revealed the presence of caffeic acid, chlorogenic acid, ferulic acid and gallic acid. Conclusions The data suggest that yacon extract reduces hyperglycemia, possibly by improving insulin sensibility through its phytochemicals constituents (phenolic compounds).

445-452

Authors: Marta Giovanetti, Teresa Milano, Luiz Carlos Alcantara, Laura Carcangiu, Eleonora Cella, Alessia Lai, Alessandra Lo Presti, Stefano Pascarella, Gianguglielmo Zehender, Silvia Angeletti, Massimo Ciccozzi

Objective To investigate the genetic diversity of Zika Virus (ZIKV) and the relationships existing among these circulating viruses worldwide. To evaluate the genetic polymorphisms harbored from ZIKV that can have an influence on the virus circulation. Methods Three different ZIKV dataset were built. The first dataset included 63 E gene sequences, the second one 22 NS3 sequences and the third dataset was composed of 108 NS5 gene sequences. Phylogenetic and selective pressure analysis was performed. The edited nucleic acid alignment from the Envelope dataset was used to generate a conceptual translation to the corresponding peptide sequences through UGene software. Results The phylogeographic reconstruction was able to discriminate unambiguously that the Brazilian strains are belonged to the Asian lineage. The structural analysis reveals instead the presence of the Ser residue in the Brazilian sequences (however already observed in other previously reported ZIKV infections) that could suggest the presence of a neutralization-resistant population of viruses. Conclusions Phylogenetic, evolutionary and selective pressure analysis contributed to improve the knowledge on the circulation of ZIKV.

460-464

Authors: Dechassa Tegegne, Mukarim Abdurahaman, Tadesse Mosissa, Moti Yohannes

Objectives To assess the seroprevalence and associated risk factors of toxoplasmosis among HIV patients in Agaro Town Health Center of Jimma zone. Methods Convenient sampling was used to collect blood samples from 135 patients attending anti-retroviral therapy from February to March 2015. Serum samples were tested for anti-Toxoplasma gondii (T. gondii) antibody by using latex agglutination test. Structured questionnaire was used to collect data on socio-demographic and risk factors associated with toxoplasmosis. Results Overall seroprevalence of toxoplasmosis was 80.7% (109/135, CI: 74.04–87.36). In multivariate analysis significant association was observed between anti-T. gondii seropositivity and raw meat consumption (OR: 3.514, CI: 1.167–10.581, P = 0.025), knowledge about toxoplasmosis (OR: 5.225, CI: 1.382, P = 0.015) and sex (OR: 4.023, CI: 1.382–19.762, P = 0.015). Conclusions Immuno compromised patients showed high rate of seropositivity and thus, it is highly advisable to introduce routine anti-T. gondii antibodies serological screening test prior to ART commencement.

470-473

Authors: Ying Kuang, Ying-Jie Nie

Objective To study the regulatory effects of miR-21 on breast cancer cell line proliferation and invasion as well as the downstream target genes. Methods Breast cancer cell lines MCF-7 were cultured and transfected with miR-21 mimics and the corresponding negative control mimics (NC mimics), and then MTS kits were used to detect cell viability. Transwell experiment was used to detect cell invasion ability, and fluorescence quantitative PCR was used to detect the expression of proliferation and invasion-related genes in cells. Results 24 h after transfection of miR-21 mimics and NC mimics, cell OD value and the number of invasive cells of miR-21 group were significantly higher than those of NC group, and mRNA contents of PDCD-4, FasL, PTEN, RhoB, Maspin, TIMP3 and RECK in cells were significantly lower than those of NC group. Conclusion miR-21 can promote the proliferation and invasion of breast cancer cell lines, and its downstream target genes include PDCD-4, FasL, PTEN, RhoB, Maspin, TIMP3 and RECK.

478-483

Authors: Xiao-Zhou Liu, Cheng-Jun Li, Su-Jia Wu, Xin Shi, Jian-Ning Zhao

Objective To investigate the role of survivin in osteosarcoma metastasis. Methods Small interfering RNA (siRNA) was used to knockdown the expression of survivin and α5 integrin in the human osteosarcoma cell line MG63. Western blotting and immunostaining methods were used to assessed the effect of survivin knockdown on the expression of α5 integrin through flow cytometry and fluorescence microscopy detection. Meanwhile, the invasion and migration of transfected cells in Transwell and wound healing assays were probed, and the growth situation of these cells transplanted into nude mice was monitored. Results Knockdown of survivin expression could inhibit the invasion and migration of osteosarcoma MG64 cells in vitro and the expression of α5 integrin on osteosarcoma MG64 cell surface, suggesting that survivin can inhibit the invasion and migration of osteosarcoma cells through downregulation of α5 integrin. Anti-α5 integrin antibody could also markedly decrease the capability of invasion and migration of osteosarcoma MG64 cells. Additionally, knockdown of survivin expression could slow the growth of osteosarcoma MG63 cells transplanted into nude mice. Conclusions Survivin-directed anti-tumor strategies might be an effective method in the treatment of osteosarcoma.

489-493

Authors: Zi-Shu Wang, Hong Jin, Dong-Ming Wang

Objective To observe the influence of different concentrations of homocysteine (Hcy) and hydrogen sulfide (H2S) on the secretion and activation of matrix metalloproteinase-2 (MMP-2) in cardiocytes so as to search for new ways to fight against myocardial tissue fibrosis. Methods Cardiocytes H9C2 was cultured in vitro and different concentrations of Hcy and H2S were added for 6-h and 24-h cultivation. MTT cell proliferation assay was applied to test the activation change of cardiocytes H9C2 after affecting by different concentrations of Hcy and H2S. ELISA and MTT were employed to detect the expression and enzymatic activity of MMP-2. Results The H9C2 cell inhibition of activity was more significant with 1 000 μmol/L of Hcy as compared with other concentrations (P < 0.001). With 2.5–100.0 μmol/L Hcy and 0.1, 1.0 and 10.0 mmol/L H2S, the activity of H9C2 did not change significantly (P > 0.05). Hcy with concentrations of 10, 50 and 100 μmol/L could increase the quantity of MMP-2 secreted by cardiocytes H9C2, and the interaction strength was concentration-dependent (P < 0.05). After interacting with 100 μmol/L of Hcy for 6 h, the zymogen activation effect of MMP-2 was stronger than that of the 2.5–25 μmol/L group (P < 0.05). After interacting with Hcy and H2S (1.0 mmol/L) for 6 h and 24 h, the activation effect of MMP-2 was stronger than those interacted with 10, 25, 50 and 100 μmol/L of Hcy (P < 0.05). Conclusions Hcy can increase the production of MMP-2 secreted by H9C2 cell and improve its zymogen activation. Besides, the interaction strength is concentration-dependent; while H2S can up-regulate the activation of MMP-2 and co-promote the activation of MMP-2 with Hcy as well.

499-502

Authors: Dong Guo, Ye-Wei Wang, Ji Ma, Lei Yan, Teng-Fei Li, Xin-Wei Han, Shao-Feng Shui

Objective To investigate the correlation between JNK signal and the apoptosis of VSMC as well as the expression of Cathepsin B and to explore the role of JNK signal in the development of cerebral aneurysm. Methods Rat models of cerebral aneurysm were established and histopathologic changes of cerebral aneurysm and the apoptosis of VSMC were analyzed. Rat models were respectively subject to subcutaneous injection of Cathepsin B siRNA and JNK inhibitor SP600125. Western blot technique was used to detect the expression of proteins like Cathepsin B, Caspase-3, and p-JNK. Spearman's rho was used to examine the correlation between p-JNK and Cathepsin B, as well as the expression of relevant proteins. Results The success rate of modeling rats with cerebral aneurysm was 88.75%. After the respective injection of Cathepsin B siRNA, SP600125 and their combination, the cell densities of VSMC of rats with cerebral aneurysm all increased significantly (P < 0.05 or P < 0.01), but the apoptosis rate of VSMC decreased significantly (P < 0.01). Compared with normal rats, the expression of Cathepsin B, Caspase-3 and p-JNK in Cerebral aneurysm models increased significantly. Effectively intervening Cathepsin B genes with Cathepsin B siRNA could significantly inhibit the expression of Cathepsin B and Caspase-3, but hardly influence the expression of p-JNK. JNK inhibitor SP600125 had no influence on the expression of Cathepsin B and Caspase-3, but effectively inhibited the expression of p-JNK. In cerebral aneurysm tissues, positive correlation was observed between the expression of p-JNK and Cathepsin B, the correlation coefficient was r = 0.640. Conclusion After the attack of cerebral aneurysm, proteins like Cathepsin B, Caspase-3 and p-JNK are all involved in the apoptosis of VSMCs. This process may be realized by Cathepsin B which activates the apoptosis mechanism of Caspase-3 and mediate the apoptosis of VSMC through the JNK signaling pathway. Therefore, silencing Cathepsin B gene or inhibiting the conduction through JNK signaling pathway can mitigate the apoptosis of vascular smooth muscle cells in cerebral aneurysm.

508-512

Authors: Ting-Ting Ji, Xuan Huang, Jie Jin, Sheng-Hua Pan, Xiao-Ju Zhuge

Objective To discuss the expression of long noncoding RNA TUG1 (lncRNA-TUG1) in gastric carcinoma (GC) and its effects on the transferring and invading capacity of gastric carcinoma cells. Methods Forty cases of carcinoma tissue and para-carcinoma tissue were selected from GC patients who underwent surgical removal in Zhejiang Provincial Hospital of Chinese Traditional Medicine and Wenzhou Central Hospital from January, 2013 to December, 2014; the expressing level of lncRNA-TUG1 in GC and para-C tissues was detected by applying the qRT-PCR technique. The correlation between lncRNA-TUG1 expression and patients' clinical data was classified and analyzed. SGC-7901 cells were transfected using lncRNA-TUG1 specific siRNA. Changes of the transferring and invading capacity of siRNA-transfected SGC-7901 cells were scratch-tested and transwell-detected. qRT-PCR was applied to detect the expression level of microRNA-144 after lncRNA-TUG1 was silenced. Changes of c-Met mRNA and protein expressions was detected by qRT-PCR and western-blot test. Results The expression level of lncRNA-TUG1 in GC tissue was significant higher than that in para-C tissue (P < 0.05) and the high expression level of lncRNA-TUG1 in GC tissue was significantly correlated with tumor lymph nodes metastasis and advance TNM phasing (P < 0.05). The transferring and invading capacity of SGC-7901 cells was highly inhibited after being transfected by lncRNA-TUG1 specific siRNA (P < 0.05). The results of qRT-PCR and western-blot proved that the expression of microRNA-144 was significantly boosted and the expression level of c-Met mRNA and protein was inhibited after lncRNA-TUG1 was silenced (P < 0.05). Conclusions lncRNA-TUG1 shows an up-regulated expression in GC tissue and that bears a correlation with clinicopathological features of malignant tumor. lncRNA-TUG1 can promote the transferring and invading capacity of GC by inhibiting the pathway of microRNA-144/c-Met.

1136-1140

Authors: M. Zamri-Saad, M.I. Kamarudin

Brucellosis is a zoonotic disease characterized by reproductive failure in animals and undulent fever in humans. In cattle, it is caused by Brucella abortus while in goats by Brucella melitensis, the main cause of brucellosis in humans. Brucellosis in livestock has been associated with importation of animals from breeder herd of unknown disease status. The prevalence of bovine brucellosis Brucella abortus in 2014 ranged between 1% and 2% in Thailand and Indonesia, and 4%–5% in Malaysia and Myanmar. Prevalence of goat brucellosis Brucella melitensis is approximately 1% in Malaysia and Thailand. ‘Test-and-slaughter’ is the general policy against brucellosis adopted by most ASEAN countries to eradicate the disease. Under this program, the Rose Bengal Plate Test (RBPT) is used as the screening test to identify infected farm/herd while the complement fixation test (CFT) is the confirmatory test. The test-and-slaughter eradication strategy that was implemented since 1979 had managed to keep the prevalence rate to less than 5%, from 3.3% in 1979, 0.23% in 1988, 1% in 1998 and 5% in 2016. The test-and-slaughter program seemed effective in reducing the prevalence of brucellosis but was unable to eradicate the disease due to several factors, which include failure to locate and identify the remaining affected animals and to control their movement, importation of breeder animals from non-brucellosis free countries and lack of participation by the farmers following unreliable test results. To support the eradication policy, research activities since 1980s have suggested combinations of serological tests to improve diagnosis while surveillance should be focused on hotspots areas. The prevalence can be further reduced by strictly sourcing breeder animals from brucella-free areas or countries.

1150-1157

Authors: Eyad M.A. Alshammari, Raju K. Mandal, Mohd Wahid, Sajad A. Dar, Arshad Jawed, Mohammed Y. Areeshi, Saif Khan, Md. Ekhlaque Ahmed Khan, Aditya K. Panda, Shafiul Haque

Objective To summarize the precise association between pulmonary tuberculosis (PTB) and P2x7 A1513C gene polymorphism. Methods PubMed and Google Scholar web-databases were searched for the studies reporting the association of P2x7 A1513C polymorphism and PTB risk. A meta-analysis was performed for the selected case–control studies and pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for all the genetic models. Results Eleven studies comprising 2 678 controls and 2 113 PTB cases were included in this meta-analysis. We observed overall no significant risk in all the five genetic models. When stratified population by the ethnicity, Caucasian population failed to show any risk of PTB in all the genetics models. In Asian ethnicity, variant allele (C vs. A: P = 0.001; OR = 1.375, 95% CI = 1.159–1.632) and heterozygous genotype (AC vs. AA: P = 0.001; OR = 1.570, 95% CI = 1.269–1.944) demonstrated significant increased risk of PTB. Likewise, recessive genetic model (CC + AC vs. AA: P = 0.001; OR = 1.540, 95% CI = 1.255–1.890) also demonstrated increased risk of PTB in Asians. Conclusions Our meta-analysis did not suggest the association of P2x7 A1513C polymorphism with PTB risk in overall or separately in Caucasian population. However, it plays a significant risk factor for predisposing PTB in Asians. Future larger sample and expression studies are needed to validate this association.

1165-1171

Authors: Pramote Sriwanitchrak, Atchara Paemanee, Sittiruk Roytrakul, Vithoon Viyanant, Kesara Na-Bangchang

Objective To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini (OV)-associated CCA in OV/dimethylnitrosamine (DMN)-induced CCA hamster model. Methods Nine Syrian hamsters were divided into 3 groups as follows (n = 3 each): normal (healthy control group); OV group; and OV/DMN group (CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavalin A (ConA)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LC-MS/MS. Results Among the 24 ConA-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1 (NDRG1) and fetuin-B (FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN (CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform ×3 (NSFL1C) was found only in the samples from OV/DMN (CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups. Conclusions NDRG1, FETUB and NSFL1C glycoproteins isolated by ConA-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.

1181-1194

Authors: Omar M.E. Abdel-Salam, Eman R. Youness, Nadia A. Mohammed, Noha N. Yassen, Yasser A. Khadrawy, Safinaz Ebrahim El-Toukhy, Amany A. Sleem

Objective To study the effect of citric acid given alone or combined with atropine on brain oxidative stress, neuronal injury, liver damage, and DNA damage of peripheral blood lymphocytes induced in the rat by acute malathion exposure. Methods Rats were received intraperitoneal (i.p.) injection of malathion 150 mg/kg along with citric acid (200 or 400 mg/kg, orally), atropine (1 mg/kg, i.p.) or citric acid 200 mg/kg + atropine 1 mg/kg and euthanized 4 h later. Results Malathion resulted in increased lipid peroxidation (malondialdehyde) and nitric oxide concentrations accompanied with a decrease in brain reduced glutathione, glutathione peroxidase (GPx) activity, total antioxidant capacity (TAC) and glucose concentrations. Paraoxonase-1, acetylcholinesterase (AChE) and butyrylcholinesterase activities decreased in brain as well. Liver aspartate aminotransferase and alanine aminotransferase activities were raised. The comet assay showed increased DNA damage of peripheral blood lymphocytes. Histological damage and increased expression of inducible nitric oxide synthase (iNOS) were observed in brain and liver. Citric acid resulted in decreased brain lipid peroxidation and nitric oxide. Meanwhile, glutathione, GPx activity, TAC capacity and brain glucose level increased. Brain AChE increased but PON1 and butyrylcholinesterase activities decreased by citric acid. Liver enzymes, the percentage of damaged blood lymphocytes, histopathological alterations and iNOS expression in brain and liver was decreased by citric acid. Meanwhile, rats treated with atropine showed decreased brain MDA, nitrite but increased GPx activity, TAC, AChE and glucose. The drug also decreased DNA damage of peripheral blood lymphocytes, histopathological alterations and iNOS expression in brain and liver. Conclusions The study demonstrates a beneficial effect for citric acid upon brain oxidative stress, neuronal injury, liver and DNA damage due to acute malathion exposure.

1200-1211

Authors: Azza A. Matloub, Hadeer A. Aglan, Sahar Salah Mohamed El Souda, Mona Elsayed Aboutabl, Amany Sayed Maghraby, Hanaa H. Ahmed

Objective To explore the in vivo anticancer, anti-angiogenesis and immunomodulatory efficacies of the bioactive polysaccharide isolated from cold aqueous extract of Jania rubens (JCEM) and Pterocladia capillacea (PCEM) as well as hot aqueous extract of Enteromorpha intestinalis (EHEM) against hepatocellular carcinoma rat model (HCC) and to study their chemical composition. Methods The sugars and amino acids composition of the bioactive polysaccharides of JCEM, PCEM and EHEM were determined using gas liquid chromatography and amino acid analyzer, respectively. These polysaccharide extracts (20 mg/kg b.wt. for 5 weeks) were assessed on hepatocarcinogenesis in rats and α-fetoprotein (AFP), carcinoembryonic antigen (CEA), glypican-3 (GPC-3), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) and Ig G levels were evaluated. Results The GLC analysis of JCEM, PCEM and EHEM polysaccharide revealed the presence of 10, 9 and 10 sugars, in addition the amino acid analyzer enable identification of 16, 15 and 15 amino acids, respectively. These polysaccharide extracts of JCEM, PCEM and EHEM produced significant decrease in serum AFP, CEA, GPC-3, HGF and VEGF compared with untreated HCC group. JCEM, PCEM and EHEM had an immunostimulatory responses by increasing the IgG levels as compared by naïve value (1.23, 1.53 and 1.17 folds), respectively. The bioactive polysaccharides in HCC induced rats improved the humoral immune response. The photomicrographs of liver tissue sections of the groups of HCC treated with polysaccharide extracts of Jania rubens and Enteromorpha intestinalis showed intact histological structure. Moreover, fractions HE1, HE4, HE7 obtained from polysaccharide of EHEM showed moderate cytotoxic activity against HepG2 in vitro with IC50 73.1, 42.6, 76.2 μg/mL. However, fractions of PCEM and JCEM show no or weak cytotoxicity against HepG2 in vitro where the cytotoxic activity of their crude polysaccharide extract proved synergetic effect. Conclusions The pronounced antitumor activity of sulfated polysaccharide-protein complexes of JCEM and EHEM is due to direct cytotoxic activity, anti-hepatocarcinogenesis, and anti-angiogenesis. In addition, JCEM, PCEM and EHEM had an immunostimulatory response and improved the humoral immune response in HCC induced rats.

1215-1217

Authors: Tatyana Vladimirovna Moskvina, Albina Dmitrievna Bartkova, Alexey Vasil'evich Ermolenko

Objective To detect nematode eggs in sandpits in urban area in Vladivostok. Methods Totally 60 samples were collected from five districts. Sand samples were analysed using combined flotation-sedimentation method. In result, 18 sand samples were positive for at one or two parasites species. Results Overall, only 3 genera of helminths were recovered. Most samples were positive for Toxocara spp. eggs. Toxocara spp. eggs were found in each district. More samples with Toxocara spp. eggs were found in Pervomaiskii district. Ascaris spp. eggs were also appeared in each district, however Ascaris eggs rate of contamination was less than rate of Toxocara spp. contamination. Toxascaris leonina eggs were the less frequent species in sandpits, it was found only in Sovetskii and Pervomaiskii districts. Ascaris eggs rate of contamination was less than rate of Toxocara spp. contamination. Toxascaris leonina eggs were the less frequent species in sandpits, it was found only in two districts. No correlation between districts and helmiths eggs present in soil samples was found (P > 0.05). Conclusions Considering the high sand contamination with parasite infective elements of both human and animal origin, measures to improve environmental and sanitary conditions are indicated, as well as promoting the concept of responsible pet ownership.

1222-1225

Authors: Claudia Weilg, Olguita Del Aguila, Fernando Mazulis, Wilmer Silva-Caso, Carlos Alva-Urcia, Rosario Cerpa-Polar, Erick Mattos-Villena, Juana Del Valle Mendoza

An 11 year old, hispanic girl with a history of B-cell acute lymphoblastic leukemia was admitted to the hospital for symptoms compatible with Bartonella henselae infection. The first molecularly diagnosed case of disseminated Bartonella henselae infection was reported in an immunocompromised patient in Lima, Peru. The analysis was confirmed by Polymerase Chain Reaction and automated sequencing of a liver biopsy sample, even though the serologic tests were negative. In conclusion, Bartonella spp. infection should have a particular diagnostic consideration in immunocompromised patients with fever of unknown origin and further investigation regarding the patient's past exposures with cats should also be elicited.

1035-1043

Authors: Kamonlak Leecharoenkiat, Pathrapol Lithanatudom, Wannapa Sornjai, Duncan R. Smith

Iron deficiency anemia and iron overload conditions affect more than one billion people worldwide. Iron homeostasis involves the regulation of cells that export iron into the plasma and cells that utilize or store iron. The cellular iron balance in humans is primarily mediated by the hepcidin–ferroportin axis. Ferroportin is the sole cellular iron export protein, and its expression is regulated transcriptionally, post-transcriptionally and post-translationally. Hepcidin, a hormone produced by liver cells, post-translationally regulates ferroportin expression on iron exporting cells by binding with ferroportin and promoting its internalization by endocytosis and subsequent degradation by lysosomes. Dysregulation of iron homeostasis leading to iron deposition in vital organs is the main cause of death in beta-thalassemia patients. Beta-thalassemia patients show marked hepcidin suppression, ineffective erythropoiesis, anemia and iron overload. Beta-thalassemia is common in the Mediterranean region, Southeast Asia and the Indian subcontinent, and the focus of this review is to provide an update on the factors mediating hepcidin related iron dysregulation in beta-thalassemia disease. Understanding this process may pave the way for new treatments to ameliorate iron overloading and improve the long term prognosis of these patients.

1048-1054

Authors: Theerarat Kochakarn, Namfon Kotanan, Krittikorn Kümpornsin, Duangkamon Loesbanluechai, Monta Thammasatta, Prasert Auewarakul, Prapon Wilairat, Thanat Chookajorn

Objective To understand the cause for the differences between potentially mild Southeast Asian and the more pathogenic ZIKV in South America. Methods A comparative genomic analysis was performed to determine putative causations stemming from ZIKV. Results Phylogenetic analyses integrating geographical and time factors revealed that Southeast Asian ZIKV might not be the direct source of South American outbreaks as previously speculated. Amino acid residues unique to South American ZIKV isolates at the envelope, pr and NS1 proteins are listed and shown in the structural context. These unique residues on external viral proteins are not found in Southeast Asian ZIKV and could be responsible for the ongoing outbreak either via an intrinsic property of the virus or interactions with human immunity. Only a selected few primer/probe sets currently in clinical use were identified of being capable of detecting ZIKV strains worldwide. The envelope proteins of dengue virus (DENV) and ZIKV also showed a remarkable degree of similarity especially at the surface residues. Conclusions These findings may help explain the cross-reactivity of DENV antibodies to ZIKV. Thus, major caveats must be exercised in using existing diagnostic tools for ZIKV.

1062-1068

Authors: Mojtaba Shahnazi, Abbas Azadmehr, Ammar Andalibian, Reza Hajiaghaee, Mehrzad Saraei, Mahmood Alipour

Objective To evaluate the scolicidal and immunomodulatory effect of the Ziziphora tenuior (Z. tenuior) extract and its fractions. Methods Protoscolices were treated with six concentrations (3, 5, 10, 25, 50, and 100 mg/mL) of Z. tenuior extract and its fractions (ethanol, petroleum ether, ethyl acetate and chloroform) in periods of 10, 20, 30, 40, 50 and 60 min, and viability of protoscolices was evaluated using the 1.0% eosin. To examine the immunomodulatory effects of Ziziphora and its fractions on macrophage cells, the non-toxic concentration of extract and different fractions determined by MTT assay, and the Griess reaction was used to measure the level of nitrite as an indicator of nitric oxide by the macrophage cells in 10, 100 and 200 μg/mL in 24 h at 37 °C. Results In this study, the Z. tenuior extract at 10 mg/mL concentration was able to kill all protoscolices during 20 min. By increasing the concentration to 25 mg/mL, the scolicidal time reduced to 10 min. Regarding the effect of different fractions of Z. tenuior, the ethanolic fraction showed the highest scolicidal activity. The extract demonstrated an inhibitory effect on the activity of macrophages and reduced nitric oxide production. Although the petroleum ether and ethanolic fractions of the extract reduced nitric oxide production, nevertheless, this effect was only significant at 10 and 100 μg/mL concentrations (P < 0.05). Conclusion The Z. tenuior extract and its fractions were effective against protoscolices yet the effect of total extract was considerable. Our findings indicates that the extract and its ethanolic and petroleum ether fractions could have anti-inflammatory properties.

1075-1077

Authors: Alireza Ahmadi, Mehrdad Hajilooi, Ghasem Solgi, Mohammad Abasi, Ahad Bazmani, Alireza Khalilian, Mohammad Matini, Khosro Sardarian

Objective To evaluated the relationship between the genetic variations at IL-8 +2767 position with VL pathogenesis among Iranian patients. Methods Three groups including patients with VL clinical presentation and leishmania seropositive (n = 124), patients seropositive but without clinical presentation (n = 82) and healthy controls (n = 63) were selected to conduct this cross-sectional study. Polymorphism at +2767 position of IL-8 was investigated using PCR-RFLP techniques. Anti-leishmania antibody titration was evaluated by the immunoflorescence technique. Results We observed higher significant frequencies +2767 A/A and A/T genotypes in Group 1 compared to Group 2 and healthy controls (P = 0.001). Also, patients in Group 1 carrying A/A genotype showed higher titer of anti-leishmania antibody than patients with A/T and T/T genotypes (P = 0.05). The validity of the data was analyzed using Hardy–Weinberg equilibrium and one way analysis of variance (ANOVA), as well as χ2 tests. Conclusions Our findings indicate that the IL-8 +2767 polymorphism is significantly involved in impaired immune responses against VL and it could be considered as a risk factor for the VL progress.

1084-1088

Authors: Qi Li, Ke Hu, Si Tang, Li-Fang Xu, Yu-Chuan Luo

Objective To explore the anti-tumor activity of tanshinone IIA in combined with cyclophosphamide against Lewis mice with lung cancer and the effect on cellular immune function. Methods Lewis tumor cells were inoculated subcutaneously into the right armpit of mice in each group (n = 20) to establish Lewis lung cancer mice model. After model establishment, mice in the model group were given normal saline by lavage, qd. Mice in treatment I group were given intraperitoneal injection of Tan IIA, 15 mg/kg, qd. Mice in treatment II group were given intraperitoneal injection of CTX, 25 mg/kg, qd. Mice in treatment III group were given intraperitoneal injections of Tan IIA and CTX, in which the administration method of Tan IIA was the same as in treatment I group, continuously for 2 weeks, and the dosage of CTX was the same as in treatment II group, 24 h after model establishment, every other day. Mice were sacrificed 2 weeks after establishment. The tumor tissues were collected to calculate the anti-tumor rate. Immunohistochemistry was used to detect the expressions of Bcl-2, Bax, VEGF, Angiostatin, and Endostatin. FCM was used to detect T lymphocyte subsets in spleen and liver of mice. Results The tumor weight in treatment I, II, and III groups was significantly lower than that in the model group (P < 0.05). The tumor weight in treatment III group was significantly lower than that in treatment I and II groups (P < 0.05). The anti-tumor rate in treatment II and III groups was significantly higher than that in treatment I group (P < 0.05). Bcl-2 expression in the tumor tissues of treatment I, II, and III groups was significantly lower than that in the model group (P < 0.05), while Bax expression was significantly higher than that in the model group (P < 0.05). Bcl-2 expression in the tumor tissues of treatment I and II groups was significantly higher than that in treatment III group (P < 0.05), while Bax expression was significantly lower than that in treatment III group (P < 0.05). CD4+ and CD4+/CD8+ in treatment I, II, and III groups were significantly higher than those in the model group (P < 0.05). CD4+ in treatment III group was significantly higher than that in treatment I and II groups (P < 0.05), while CD4+/CD8+ was significantly higher than that in treatment II group (P < 0.05). The comparison of CD8+ among each group was not statistically significant (P > 0.05). NK cell activity in treatment I, II, and III groups was significantly higher than that in the model group (P < 0.05). NK cell activity in treatment III group was significantly higher than that in treatment I and II groups (P < 0.05). Conclusions Tan IIA in combined with CTX can down regulate Bcl-2 expression in lung cancer tissues, up regulate Bax expression, inhibit the neovascularization of tumor tissues, and enhance the immunological function, with a significant anti-tumor activity.

1095-1100

Authors: Wei Chen, Yue-Qiang Hu, Ling-Fei Jiang, Lin Wu

Objective To explore the protective effect and possible mechanism of action of Zhuyu Annao pill in mice with intracerebral hemorrhage (ICH). Methods Sixty mice were divided into the control group, hemorrhage group, drug-treated group (after hemorrhage), TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group (after hemorrhage) with 12 in each group. Model of autologous ICH was established in all groups. After drilling and 12 h of fasting, models in the control group hemorrhage group and TLR4-knockout hemorrhage group were all drenched with 10 mL/kg distilled water by intragastric administration. Models in the drug-treated group and TLR4-knockout hemorrhage + drug-treated group were drenched with 6.25 g/kg of Zhuyu Annao pill. All groups were treated for 7 d. Longa scoring method was used to measure the neurological defect scores and determine the brain water contents of all groups; ELISA was employed to detect the inflammatory factor interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and IL-1β in brain tissues; and Western blot was applied to test the expression quantities of apoptotic protein Bax and anti-apoptotic protein Bcl-2 in brain tissues. Results At day 3 and 7, compared with the hemorrhage group, the neurological defect scores of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group decreased significantly (P < 0.05). Compared with the hemorrhage group, the brain water contents of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group reduced significantly (P < 0.05). Compared with the hemorrhage group, the inflammatory factor IL-6, TNF-α and IL-1β of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group decreased significantly (P < 0.05). Compared with the hemorrhage group, the expression of apoptotic protein Bax of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group decreased significantly and the expression of anti-apoptotic protein Bcl-2 increased significantly (P < 0.05). Conclusions Zhuyu Annao pill can alleviate encephaledema for mice with ICH and reduce inflammatory responses and nerve cell apoptosis. TLR4 can mediate inflammatory injury induced by ICH. Thus, Zhuyu Annao pill can play a protective role for brains by decreasing the expression of TLR4.

1105-1110

Authors: Bo Yang, Sheng-Ze Li, Ling Ma, Hong-Li Liu, Jian Liu, Jun-Jun Shao

Objective To explore the expression, biological function and possible mechanism of action of microRNA molecular-196a (miR-196a) in epithelial ovarian cancer. Methods RT-PCR was used to detect the expression quantities of epithelial ovarian tissue, benign ovarian tissue, normal ovary epithelial tissue, ovarian cancer cell lines and miR-196a in normal ovarian epithelial cells to analyze the relationship between the expression of miR-196a and the clinical pathologic parameters of ovarian cancer. Among those cell lines, the cell line of which miR-196a expressed the most or least was selected and transfected the ovarian cancer cell line by using negative control plasma and miR-196a inhibitor. After transfection, RT-PCR was used to test the expression quantity of miR-196a, Transwell chamber method was applied to determine the migration and invasion abilities of ovarian carcinoma cells and Western blot was employed to detect the expression of HOXA10 protein. Results The relative expression quantities of miR-196a in ovarian cancer tissue and benign ovarian tissue were significantly higher than that in normal ovarian epithelial tissue, and the expression quantity of miR-196a in ovarian cancer tissue was distinctively higher than that in benign ovarian tissue (P < 0.05). Among 78 cases of epithelial ovarian cancer, the expression quantities of miR-196a in patients with low differentiation were all significantly higher than those in patients with high differentiation (P < 0.05). The expression of miR-196a showed no significant relation with age, clinical stage and whether CA125 was positive or not in patients (P > 0.05). Compared with normal ovarian epithelial cell line IOSE80, the expression quantities of miR-196a of all ovarian cancer cell lines increased obviously and differences were statistically significant (P < 0.05). Among them, the expression of miR-196a of ovarian cancer cell line SKOV3 was the highest, while it decreased significantly (4.678 ± 0.785 vs. 2.131 ± 0.345, t = 2.938, P < 0.05) after the ovarian cancer cell line SKOV3 was transfected by miR-196a inhibitor. The results of Transwell chamber method showed that the migration and invasion abilities of ovarian cancer cells SKOV3 were declined significantly after the expression of miR-196a was down-regulated and the difference showed statistical significance (P < 0.05). The results of Western blot revealed that the relative expression of HOXA10 decreased distinctly after the expression of miR-196a was down-regulated and also the difference showed statistical significance (P < 0.05). Conclusions The miR-196a might serve as a cancer-promoting gene to promote the migration and invasion of epithelial ovarian cancer by downstream target gene HOXA10.

1115-1122

Authors: Harapan Harapan, Samsul Anwar, Aslam Bustaman, Arsil Radiansyah, Pradiba Angraini, Riny Fasli, Salwiyadi Salwiyadi, Reza Akbar Bastian, Ade Oktiviyari, Imaduddin Akmal, Muhammad Iqbalamin, Jamalul Adil, Fenni Henrizal, Darmayanti Darmayanti, Rovy Pratama, Jhony Karunia Fajar, Abdul Malik Setiawan, Mandira Lamichhane Dhimal, Ulrich Kuch, David Alexander Groneberg, R. Tedjo Sasmono, et al.

Objective To explore and understand the attitude towards dengue vaccination and its modifiable determinants among inhabitants of Aceh (northern Sumatra Island, Indonesia), the region that was most severely affected by the earthquake and tsunami of 26 December 2004. Methods A community-based, cross-sectional study was conducted among 535 healthy inhabitants in nine regencies (Kabupaten or Kotamadya) of Aceh that were selected randomly from November 2014 to March 2015. A set of validated, pre-tested, structured questionnaires was used to guide the interviews. The questionnaires covered a range of explanatory variables and one outcome variable (attitude to dengue vaccination). Multi-step logistic regression analysis and Spearman's rank correlation were used to test the role of explanatory variables for the outcome variable. Results More than 70% of the participants had a poor attitude towards dengue vaccination. Modifiable determinants associated with poor attitude to dengue vaccination were low education level, working as farmers and traditional market traders, low socioeconomic status and poor knowledge, attitude and practice regarding dengue fever (P < 0.05). The KAP domain scores were correlated strongly with attitude to dengue vaccination, rs = 0.25, rs = 0.67 and rs = 0.20, respectively (P < 0.001). Multivariate analysis found that independent predictors associated with attitude towards dengue vaccination among study participants were only sex and attitude towards dengue fever (P < 0.001). Conclusions This study reveals that low KAP regarding dengue fever, low education level and low socioeconomic status are associated with a poor attitude towards dengue vaccination. Therefore, inhabitants of suburbs who are working as farmers or traditional market traders with low socioeconomic status are the most appropriate target group for a dengue vaccine introduction program.

925-932

Authors: A. Oryan, M. Akbari

Recently, vector-borne parasitic diseases such as leishmaniasis have been emerged or re-emerged in many geographical areas and resulted in global health and economic concerns that involve humans, domestic animals and wild life. The ecology and epidemiology of leishmaniasis are affected by the between host, reservoir and vector (human, animal and sandfly) and the environment. Important drivers for the emergence and spread of leishmaniasis include environmental factors such as alterations in temperature and water storage, irrigation habits, deforestation, climate changes, immunosuppression by HIV or organ transplant, development of drug resistance, increase traveling to endemic regions and dog importation. War, poor socio-economic status and low level household are also major contributors to the spread of this disease. Health education via the public media and training should be implemented by international organizations and governmental agencies in collaboration with research institutions. Fully protection during transmission season, using bednets and insecticides and reservoirs' control should be also mentioned in the planning. Based on the findings of the recent studies and high prevalence of leishmaniasis, it is concluded that serious public health monitoring should be considered.

938-946

Authors: Suryasnata Das, Reena Parida, I. Sriram Sandeep, Sanghamitra Nayak, Sujata Mohanty

Betelvine (Piper betle L.) is cultivated for its deep green heart shaped leaf for (15–20) million Indian and 2 billion foreign consumers annually. The crop provides Rs (6 000–7 000) million of national income per year and at the same time leaves worth Rs (30–40) million is exported to other countries. The leaves are not only used directly for chewing purposes but also possesses antioxidant, anti-inflammatory, anti-apoptotic, anti-cancer and anti-microbial properties. Besides, the leaves also contain eugenol rich essential oil (1%–3%) which is the source for medicine, stimulant, antiseptic, tonic and other ayurvedic formulations. The essential oil also contains chavibetol, caryophyllene and methyl eugenol which are the potent source for preparation in ayurvedic medicine and herbal products. Cost of betelvine essential oil is 10$ per 5 mL. In spite of its great economical and medicinal importance betelvine is still neglected by the researchers for proper characterization and authentication for selection of elite landraces. Lack of awareness among people, use of same planting material for many generations, existing of many synonyms for a single landraces, no proper characterization of available landraces are some of the significant constraints for its commercialization. Our review endeavours a complete advance in the research on betelvine, existing lacunae for its proper characterization and commercial cultivation. It also attempts to provide a comprehensive account on biotechnological interventions made in betelvine aimed at complementing conventional programmes for improvement of this nutraceutically important cash crop.

954-961

Authors: Suleman Atique, Shabbir Syed Abdul, Chien-Yeh Hsu, Ting-Wu Chuang

Objective To identify the influences of local and regional climate phenomena on dengue transmission in Lahore District of Pakistan, from 2006 to 2014. Methods Time-series models were applied to analyze associations between reported cases of dengue and climatic parameters. The coherence trend of regional climate phenomena (IOD and ENSO) was evaluated with wavelet analysis. Results The minimum temperature 4 months before the dengue outbreak played the most important role in the Lahore District (P = 0.03). A NINO 3.4 index 9 months before the outbreaks exhibited a significant negative effect on dengue transmission (P = 0.02). The IOD exhibited a synchronized pattern with dengue outbreak from 2010 to 2012. The ENSO effect (NINO 3.4 index) might have played a more important role after 2012. Conclusions This study provides preliminary results of climate influences on dengue transmission in the Lahore District of Pakistan. An increasing dengue transmission risk accompanied by frequent climate changes should be noted. Integrating the influences of climate variability into disease prevention strategies should be considered by public health authorities.

968-972

Authors: Aletheia Blasi, Alessandra Lo Presti, Eleonora Cella, Silvia Angeletti, Massimo Ciccozzi

Objective To estimate the genetic diversity of Kokobera virus, the date of origin and the spread among different viruses in the endemic regions of Australia. Methods Two datasets were built. The first consisting of 29 sequences of the NS5/3′ UTR region of Kokobera group downloaded from GenBank, the second including only 24 sequences of Kokobera viruses, focus is on this group. Results Bayesian time analysis revealed two different entries in Australia of Kokobera virus in the 50s years with the dated ancestor in 1861 year. Clades A and B showed a clear separation of the Kokobera sequences according to the geographic region. Conclusions Data from the study showed as Kokobera virus, despite of its ancient origin and its circulation before the European colonization, remained limited to the Australian country and nowadays limited mostly to the regions were Australian marsupials are mostly found.

979-984

Authors: Maureen Leyva, Leidys French-Pacheco, Felipe Quintana, Domingo Montada, Mayda Castex, Ariel Hernandez, María del Carmen Marquetti

Objective To evaluate an essential oil with larvicide, adulticide and growth inhibitory activity against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes, of medical importance. Methods Standardized methodology by WHO was used to determine the levels of susceptibility of mosquito larvae exposed to the essential oil. To evaluate the adulticide activity with the essential oil at different doses, bottles were impregnated according to the methodology CDC. To determine the development inhibitory activity of Melaleuca quinquenervia (M. quinquenervia) oil in three mosquito species, third instar larvae were exposed to the LC50 and LC90 dose (calculated for each population) of M. quinquenervia oil in glass containers with a capacity of 500 mL. After 24 h exposure, the dead larvae were discarded. The mortality of larvae and pupae were recorded on a daily basis. Results The calculated LC50 indicates an order of effectiveness of preferential oil for Culex quinquefasciatus (LC50 = 0.002 1%), Aedes aegypti (LC50 = 0.004 7%) and Aedes albopictus (LC50 = 0.004 9%). Conclusions The adulticide activity was achieved with impregnated bottles at 40 and 50 mg/mL with the three mosquitoes species. In larvae, a growth inhibition was detected when exposed to sublethal doses. The results indicate that M. quinquenervia is a plant with promising environmentally sustainable source for vector control.

991-994

Authors: Wattana Sukeepaisalncharoen, Udomlack Peansukwech, Kittisak Sawanyawisuth

Objective To find additional factors suggestive of hepatitis C virus (HCV) infection in the general population by using data from a hepatitis virus survey. Methods This study collected data of HCV infection from a hepatitis virus survey. The survey was conducted in 13 provinces in the northeast Thailand in 2014 and 2015. During the survey, a blood test was performed to screen for HCV. A questionnaire was also distributed to all participants asking about baseline characteristics, risk factors for HCV infection, and daily life activities. Risk factors for HCV infection were executed. Results There were 2 112 participants for the survey. Of those, 110 participants (5.21%) tested positive for HCV infection. After adjustment by multivariate logistic regression, three factors were significantly associated with HCV infection, namely male gender, age, and family history of liver cancer. The adjusted ORs and 95% CI of these factors were 3.14 (1.50, 6.56), 3.78 (1.12, 12.76), and 2.28 (1.08, 4.80), respectively. Conclusions Male gender, increasing age, and family history of liver cancer are predictors of HCV infection in endemic areas. Males with a family history of liver cancer in their first-degree relatives should be tested for HCV infection regardless of symptoms.

1002-1006

Authors: So Jung Kim, So Yun Park, Sun-Mee Hong, Eun-Hye Kwon, Taek-Kyun Lee

Objective To determine skin whitening and wrinkle improvement efficacy, glycoprotein fractions were extracted from liquid extracts of boiled sea cucumber and their effects on tyrosine and elastase inhibitory activities were assayed. Methods Fractions above and below 50 kDa (>50 kDa and <50 kDa) were extracted via a series of steps involving: boiling, filtering, desalting and freeze drying. Cytotoxicity, skin whitening and wrinkle-removing effects of boiled liquid were determined. Results Our MTT data showed that neither glycoprotein fraction of boiled liquid induces cellular cytotoxicity up to a concentration of 10 mg/mL treatment of the mouse melanoma cell line, B16F10, with 10 mg/mL >50 kDa enhanced tyrosinase and elastase inhibitory activities by 50.84% and 28.78%, respectively. Correlations of the >50 kDa concentration with tyrosinase inhibitory (R2 = 0.968) and elastase inhibitory (R2 = 0.983) efficacy were significant. Conclusions >50 kDa glycoprotein fraction isolated from liquid extracts of boiled sea cucumber, which can serve as a functional cosmetic ingredient for whitening and wrinkle improvement of skin.

1013-1018

Authors: Bang-Jiang Fang, Jun-Yi Shen, Hua Zhang, Shuang Zhou, Chuan-Zhu Lyu, Yi-Qiang Xie

Objective To study the effect of emodin on protein and gene expressions of the massagers in mobility signal transduction system of cholecyst smooth muscle cells in guinea pig with cholesterol calculus. Methods The guinea pigs were randomly divided into 4 groups, such as control group, gall-stone (GS) group, emodin group and ursodeoxycholic acid (UA) group. Cholesterol calculus models were induced in guinea pigs of GS, emodin and UA groups by lithogenic diet, while emodin or UA were given to the corresponding group for 7 weeks. The histomorphological and ultrastructure change of gallbladder were detected by microscope and electron microscope, the content of plasma cholecystokinin (CCK) and [Ca2+]i were analyzed successively by radioimmunoassay and flow cytometry. The protein and mRNA of Gsα, Giα and Cap in cholecyst cells were determined by western blotting and real time polymerase chain reaction (RT-PCR). Results Emodin or UA can relieve pathogenic changes in epithelial cells and muscle cells in gallbladder of guinea pig with cholesterol calculus by microscope and transmission electron microscope. In the cholecyst cells of GS group, CCK levels in plasma and [Ca2+]i decreased, the protein and mRNA of GS were down-regulated, the protein and mRNA of Gi and Cap were up-regulated. Emodin significantly decreased the formative rate of gallstone, improved the pathogenic change in epithelial cells and muscle cells, increased CCK levels in plasma and [Ca2+]i in cholecyst cells, enhanced the protein and mRNA of Gs in cholecyst cells, reduced the protein and mRNA of Gi and Cap in cholecyst cells in guinea pig with cholesterol calculus. Conclusion The dysfunction of gallbladder contraction gives rise to the disorders of mobility signal transduction system in cholecyst smooth muscle cells, including low content of plasma CCK and [Ca2+]i in cholecyst cells, abnormal protein and mRNA of Gs, Gi and Cap. Emodin can enhance the contractibility of gallbladder and alleviate cholestasis by regulating plasma CCK levels, [Ca2+]i in cholecyst cells and the protein and mRNA of Gs, Gi and Cap.

1022

Authors: Somsri Wiwanitkit, Viroj Wiwanitkit

None

832-838

Authors: Walter Chingwaru, Richard H. Glashoff, Jerneja Vidmar, Petrina Kapewangolo, Samantha L. Sampson

Mycobacterium tuberculosis and human immunodeficiency virus (HIV) co-infections have remained a major public health concern worldwide, particularly in Southern Africa. Yet our understanding of the molecular interactions between the pathogens has remained poor due to lack of suitable preclinical models for such studies. We reviewed the use, this far, of mammalian cell culture models in HIV–MTB interaction studies. Studies have described the use of primary human cell cultures, including (1) monocyte-derived macrophage (MDM) fractions of peripheral blood mononuclear cell (PBMC), alveolar macrophages (AM), (2) cell lines such as the monocyte-derived macrophage cell line (U937), T lymphocyte cell lines (CEMx174, ESAT-6-specific CD4+ T-cells) and an alveolar epithelial cell line (A549) and (3) special models such as stem cells, three dimensional (3D) or organoid cell models (including a blood–brain barrier cell model) in HIV–MTB interaction studies. The use of cell cultures from other mammals, including: mouse cell lines [macrophage cell lines RAW 264.7 and J774.2, fibroblast cell lines (NIH 3T3, C3H clones), embryonic fibroblast cell lines and T-lymphoma cell lines (S1A.TB, TIMI.4 and R1.1)]; rat (T cells: Rat2, RGE, XC and HH16, and alveolar cells: NR8383) and primary guinea pigs derived AMs, in HIV–MTB studies is also described. Given the spectrum of the models available, cell cultures offer great potential for host-HIV–MTB interactions studies.

844-850

Authors: Hamza Dar, Tahreem Zaheer, Muhammad Talha Rehman, Amjad Ali, Aneela Javed, Gohar Ayub Khan, Mustafeez Mujtaba Babar, Yasir Waheed

Objective To predict immunogenic promiscuous T cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools. To date, no epitope data are available for the Zika virus in the IEDB database. Methods We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks. A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using PorPred1 and ProPred immunoinformatic algorithms respectively. The antigenicity predicted score was also calculated for each predicted epitope using the VaxiJen 2.0 tool. Results By using ProPred1, 23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus. The greatest number of MHC class I binding epitopes were projected within the NS5 (21%), followed by Envelope (17%). For MHC class II, greatest number of predicted epitopes were in NS5 (19%) followed by the Envelope, NS1 and NS2 (17% each). A variety of epitopes with good binding affinity, promiscuity and antigenicity were predicted for both the HLA classes. Conclusion The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates, which will be able to induce a broad range of immune responses in a heterogeneous HLA population. However, our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.

856-859

Authors: Pimwan Thongdee, Jiraporn Kuesap, Raewadee Wisedpanichkij, Kesara Na-Bangchang

Objective To preliminarily investigate the possible role of prostaglandin D2 (PGD2) in malaria infections. Methods Blood and urinary samples (n = 120 each) were collected from Thai patients with Plasmodium falciparum (P. falciparum) with moderate (n = 26) and high (n = 4) parasitemia, patients with Plasmodium vivax (P. vivax) (n = 30), patients with fever associated with other infections (n = 30), and healthy subjects (n = 30). PGD2 concentrations in plasma and urinary samples of healthy subjects, patients with fever associated with other infections and patients with malaria were determined using Prostaglandin D2-MOX express EIA kit (Cayman Chemical, USA). Results The possible association between PGD2 and malaria infections is clearly demonstrated with PGD2 concentration in urine. The urinary PGD2 concentrations were relatively high (about 5-fold) in patients with P. falciparum with moderate parasitemia and P. vivax infections compared with other groups. Furthermore, the concentration in patients with P. falciparum with moderate parasitemia and P. vivax infection were significantly higher than that in healthy subjects and patients with fever associated with other infections. Conclusions Urinary PGD2 concentrations may offer a more dependable and useful tool for predicting malaria severity. Confirmation is this preliminary finding is required with a larger sample size.

866-871

Authors: Tooba Mahboob, Abdul-Majid Azlan, Tian-Chye Tan, Chandramathi Samudi, Shamala Devi Sekaran, Veeranoot Nissapatorn, Christophe Wiart

Objective To examine the acanthamoebicidal effects of ethyl acetate, aqueous and butanol fractions of dried flower buds of Lonicera japonica (L. japonica) Thunb. (Flos Lonicerae) in vitro. Methods Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR. They were exposed to ethyl acetate, water and butanol fractions of L. japonica Thunb. at concentrations ranging from 0.5 mg/mL to 1.5 mg/mL. The extracts were evaluated for growth inhibition at 24, 48 and 72 h, respectively. Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment. Results Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h, 49.2% after 48 h and 33.7% after 72 h chlorogenic acid, the major active constituent of L. japonica Thunb. at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h, 84.0% after 48 h and 72.3% after 72 h. This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h. Conclusions Results obtained from this study show that ethyl acetate fraction at 1.5 mg/mL is the most potent fraction of L. japonica Thunb. and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

877-881

Authors: Bang-Jiang Fang, Jun-Yi Shen, Hua Zhang, Shuang Zhou, Chuan-Zhu Lyu, Yi-Qiang Xie

Objective To study the mechanism of insulin resistance in the cholesterol gallstone formation from insulin signal transduction pathway so as to reveal the possible mechanism and the effective role of Albiflorin Granule on preventing the cholesterol gallstones. Methods Serum triglycerides (TG), free fatty acid (FFA), and total cholesterol (TC) from different groups were measured and liver cells InsR, PKB, IKK-β protein expression levels were detected by western blotting. Results Albiflorin significantly decreased the cholesterol gallstone formation rate, increased glucose infusion rate in gallstone guinea pigs and improved insulin resistance. Compared with the normal group, insulin receptor and PKB protein expression in GS group were significantly reduced. IKK-β protein in the GS group increased significantly and Albiflorin could reduce IKK-β protein expression in guinea pig liver cells. Conclusions The model of insulin resistance in cholesterol gallstone guinea pig was successfully established, which plays an important role in the cholesterol gallstone formation. All aspects of insulin signaling pathway are involved in gallstone formation. Albiflorin can regulate various aspects of insulin signal transduction pathway to prevent the formation of gallbladder.

887-892

Authors: Tahereh Mikaeili Galeh, Abdolhassan Kazemi, Mahmoud Mahami-Oskouei, Behzad Baradaran, Adel Spotin, Seddigheh Sarafraz, Majid Karamat

Objective To identify the frequencies (F) of ferredoxin and nitroreductase mutations on Iranian clinical isolates of Giardia lamblia in order to predict whether the nitazoxanide can be prescribed as suitable drug for symptomatic to metronidazole-resistant giardiasis. Methods Forty Giardia lamblia isolates as of 38 symptomatic and two metronidazole-resistant patients were collected from Iran. DNAs were extracted and amplified by targeting ferredoxin and GlNR genes. The amplicons were directly sequenced to determine gene mutations. Results The various amino acid substitutions (F: 20%, Haplotype diversity: 0.891, Tajima's D: −0.440 13) were identified by analyzing ferredoxin gene in four symptomatic and two resistant isolates. Only two haplotypes (F: 5%, HD: 0.345; Tajima's D: 0.778 15) characterized in metronidazole-resistant isolates of GlNR, however, no point mutations was found in symptomatic isolates. Conclusions Non-synonymous mutations of ferredoxin oxidoreductase gene reduce translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression and its activity. This leads to decrease in metronidazole drug delivery into the cells. Mutations in these isolates may lead to their resistance to metronidazole. No to low synonymous mutations of GlNR demonstrates that nitazoxanide can be prescribed as promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in Iranian clinical isolates.

898-902

Authors: Chang-Jiang Lei, Chun Yao, De-Ke Li, Zhi-Xiong Long, Yuan Li, Dan Tao, Yan-Ping Liou, Jiang-Zhou Zhang, Ning Liu

Objective To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7. Methods Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132, respectively or together. The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry, and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR. Results Compared with the control group, the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly, and the apoptosis ratio increased distinctly (P < 0.05). Besides, the effect of the co-transfection group was better than that of the single transfection group. The protein levels of GPC3 (Glypican-3) and YAP (Yes-associated protein), the target genes transfected only by miR-520c-3p and miR-132, respectively, reduced obviously (P < 0.05), which was similar with the co-infected cells, but cells transfected by miR-132 only showed a decrease of YAP. Conclusions The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP, enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.

909-912

Authors: Arshad Javaid, Mazhar Ali Khan, Mir Azam Khan, Sumaira Mehreen, Anila Basit, Raza Ali Khan, Muhammad Ihtesham, Irfan Ullah, Afsar Khan, Ubaid Ullah

Objective To assess the profile of TB/multidrug-resistant TB (MDR-TB) among household contacts of MDR-TB patients. Methods Close contacts of MDR-TB patients were traced in the cross-sectional study. Different clinical, radiological and bacteriological were performed to rule out the evidence of TB/MDR-TB. Results Between January 2012 and December 2012, a total of 200 index MDR-TB patients were initiated on MDR-TB treatment, out of which home visit and contacts screening were conducted for 154 index cases. Of 610 contacts who could be studied, 41 (17.4%) were diagnosed with MDR-TB and 10 (4.2%) had TB. The most common symptoms observed were cough, chest pain and fever. Conclusions The high incidence of MDR-TB among close contacts emphasize the need for effective contact screening programme of index MDR-TB cases in order to cut the chain of transmission of this disease.

916-919

Authors: Adil Coskun, Erdogan Malatyali, Hatice Ertabaklar, Mustafa B. Yasar, Ali O. Karaoglu, Sema Ertug

Objective To determine Blastocystis frequency and subtypes (ST) in ulcerative colitis (UC) patients and analyse some laboratory findings between Blastocystis positive and negative cases. Methods Faecal samples from 150 UC patients in Adnan Menderes University, Training and Research Hospital were examined by direct microscopy and cultivated in Jones medium. Blastocystis positive cultures were subjected to DNA isolation and subtypes were identified by sequencing of barcode region. A retrospective analysis was conducted on C reactive protein (CRP), leucocyte counts (WBC), neutrophil counts, and sedimentation rates. Results The overall positive rate of Blastocystis was 8% (12 patients) and the most abundant subtype was ST3 (eight isolates, 66.7%), followed by ST1, ST2 and ST7. Laboratory findings between Blastocystis infected and non-infected UC patients were not significantly different. Blastocystis frequency was 3.8% among the patients in active stage, while it was 11.8% among the patients in remission stage. Conclusions The present study confirms previous findings that have indicated the predominance of Blastocystis ST3 in humans and contributes additional evidence that suggests the low colonisation of Blastocystis infection in ulcerative colitis patients during active stage.