Asian Pacific Journal of Tropical Medicine

320-329

Authors: Meghan Tipre, Paul Vijay Kingsley, Tamika Smith, Mark Leader, Nalini Sathiakumar

Objective: To conduct an epidemiological and clinical review of published case reports of melioidosis from India and Bangladesh. Methods: Data from published case reports were abstracted and summarized. We further compared the clinical epidemiology of the melioidosis cases in India with case series from highly endemic areas in Northern Australia and Southeast Asia to elucidate any differences in presentations and risk factors between the regions. Results: We identified a total of 99 cases published between 1953 and June 2016, originating from India (n=85) or Bangladesh (n=14). Cases were predominantly male and ranged in age from 1 month to 90 years. Diabetes mellitus was the most common risk factor reported (58%). About 28% of the cases had history of exposure via high-risk occupations or exposure to contaminated water. The overall case fatality rate (CFR) was 26%. Factors influencing mortality included the occurrence of septic shock (CFR, 80%), environmental exposure (CFR, 39%), primary presentation of pneumonia (CFR, 38%), misdiagnosed and/or mistreated cases (CFR, 33%) or the presence of a risk factor (CFR, 29%). Because of the small number of cases in Bangladesh, pattern of clinical epidemiology is limited to India. Soft tissue abscess (37%) was the most common clinical presentation reported from India followed by pneumonia (24%) and osteomyelitis/septic arthritis (18%). Neurological melioidosis (n=10, 12%) presented as pyemic lesions of the brain or meninges. A few cases of prostatic abscess (n=4) in men and parotid abscess (n=4) were also noted. The above patterns were consistent with case series from Southeast Asia and Northern Australia for the most part, in terms of risk factors associated with infection and factors influencing mortality. Differences included clinical presentation of pneumonia which was notably lower than that reported in Southeast Asia and Northern Australia; a higher proportion of neurological and parotid abscess presentation; and a lower CFR compared to that reported in case series in Southeast Asia. About 39% of the cases were misdiagnosed and/or mistreated, suggesting underreporting and under estimation of the true disease burden. Conclusions: The concentration of melioidosis cases in southern and eastern states in India and in Bangladesh, which share climatic conditions and rice farming activities with known endemic areas in Southeast Asia, suggests an endemicity of melioidosis in this region. Thus, increased awareness among healthcare personnel, particularly among clinicians and nurses practicing in rural areas, and improved surveillance through case registries is essential to guide early diagnosis and prompt treatment.

336-341

Authors: Maryam Homayoon, Yahya Tahamtan, Mohammad Kargar, Seyed Mohammad Hossein Hosseini, Abbas Akhavan Sepahy

Objective: To evaluate the effects of Pasteurella multocida (P. multocida) vaccines on the expression and release of antibodies, interleukin (IL)-6 and IL-12 by serum. Methods: Balb/c mice were immunized with two formalin and iron inactivated vaccine doses within 2 weeks. The vaccines were adjuvant with P. multocida A strain, P. multocida B strain and Salmonella typhimurium bacterial DNA (AbDNA, BbDNA and SbDNA for short, respectively). The animals were challenged 4 weeks after immunization. Blood of mice was collected to detect the change of specific antibody, IL-6, and IL-12 using ELISA. Results: The specific antibody and interleukins in the immunized group increased significantly compared to the control mice after vaccination and challenge (P<0.05). The highest release of these cytokines was obtained by P. multocida inactivated with iron and adjuvant with AbDNA at a concentration of 25 μg/mL. The antibody titer peak was 0.447 in mice vaccinated with iron-killed whole-cell antigen adjunct with AbDNA. The time-courses of release showed that bacterial DNA was able to stimulate IL-6 and IL-12 production more than alum (P<0.05). Conclusions: Our findings introduce that bacterial DNA is capable of releasing an immunological response with several cytokines. These indicate that bacterial DNA entrapped with killed P. multocida antigen is a new and effective adjuvant to enhance specific immunity and resistance of animal against the infectious pathogen, which could simplify the development of highly promising strong adjuvant.

350-354

Authors: Tauhidur Rahman Nurunnabi, Shaymaa Al-Majmaie, Ismini Nakouti, Lutfun Nahar, S. M. Mahbubur Rahman, Md. Hossain Sohrab, Md. Morsaline Billah, Fyaz M. D. Ismail, George P Sharples, Satyajit D Sarker

Objective: To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate (EtOAc) extract of endophytic fungus Colletotrichum gloeosporioides (C. gloeosporioides) isolated from Sonneratia apetala. Methods: Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Gram-positive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay. Results: The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites. Kojic acid (1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1D, 2D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms. Whilst the minimum inhibitory concentration (MIC) values from the EtOAc extract ranged between 2.4× 10-4 mg/mL and 2.5 mg/mL, those of kojic acid (1) were between 0.125 mg/mL and 1 mg/mL. The EtOAc extract and kojic acid (1) were most active against Pseudomonas aeruginosa (MIC = 2.4×10-4 mg/mL) and Micrococcus luteus (MIC = 0.125 mg/mL), respectively. Conclusions: The results revealed that the endophytic fungus C. gloeosporioides could be a good source of commercially important kojic acid, which exhibited antimicrobial properties.

272-279

Authors: Omar M.E. Abdel-Salam, Amany A Sleem, Eman R Youness, Fatma A. Morsy

Objective: To investigate the effect of Cannabis sativa extract on the development of neuroand hepato-toxicity caused by malathion injection in rats. Methods: The extract of Cannabis sativa was obtained from the plant resin by chloroform treatment. Δ9-Tetrahydrocannabinol content of the extract (20%) was quantified using gas chromatography–mass spectrometry. The doses of cannabis extract were expressed as Δ9-tetrahydrocannabinol content of 10 or 20 mg/kg. Malathion (150 mg/kg) was intraperitoneally administered followed after 30 min by the cannabis extract (10 or 20 mg/kg, subcutaneously). Rats were euthanized 4 h later. Malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide and paraoxonase-1 (PON-1) activity were determined in brain and liver. Brain 5-lipoxygenase and butyrylcholinesterase (BChE) activity were measured as well. Histopathological examination of brain and liver tissue was also performed. Results: Compared to controls, malathion resulted in increased oxidative stress in brain and liver. MDA and nitric oxide concentrations were significantly increased (P<0.05) and GSH significantly decreased with respect to control levels (P<0.05). Malathion also significantly inhibited PON-1 and BChE activities but had no effect on brain 5-lipoxygenase. Brain MDA concentrations were not altered by cannabis treatment. Cannabis at 20 mg/kg, however, caused significant increase in nitric oxide and restored the GSH and PON-1 activity. Brain BChE activity significantly decreased by 26.1% (P<0.05) after treatment with 10 mg/kg cannabis. Cannabis showed no effect on brain 5-lipoxygenase. On the other hand, rats treated with cannabis exhibited significantly higher levels of liver MDA, nitric oxide and PON-1 activity compared with the malathion control group. Rats treated with only malathion exhibited spongiform changes, neuronal damage in the cerebral cortex and degeneration of some Purkinje cells in the cerebellum. There were also hepatic vacuolar degeneration and dilated and congested portal vein. These histopthological changes induced by malathion in brain and liver were reduced to great extent by cannabis administration at 20 mg/kg. Conclusions: Our data suggest that acute treatment with cannabis alleviates the malathion-induced brain and hepatic injury in rats possibly by maintaining the levels of GSH and PON-1 activity.

285-291

Authors: Ahlem Ben Sassi, Sana Amroussi, Malek Besbes, Mahjoub Aouni, Fethia Skhiri

Objective: To investigate the antioxidant and α-glucosidase properties and phytochemical constituents of roots, stems, leaves and flowers extracts and aerial parts oil of Chrysanthoglossum trifurcatum (Desf.) (C. trifurcatum). Methods: For extraction from roots, stems, leaves and flowers of C. trifurcatum, methanol, ethyl acetate and petroleum ether solvents were used. Phenols, flavonoids, flavonols and tannins contents were evaluated. More, C. trifurcatum aerial parts oil composition was determined using chromatography/mass spectrometry. The antioxidant effect was estimated by DPPH, ABTS and reducing power test systems. The α-glucosidase inhibition was determined by colorimetric assay using the enzyme from Aspergillus niger and the p-nitrophenyl glucopyranoside (pNPG) as substrate. Results: The highest amounts of polyphenols, flavonoids, flavonols and tannins were shown by the methanolic extract of leaves. The main components of the aerial parts oil were limonene (29.21%), γ-terpinene (12.96%), 4-terpenyl acetate (12.18%) and α-pinene (5.76%). The activity evaluated by DPPH, ABTS and reducing power tests was important for stems (IC50=0.68 mg/mL) and flowers (IC50=0.67 mg/mL) methanolic extracts and essential oil (IC50=0.72 mg/mL). Findings of α-glucosidase activity revealed that petroleum ether extracts of leaves and roots together with aerial parts oil showed a highest activity with IC50 of 0.044, 0.045 and 0.049 mg/mL, respectively. Conclusions: Observed antioxidant and α-glucosidase activities of oil and extracts are attributed to the presence of the active phytochemicals in C. trifurcatum organs. Thus, the C. trifurcatum can be used as a source of antioxidant compounds and dietary supplement to treat patients with type 2 diabetes.

297-304

Authors: Jing Pan, Xing-Hua Han, Wei Wang, Yue-Yin Pan

Objective: To explore the expression level and prognostic significance of erythropoietin receptor (EPOR) in patients with breast cancer (BRCA) based on estrogen receptor (ER) status and different molecular subtypes. Methods: The Cancer Genome Atlas (TCGA) and GTEx data were collected in GEPIA initially to identify the dysregulated genes. Further, bc- GenExMiner 4.1 online bioinformatics tool was used to evaluate EPOR mRNA differential expression level according to different classification of clinicopathologic parameters in patients with breast cancer. Additionally, the prognostic value between EPOR mRNA expression and free survival of metastatic relapse (MR) or any event (AE, namely any relapse or death) in patients with breast cancer was done. Results: EPOR mRNA was significantly downregulated in BRCA (1 085 cases) compared to normal tissues (291 cases) (P<0.05). Univariate Cox analysis revealed that high EPOR mRNA expression was remarkably correlated to a decreased risk of MR (HR: 0.79, P<0.000 1) and AE (HR: 0.87, P=0.000 7) especially in breast cancer patients with ER+. Besides, high EPOR mRNA level also associated with a favorable MRfree survival (HR: 0.81, P=0.007 2) and AE-free survival (HR: 0.88, P =0.029 9) in ER+ breast cancer patients. However, no similar above phenomenon was detected in ER- patients. Moreover, the subsequent prognostic adjusted analyses and univariate Cox analysis of AE based on SSP or SCM molecular subtypes validated the above results. Conclusion: EPOR mRNA level is a valuable prognostic indicator for patients with ER+ breast cancer.

309-312

Authors: Lin Huang, Dan-Xin Wang, Li-Min Zhou, Tao Wang, Hai-Ying Zhang, Yun-Xia Zhang, Yin-Dong Zhang

Objective: To investigate the relationship between polymorphisms of rs1532624 and rs289741 loci in cholesteryl ester transfer protein (CETP) genes and atherosclerotic cerebral infarction (ACI). Methods: The CETP gene rs1532624 and rs289741 in 95 patients with ACI and 177 healthy subjects were genotyped by MassARRAY mass spectrometry. Each locus genotype and allele frequency distributions were compared. Results: The difference of allele frequency distribution between the rs1532624 (χ2=1.723, P=0.189) and rs289741 (χ2=2.466, P=0.116) were not statistically significant. The frequency distribution of rs1532624 genotype between the cerebral infarction group and healthy control group was statistically significant (χ2=7.096, P=0.029), while rs289741 genotype frequency distribution between the two groups was not statistically significant (χ2=2.906, P=0.234). Conclusion: ACI have a positive correlation with rs1532624 polymorphism, and AA genotype may be susceptible factors of ACI.

186-193

Authors: Javed Iqbal, Banzeer Ahsan Abbasi, Ali Talha Khalil, Barkat Ali, Tariq Mahmood, Sobia Kanwal, Sayed Afzal Shah, Wajid Ali

Breast cancer is a frightful disease and serious concern in women around the world causing significant health care burden in both developed and developing countries. Extensive research work has shown that breast cancer provides strong resistance to chemical agents, UV radiation, and hormonal treatments. It is generally accepted that cell genetics is not the only main reason for breast cancer and genetic risk factors, for example, mutations in BRCA1 and BRCA2 genes constitute 5%-10% of all breast cancer rates. Other related factors include age, gender, race, ethnicity, weight, reproductive factors, exo- and endogenous hormonal exposures, oral contraceptives use, ultraviolet radiation, diet, and night work (circadian disruption). Many studies have revealed that dietary isoflavones regulate breast cancer occurrence, recurrence and prognosis. Dietary isoflavones have long been part of Asian population diet and there is a significant increase as compared to dietary isoflavones intake among other populations. Dietary isoflavones are natural phytoestrogens having both estrogenic and anti-estrogenic potentials on breast cancer cells in culture, animal models and in experimental trials. This literature survey provides a comprehensive overview on the tumor preventive and tumor promoting potentials of dietary isoflavones on breast cancer. In addition, this paper provides a literature review of dietary isoflavones and their effects on up-regulation and down-regulation of different signaling pathways, genes and proteins. Finally, future perspectives of dietary isoflavones and breast cancer researchers are also critically discussed, which will provide a deeper insight regarding the inner molecular mechanisms of action.

202-208

Authors: Tatsuya Fuji, Tatsurou Inoue, Yasushi Hasegawa

Objective: To investigate whether the extract from the nacreous layer of pearl oysters (nacre extract) improves impairments in memory caused by scopolamine administration in rodents. Methods: Nacre extract was prepared from the inner shell layer of pearl oyster. Effects of nacre extract on scopolamine-induced memory impairment were estimated using novel object recognition test, Y-maze test, and Barnes maze test. Effect of nacre extract on mRNA expressions which are genes associated with memory in the hippocampus was investigated using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Administration of nacre extract led to the protection against scopolamine-induced impairments in object recognition, short-term memory, and spatial memory. Treatment with nacre extract reversed the mRNA expression of brain-derived neurotrophic factor (BDNF) and Homer protein homolog 1 (Homer-1a) in the hippocampus, which decreased with the treatment of scopolamine. Conclusions: These results suggest that nacre extract has attenuating effects on memory impairments induced by scopolamine through the increase in mRNA expression of BDNF and Homer-1a.

214-221

Authors: Swati Gupta, Kamla Prasad Mishra, Paban Kumar Dash, Manmohan Parida, Lilly Ganju, Shashi Bala Singh

Objective: To investigate the therapeutic efficacy of andrographolide, a plant derived compound, against chikungunya virus (CHIKV) infection. Methods: Using flow cytometry and immunoblotting assay, in vitro viral protein expression was studied in THP-1 cells line. In Balb/c mouse neonates, viral RNA copy number was determined by real time PCR. Results: The results showed reduced CHIKV protein expression on andrographolide treatment in CHIKV-infected human peripheral blood mononuclear cells, Vero cells and THP-1 cell line. In vivo, andrographolide treatment to CHIKV-infected neonates reduced viral RNA copy number. Further, andrographolide also increased cytotoxic T lymphocytes both in vitro and in vivo. Andrographolide also activated host innate immune pathways, viz., protein kinase R, phosphorylated eukaryotic initiation factor 2α, retinoic acid inducible gene-I and interferon regulatory factor 3/7, thereby increasing IFN-α secretion. CHIKV-induced nuclear factor κ light chain enhancer of activated B cells and tumor necrosis factor-α was also reduced on andrographolide treatment. Conclusion: Andrographolide inhibits CHIKV by suppressing viral protein expression and up-regulating host innate immunity and hence could be an effective therapeutic agent against CHIKV infection.

227-234

Authors: Aníbal E Carbajo, Alejandra Rubio, María J Viani, María R Colombo

Objective: To analyze the largest outbreak of dengue in Argentina in the municipality of Tigre during 2016, through detailed spatial analyses of the occurrence of cases in relation to demographic factors and vector control actions. Methods: Detailed and georreferenced data on dengue cases with laboratory results (NS1 or IgM) were analyzed. The occurrences of imported and autochthonous cases by census tract were modeled using demographic variables (population by age class, proportion of foreigners, proportion with university grade, proportion of males), dwelling variables (number of homes, proportion of dwellings with latrine, number of dwellings, proportion of houses, proportion of flats, proportion of slums) and census tract area as explanatory variables. The probability of occurrence of autochthonous and imported cases was modeled separately. The spatio-temporal occurrence of cases was studied in relation to focal and perifocal control actions (involving education campaigns, removal of Aedes aegypti breeding sites and exhaustive insecticide spraying) to assess the efficiency in stopping autochthonous cases spreading. Results: All autochthonous cases occurred in the urban environment with no sylvan cases. The majority of the imported cases registered came from Paraguay and Northeastern Argentina. The age structure of imported and autochthonous cases did not differ from the age structure of the municipality, while that of the negative cases did. When studied spatially, the occurrence of imported cases by census tract was mildly associated with a higher proportion of foreign population and more people at active age, while occurrence of autochthonous cases was not significantly associated with any of the studied variables. For census tracts with laboratory confirmed results, the models showed higher probability of autochthonous cases related to higher population density and population age structure. The clustering of autochthonous cases was generally mild, with prevailing isolated cases and a weak spread inside the municipality. The biggest outbreak focus was associated with a delay in the focal vector control. Conclusions: Results confirmed the virus pressure coming from neighboring countries and related to population movement by workers. All autochthonous cases occurred in the urban environment with no sylvan cases. The susceptibility of residents to dengue may be similar among age classes and the laboratory tests were performed more frequently in the younger. Autochthonous cases incidence was low and spatio-temporal clustering of cases weak, suggesting that control measures were effective when no delay occurred in their application.

240-244

Authors: You-Wei Huang, Jia-Hong Chen, Zi-Xi Qin, Jie-Kun Chen, Ren-Dong Hu, Zheng Wu, Xi Lin

Objective: To investigate the role of ClC-3 chloride channel in the proliferation of breast cancer cell line Mcf-7 treated with curcumin and its specific mechanism. Methods: MTT assay was used to detect the effect of chloride channel blocker (DIDS) and curcumin on Mcf-7 and human normal cell viability. Patch-clamp technique was used to determine the current density before and after drug treatment. Apoptosis assay by flow cytometry was performed for further examination of cell apoptosis. Results: Curcumin had toxicity on Mcf-7 and HUVEC cells and DIDS reduced the survival rate of Mcf-7 cells by inhibiting proliferation. Curcumin could activate the chloride ion current on MCF-7 cell membrane, which would be inhibited by DIDS. Finally, curcumin in low concentration combined with DIDS could significantly promote the MCF-7 cells apoptosis. Conclusions: Our results suggest that ClC-3 protein is involved in the regulation of curcumin induced proliferation inhibiting in breast cancer cells through inducing cell apoptosis. ClC-3 may be a potential target of tumor therapy.

251-254

Authors: Shao-Wen Cheng, Li-Na Xian, Zhi-Xing Lin, Xue Ao, Jun-Yi Yao, Ying-Qing Li, Yong-Yan Li, Xiu-Ru Li, Wei-Cheng Wang, Chuan-Zhu Lyu,Ying Li

Objective: To detect the clinical relevance of mannose-binding lectin 2 (MBL2) gene polymorphism and sepsis in Chinese lived in Hainan island. Methods: Blood samples from 57 patients with sepsis and 69 patients without sepsis were collected in the ICU of several large hospitals in Hainan province. Genomic DNA was extracted from whole blood and then PCR purification product was sequenced and typed by 3730 sequencing analyzer. The concentration of MBL2 in serum was detected by ELISA. Results: We found that genotype and allele distributions in two groups were in accordance with the Hardy-Weinberg Equilibrium. The frequency of GA genotype was significantly higher than that in non-sepsis group (P=0.013). A allele frequency in sepsis group was also much higher than that in non-sepsis group (P=0.028). Logister regression analysis showed that the patients who carried A allele were more prone to get sepsis than G allele carrier (P=0.014, OR=2.550, 95%CI=1.207-5.386). The MBL2 level in serum of sepsis patients with genotype GG and GA was significantly lower than that in non-sepsis group (P<0.05). In sepsis group, the MBL2 serum level of patients with genotype GA was obviously lower than that in patients with genotype GG (P<0.05). Conclusions: The variation of rs1800450 G→A increased the incidence of sepsis and decreased the level of MBL2 in serum.

260-264

Authors: Hou-Cong Li, Shu Wang

Objective: To investigate the effects of steroidal alkaloids and sarcovagine D isolated from Sarcococca hookeriana var. digyna on cell-proliferation and secretion of inflammatory cytokines in TNF-α-induced human RA-FLS MH7A in vitro, and against complete Freund’s adjuvant (CFA)-induced arthritis rats in vivo. Methods: CCK-8 assay was utilized to evaluate the anti-proliferation activity in vitro. In in-vivo study, rats were randomly divided into control group, model group (CFA), steroidal alkaloids (STA) groups (5.0, 2.5 and 1.25 g/kg BW), and sarcovagine D (SD) groups (50, 100 and 200 mg/kg BW), 10 rats for each group. To evaluate the anti-inflammation effect, the histology, biochemical parameters and expression of inflammatory cytokines were detected. Results: Steroidal alkaloids and sarcovagine D showed strong anti-proliferative activity during MH7A cell culture proliferation and downregulated NO levels, and inflammatory cytokines (IL-1β, IL-6 and PGE2) in vitro. The rats treated with CFA induced ankle inflammation and lymphocyte infiltration, upregulated IL-1β, TNF-α, IL-6, PGE2, MDA and NO levels, and downregulated IL-10 and SOD. On the contrary, steroidal alkaloids ameliorated all the changes induced by CFA in rats, suggesting the antiinflammatory effect of steroidal alkaloids and sarcovagine D. Conclusions: The results suggest that Sarcococca hookeriana var. digyna has anti-inflammatory effect. Steroidal alkaloids and sarcovagine D has the potential to cure RA.

98-108

Authors: Neeraj Tiwari, Dhiraj Kishore, Surabhi Bajpai, Rakesh K. Singh

Elimination of visceral leishmaniasis is a priority programme in Indian subcontinent. The World Health Organization has set a new target to eliminate kala-azar by the year 2020 as previous target elimination year (2015) has passed. The elimination programme has successfully curbed the rate of infection in endemic regions; however, there are still few challenges in its route. The current drug control regime is extremely limited and comprises only two (amphotericin B and miltefosine) drugs, which are also susceptible for parasites resistance. Moreover, these drugs do not produce sterile cure, and cured patients may develop post kala-azar dermal leishmaniasis even after a decade of cure leaving behind a potent source of parasitic reservoirs for further disease transmission. A significant proportion of endemic population remain seropositive but aymptomatic for many years without any clinical symptom that serve as latent parasitic reservoirs. The lack of tools to identify live parasites in asymptomatic infections and there association in disease transmission, parameters of sterile cure along with post kala-azar dermal leishmaniasis progression remain a major threat in its elimination. In this review, we discuss the potential of host immune inhibitory mechanisms to identify immune correlates of protective immunity to understand the mystery of asymptomatic infections, sterile cure and post kala azar dermal leishmaniasis.

116-122

Authors: Ícaro Gusmão Pinto Vieira, Francisca Noélia Pereira Mendes, Sabrina César da Silva, Raquel Teixeira Terceiro Paim, Bruno Bezerra da Silva, Stephen Rathinaraj Benjamin, Eridan Orlando Pereira Tramontina Florean, Maria Izabel Florindo Guedes

Objective: To evaluate the antidiabetic effect of galactomannans extracted from Adenanthera pavonina’s L. seeds (GAP) in streptozotocin (STZ) induced diabetic mice. Methods: The preliminary galactomannan yield from Adenanthera pavonina L. plant and extraction products composition were evaluated. Various chemical characterization methods like thin layer chromatography, Fourier transform infrared spectroscopy, 1H and 13C nuclear magnetic resonance, and molecular weight by gel permeation chromatography have been employed to characterize the extracted galactomannan. The mice were divided in four groups: Normal control, diabetic control, GAP (1% and 2%) treated and standard drug treated groups. Diabetic mice received treatment daily for 30 d. Diabetes was induced by STZ at a single dose of 120 mg/kg. Body weight, water and food intake, fasting blood glucose, total cholesterol and triglycerides were measured. Histopathological analysis of pancreas and liver were performed to evaluate STZ-induced tissue injuries. Results: The isolated and extracted galactomannan from Adenanthera pavonina was confirmed by various chemical characterization methods. GAP exhibited a 1.46:1 mannose: galactose ratio, and high molar weight. Both GAP enriched food decreased glycaemia, total cholesterol and triacylglycerol. GAP didn’t interfere on food intakes or body weight, although it increased water intake. Furthermore, the relative liver weight indicated toxic galactomannan effects on the histopathological changes of the pancreas in STZ induced diabetes. Conclusions: It is concluded that GAP is a natural product that contains potent galactomannan and is useful in preventing and treating diabetes.

131-135

Authors: Alireza Zahraei-Ramazani, Abedin Saghafipour, Yavar Rassi

Objective: To represent a new geographical record, Phlebotomus (Adlerius) kabulensis (P. kabulensis), which is suspected to be a potential vector of visceral leishmaniasis. Methods: For the first time, P. kabulensis specimens were collected using the sticky paper traps method in outdoor places in mountainous areas with vegetation coverage of three provinces in Iran. Identification of males was based on ecological, morphological, morphometric and molecular (mtDNA cytochrome b gene sequences) criteria. Generally, males have two ascoids on the 8th antennal segment and one ascoid on segments 9th to 15th, aedeagus with normal obtuse- angled sub-terminal notch and coxite with 27–50 groups of hairs on the distal half of its body. Results: Morphometric measurement revealed that P. kabulensis specimens were the same as compared with seven other morphological characters in three provinces of the country but lengths of the coxite were significantly different. The PCR result of the cytochrome b (Cyt b)- mtDNA fragment shows 550-bp length, with its special nucleotide arrangement. The male and female of P. kabulensis were newly discovered members of the subgenus Adlerius from Iran. Initial DNA analysis indicated how distinct this species is. Conclusions: The results show that the P. kabulensis female will be identified by comparing with other Adlerius female groups regarding its morphometric characters and molecular sequencing.

141-146

Authors: Ya-Chao Yao, Nan Li, Liang-Shan Hu, Ya-Hong Li, Zhi Zhang

Objective: To access the performance of the Tellgenplex human papillomavirus (HPV) DNA test compared to the polymerase chain reaction-reverse dot blot (PCR-RDB) assay for the HPV genotyping. Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay. The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively. Each sample showed discrepancy was genotyped using sequencing. Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype. This showed perfect agreement (>0.81) for high-risk HPV genotypes (35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement (>0.65) for high-risk HPV genotypes (16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis. The positive rates of the two assays for frequent HPV genotypes (16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81 (P<0.05). As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes (16, 52, and 81). All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test (HPV genotypes 44 and 55) were confirmed by sequencing. Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.

151-154

Authors: Ling Fan, Li-Feng Chen, Jing Fan, Lan-Ping Zhao, Xiao-Yun Zhang

Objective: To investigate the electrophysiology effects and mechanism of iron overload on the slow response autorhythmic cells in the left ventricular outflow tract of guinea pigs. Methods: Standard microelectrode cell recording techniques were adopted to observe the electrophysiological effects of different concentrations of Fe2+ (100 μmol/L, 200 μmol/L) on the left ventricular outflow tract autorhythmic cells. Heart tissues were perfused with FeSO4 (200 μmol/L) combing with CaCl2 (4.2 mmol/L), Verapamil, (1 μmol/L), and nickel chloride (200μmol/L) respectively to observe the influences of these contents on electrophysiology of FeSO (200μmol/L) on the left ventricular outflow tract autorhythmic cells. Results: Fe2+ 4 at both 100 μmol/L and 200 μmol/L could change the electrophysiological parameters of the slow response autorhythmic cells of the left ventricular outflow tract in a concentration- dependent manner resulting into decrease in Vmax, APA and MDP, slower RPF and VDD, and prolonged APD and APD (P all <0.05). Besides, perfusion of increased Ca2+ concentration 50 90 could partially offset the electrophysiological effects of Fe2+ (200 μmol/L). The L-type calcium channel (LTCC) blocker Verapamil (1 μmol/L) could block the electrophysiological effects of Fe2+ (200 μmol/L). But the T-type calcium channel (TTCC) blocker nickel chloride (NiCl2, 200 μmol/L) could not block the electrophysiological effects of Fe2+ (200 μmol/L). Conclusions: Fe2+ can directly change the electrophysiological characteristics of the slow response autorhythmic cells of the left ventricular outflow tract probably through the L-type calcium channel.

162-165

Authors: Fu-Mei Chen, Ke Wang, Liang Gao, Xu-Dong Yao

Objective: To assess the effectiveness and safety of lumboperitoneal shunt for treatment of post-traumatic hydrocephalus (PTH). Methods: A retrospective analysis of medical records of patients with lumboperitoneal shunts admitted in Shanghai Tenth People's Hospital from January 2014 to March 2017 was done. Experience with lumboperitoneal shunt placement for PTH was reviewed. The diagnosis of PTH was based on ventricular enlargement with the Evans’ index (EI>0.3) before shunt implantation. Patients were evaluated for improvements in Glasgow Coma Scale (GCS), Glasgow Outcome Scale (GOS), and EI after shunt placement. Results: Totally, the study included 34 PTH patients with the average age of 49.32 years (range: 9–67 years). The average follow-up period was (3.9±3.5) months. Before lumboperitoneal shunt, the GOS score was (4±1), the GCS score was (8.53±3.38), and the EI score was (0.40±0.08). After shunt implantation, the GOS score was (3±1), the GCS score was (10.29±3.15), and the EI score was (0.34±0.13), respectively (P<0.05). Before lumboperitoneal shunt, 24 (70.58%) patients had a GOS score of 4 (vegetative state), and 10 (29.42%) patients had a GOS score of 3 (severe disabled). After lumboperitoneal shunt, 18 (52.94%) patients had improvement in GOS (11 patients improve from GOS 4 to GOS 3, 5 patients from GOS 3 to GOS 2 and 2 patients from GOS 3 to GOS 1), 22 (64.71%) patients achieved improvement in their GCS (14 patients GCS improvements ≥2 and 8 patients GCS improvement=1), 21 (61.76%) patients had EI improvement (18 patients with EI<0.3). There was no complication in this study. Conclusion: Lumboperitoneal shunt placement is safe and effective for PTH, and serious complications are not observed.

171-176

Authors: Hua-Ping Huang, Guo-Sheng Yuan, Yu-Chen Zhou, Cheng-Guang Hu, Jun-Wei Liu, Shuai Yuan, Yu-Rong Qiu, Yi-Ping Li, Yong-Yuan Zhang, Yuan-Ping Zhou

Objective: To determine the frequency of anti-HCV antibody positivity in patients with non- liver disease complaints, to explore whether anti-HCV positive patients had been properly advised and visited hepatologists for further assessments, and to investigate their clinical characteristics as well as the HCV treatment status. Methods: A hospital based survey of non- liver disease patients with anti-HCV positive and their attending physicians was conducted to determine: 1. were the patients adequately advised of the implication of anti-HCV positive finding; 2. to what extent the patients were aware of potential chronic liver disease associated with HCV infection and whether they sought for further assessments and care of hepatologists. Results: A total of 295 294 non-liver disease patients were tested for anti-HCV antibody, and 2 778 of them were found to be positive (0.94%). However, only 45.10% (1 253/2 778) of the anti-HCV antibody (+) patients were referred to hepatologists and received HCV RNA test. In addition, 34.10% (312/915) and 1.42% (13/915) of them had already advanced to cirrhosis and hepatocellular carcinoma (HCC), respectively. Further analysis showed that the patients who declined antiviral therapy were older, with lower education and lower income, possessed poorer knowledge on the risk of chronic hepatitis C, and had more severe liver diseases. Surprisingly, 65% of the surveyed physicians did not know the genotype-guided treatment duration suggested by the guidelines. Alarmingly, 22% of the surveyed physicians did not know the standard assays for the diagnosis of HCV infection. Conclusions: Our findings highlight the challenge and hidden enormous burden of chronic HCV infection among patients with non-liver disease complaints in China.

653-658

Authors: Sung Min Woo, Woo Rin Choi, Dooly Jang, Chun Sik Yi, Hae Lim Kim, Kyung Hyeon Kim, et al

Objective: To investigate the effects of a herb complex extract (HCE) prepared from Cornus officinalis Sieb. Et Zucc., Eriobotrya japonica Lindley, and olive leaves on immune response of mouse spleen NK cells in vitro and in vivo analysis. Methods: The activity of natural killer (NK) cells was measured in splenocytes and YAC-1 cells. Mice were immunosuppressed using cyclophosphamide (5 mg/kg body weight). Three different doses of HCE (200, 400, and 800 mg/kg body weight) and red ginseng extract (800 mg/kg body weight) which was used as standard immunomodulatory herb were administered orally for 4 weeks. The body weight, dietary, water intake, organs (liver, thymus, and spleen) weight, completed blood count, and cytokines (tumor necrosis factor alpha, interferon gamma, and interleukin-2) production was measured. Results: At the maximum concentration of HCE, the activity of NK cells was increased by 48.5%. HCE increased liver, spleen, and thymus weights without altering numbers of white blood cells, lymphocytes, and neutrophils in a cyclophosphamide-induced immunosuppression rat model. However, HCE recovered the inhibited cytokine expression; HCE (800 mg/kg) increased cytokines levels. The results indicate the immune enhancement potential of this HCE. Conclusion: The HCE enhances immunity by increasing NK cell activity, regulating cytokine levels, and maintaining spleen weight. Therefore, it may be used as a potential immunity enhancer.

666-675

Authors: Nutan V Badgujar, Kinnari N Mistry, Dharamshibhai N Rank, Chaitanya G Joshi

Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS and 1H-NMR techniques were used to identify the active compounds present. EnzCheck caspase-3 assay kit was used to measure caspase-3 activity. Results: Methanolic extract of Vitex negundo (V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS and 1H-NMR techniques, cytotoxic compounds (luteolin and p-hydroxy benzoic acid) were identified which increased casp-3 activity in a dose and time-dependent manner in A-549 treated cells. Conclusions: It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.

682-687

Authors: Darawan Nonsaithong, Supit Yotmek, Somsri Yotmek, Hathai Nochote, Sirichit Wongkamchai, Sittiruk Roytrakul, Usa Lek-Uthai

Objective: To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR). Methods: A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected. Results: Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences. Conclusions: This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.