Antiviral activity of the dichloromethane extracts from Artocarpus heterophyllus leaves against hepatitis C virus

Objective: To determine anti-viral activities of three Artocarpus species: Artocarpus altilis, Artocarpus camansi, and Artocarpus heterophyllus (A. heterophyllus) against Hepatitis C Virus (HCV). Methods: Antiviral activities of the crude extracts were examined by cell culture method using Huh7it-1 cells and HCV genotype 2a strain JFH1. The mode of action for anti-HCV activities was determined by time-of-addition experiments. The effect on HCV RNA replication and HCV accumulation in cells were analyzed by quantitative reverse transcription-PCR and western blotting, respectively. Results: The dichloromethane (DCM) extract of A. heterophyllus exhibited strong anti-HCV activity with an inhibitory concentration (IC50) value of (1.5 ± 0.6)μg/mL without obvious toxicity. The DCM extracts from Artocarpus altilis and Artocarpus camansi showed moderate anti-HCV activities with IC50 values being (6.5 ± 0.3) μg/mL and (9.7 ± 1.1) μg/mL, respectively. A time-of-addition studies showed that DCM extract from A. heterophyllus inhibited viral entry process though a direct virucidal activity and targeting host cells. HCV RNA replication and HCV protein expression were slightly reduced by the DCM treatment at high concentration. Conclusions: The DCM extract from A. heterophyllus is a good candidate to develop an antiviral agent to prevent HCV grant reinfection following liver transplantation.

The Osteogenic Capacity of Human Amniotic Membrane Mesenchymal Stem Cell (hAMSC) and Potential for Application in Maxillofacial Bone Reconstruction <i>in Vitro</i>Study

Amniotic membrane of human placenta is a source of abundant mesenchymal stem cell (hAMSC) which makes it a potential source of allogeneic multipotent cell for bone healing. However, much has to be explored about its isolation procedure and the osteogenic differentiation potential. The aims of this study are to establish the procurement procedure of human amniotic membrane, the isolation and culture of hAMSC, the MSC phenotypic characterization, and the in vitro osteogenic differentiation of hAMSC. Results of the study are as follows. The quality of human amniotic membrane would be best if procured from Caesarean operation under highly aseptic condition to avoid fungal and bacterial contamination on the culture. Isolation procedure using modified Soncini protocol yielded large amount of MSC with high proliferative capacity in culture medium. Characterization of hAMSC showed that the majority of the target cells exhibited specific MSC markers (CD105 and CD90) with a small number of these cells expressing CD45, the marker of hematopoeitic cells. The in vitro osteogenic differentiation of hAMSC followed by Alizarin Red staining showed that osteoblastic differentiation was detected in a significantly high number of cells. This study concludes that hAMSCs isolated from human amniotic membrane have the capacity for in vitro osteogenesis which makes them be one of the potential allogeneic stem cells for application in maxillofacial bone reconstruction.

Prevalence and genotype distribution of hepatitis B virus among migrant workers in Lombok Island, Indonesia

Objective:To examine the potential risk of hepatitis B virus(HBV)spread in Indonesia by migrant workers,based on the molecular characteristics of HBV strains.Methods:Sera collected from migrant workers traveling to their destination countries(pre-migrant workers)and those returning to Indonesia(post-migrant workers)were screened for HBsAg by ELISA,followed by HBV DNA detection by PCR and(sub)genotype/subtype determination according to surface region and whole genome sequencing.Results:Of 87 pre-migrant workers,15(17.24%)were HBsAgpositive,whereas 15(12.10%)of 124 post-migrant workers were HBs Ag seropositive.HBV genotype analysis based on the S region showed that HBV-B3/adw2 was predominant(96.15%,25/26)whereas 3.85%(1/26)of isolates were HBV-C3/adrq+.Whole genome sequencing of selected strains and phylogenetic tree analysis identified subgenotype B7 in three samples previously categorized as subgenotype B3 based on S region analysis,supporting a recent argument that subgenotypes B5/B7/B8/B9 could be considered as a quasi-subgenotype of B3.Conclusions:A high prevalence of HBsAg carriers was detected among migrant workers from Lombok Island,with no significant difference in prevalence between before and after returning to Indonesia.All strains were classified into genotypes common in Indonesia,and the results suggested that migrant workers are not a risk factor for HBV transmission into Indonesia.

Molecular epidemiology of hepatitis B virus in Asia

Although safe and effective vaccines against hepatitis B virus(HBV) have been available for three decades, HBV infection remains the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma(HCC) worldwide, especially in Asian countries. HBV has been classified into at least 9 genotypes according to the molecular evolutionary analysis of the genomic DNA sequence and shown to have a distinct geographical distribution. Novel HBV genotypes/subgenotypes have been reported, especially from Southeast Asian countries. The clinical characteristics and therapeutic effectiveness of interferon(IFN) and nucleos(t)ide analogues vary among different HBV genotypes. Mutations at T1653 C in subgenotype C2 from Japan and South Korea, C/A1753 T and C1858 T in subgenotype C1 from Vietnam, and C1638 T and T1753 V in subgenotype B3 from Indonesia were reported to be associated with advanced liver diseases including HCC. Genotype distribution in Japan has been changed by an increasing ratio of subgenotype A2 in chronic hepatitis B. While a large number of epidemiological and clinical studies have been reported from Asian countries, most of the studies were conducted in developed countries such as Taiwan, China, South Korea and Japan. In this review, the most recent publications on the geographical distribution of genetic variants of HBV and related issues such as disease progression and therapy in Asia are updated and summarized.

Optimized combinations of statins and azoles against Acanthamoeba trophozoites and cysts in vitro

Objective: To evaluate the combination of several statins(atorvastatin, fluvastatin and simvastatin) and azoles(voriconazole, posaconazole and itraconazole) against Acanthamoeba spp. Methods: The efficiency of the different drug combinations against the trophozoite stage of different Acanthamoeba strains were evaluated by Alamar Blue assay. Effect on the cyst stage was observed by inverted microscope. Cytotoxicity of combinations of azoles and statins was evaluated by measuring the release of lactate dehydrogenase from a murine macrophage cell line. Results: Combinations of any of the tested statins and voriconazole or posaconazole were more efficient in inhibiting Acanthamoeba compared to statins or azoles individually. The drug combinations at the combined inhibitory concentrations 50% showed lower toxicity compared to that of the compounds alone.Conclusions: The combinations of statins together with voriconazole and posaconazole are more efficient than these drugs alone, and these combinations have lower cytotoxicity in mammalian cell lines.

Healing Mechanism and Osteogenic Capacity of Bovine Bone Mineral—Human Amniotic Mesenchymal Stem Celland Autogenous Bone Graft in Critical Size Mandibular Defect

Experiments on maxillofacial bone tissue engineering showed the promising result;however, its healing mechanisms and effectiveness had not been fully understood. The aim of this study is to compare the bone healing mechanism and osteogenic capacity between bovine bone mineral loaded with hAMSC and autogenous bone graft in the reconstruction of critical size mandibular bone defect. Critical size defects were made at the mandible of 45 New Zealand white rabbits reconstructed with BBM-hAMSC, BBM alone, and ABG, respectively. At the end of first, second, and twelfth weeks, five rabbits from each experimental week were sacrificed for histology and immunohistochemistry staining. Expressions of vascular endothelial growth factor (VEGF), bone mor-phogenic proteins-2 (BMP2), Runx2 and the amount of angiogenesis were analyzed in the first and second week groups, while expressions of Runx2, osteocalcin, collagen type-I fibres, trabecular area and bone incorporation were analyzed in the twelfth week groups. The result showed that expressions of VEGF, BMP2 and Runx2 as well as the amount of angiogenesis were higher in ABG compared with BBM-hAMSC group in the first and second weeks of healing. The result of twelfth week of healing showed that expressions of Runx2 and osteocalcin as well as the thickness of collagen type-I fibres were significantly higher in BBM-hAMSC compared to ABG group, while there was no statistically difference in trabecular area and bone incorporation between BBM-hAMSC and ABG group. This study concluded that early healing activities were higher in auto-genous bone graft than in BBM-hAMSC, while osteogenic activities in the late stage of healing were higher in BBM-hAMSC compared to autogenous bone graft. It was also concluded that the osteo-genic capacity of BBM-hAMSC was comparable to autogenous bone graft in the reconstruction of critical size defect in the mandible.

Study Biocompatibility and Osteogenic Differentiation Potential of Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) with Gelatin Solvent

The human umbilical cord is a source of numerous Mesenchymal Stem Cells (MSCs), making it as a potential source of allogeneic multipotent cell for bone tissue engineering. The aims of this study were to find: 1) Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) phenotypic characterization, 2) The in-vitro osteogenic differentiation potential of hUCMSCs, 3) The cytotoxicity of gelatin solvent to hUCMSCs using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. As a result, through characterization of hUCMSCs, the majority of target cells expressed specific MSCs markers, Cellular Differentiation (CD)73, smaller number of subpopulation expressed CD90 with only minimal subpopulation expressed CD105 and all negative MSCs markers. Osteoblastic differentiation was found in a significantly high number of cells when in vitro osteogenic differentiation of hUCMSCs with Alizarin Red staining was done. Biocompatibility analysis using the MTT assay showed that gelatin solvent and Alpha modification of minimum essential medium Eagle (α-MEM) was non-toxic for hUCMSCs in vitro. The study concluded that hUCMSCs isolated from human umbilical cord was capable of undergoing in vitro osteogenesis, indicating its potential as allogeneic stem cells for clinical application in bone tissue engineering.

Hypoxic Preconditioning Improved Neuroprotective Effect of Bone Marrow-Mesenchymal Stem Cells Transplantation in Acute Glaucoma Models

This study explored the novel strategy of hypoxic preconditioning of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) before intra vitreal transplantation to improve neuroprotective effects of Retinal Ganglion Cells (RGCs) in Acute Glaucoma Models. The methods of this research were isolated mesenchymal stem cells from the bone marrow of adult wild-type Sprague-Dawley (SD) rats. BM-MSCs were cultured under normoxic or hypoxic (1% oxygen for 24 hours) conditions. Normoxic or hypoxic BM-MSCs were transplanted intravitreally 1 week after ocular hypertension induction by acutely increasing IOP to 100 - 120 mmHg for 60 minutes. Rats were killed 4 weeks after transplanted. Apoptosis was examined by tunnel assay and expression Brn3b (Brn3b = RGCs marker) by immunohistochemical analysis of the retina. Results showed that transplantation of hypoxic preconditioning BM-MSCs in acute glaucoma models resulted in a significant apoptosis decreasing (p < 0.05) and an significant increasing in RGCs (p < 0.05), as well as enhanced mor-phologic and functional benefits of stem cell therapy versus normoxic BM-MSCs transplantation. Conclusions: Hypoxic preconditioning enhances the capacity of BM-MSCs transplantation to improve neuroprotective effects of RGCs in Acute Glaucoma Models.

Current hepatitis B virus infection situation in Indonesia and its genetic diversity

Indonesia has a moderate to high endemicity of hepatitis B virus(HBV) infection. The risk for chronic HBV infection is highest among those infected during infancy. Since 1997, hepatitis B(Hep B) vaccination of newborns has been fully integrated into the National Immunization Program. Al though HBV infection has been reduced by the universal newborn Hep B immunization program, it continues to occur in Indonesia. The low birth dose coverage and the presence of vaccine escape mutants might contribute to this endemicity among children. Although limited information is available for an analysis of occult HBV infection(OBI), several variations and substitutions in the pre-S/S region have been detected in Indonesian HBV strains. Additionally, persistent infection and disease progression of chronic hepatitis B are related to not only viral factors but also the host genome. Indonesia is one of the most ethnically heterogeneous nations, with Javanese and Sundanese as the two highest ethnic groups. This multi-ethnicity makes genomic research in Indonesia difficult. In this article, we focused on and reviewed the following aspects: the current hepatitis B immunization program and its efficacy, OBI, HBV infection among high-risk patients, such as hemodialysis patients, and research regarding the host genome in Indonesia.

Stem cell therapy and diabetic erectile dysfunction:A critical review

Erectile dysfunction(ED)has been identified as one of the most frequent chronic complications of diabetes mellitus(DM).The prevalence of ED is estimated to be about 67.4%in all DM cases worldwide.The pathophysiological process leading to ED involves endothelial,neurological,hormonal,and psychological factors.In DM,endothelial and neurological factors play a crucial role.Damages in the blood vessels and erectile tissue due to insulin resistance are the hallmark of ED in DM.The current treatments for ED include phosphodiesterase-5 inhibitors and penile prosthesis surgery.However,these treatments are limited in terms of just relieving the symptoms,but not resolving the cause of the problem.The use of stem cells for treating ED is currently being studied mostly in experimental animals.The stem cells used are derived from adipose tissue,bone,or human urine.Most of the studies observed an improvement in erectile quality in the experimental animals as well as an improvement in erectile tissue.However,research on stem cell therapy for ED in humans remains to be limited.Nevertheless,significant findings from studies using animal models indicate a potential use of stem cells in the treatment of ED.

Viral hepatitis and human immunodeficiency virus coinfections in Asia

Hepatitis B virus(HBV), hepatitis C virus(HCV),and human immunodeficiency virus(HIV) affect many people in Asian countries, although there are geographic differences. Both HBV and HIV(HBV/HIV) and HCV/HIV co-infections are highly prevalent in Asia. Hetero- and homosexual, injection drug use, and geographic area are strong predictors of HBV, HCV, and HIV serostatus. In HBV endemic regions, the prevalence and genotype distribution of HBV/HIV coinfection is almost comparable with that in the general population. In Japan, where HBV has low endemicity, the prevalence of HBV/HIV co-infection is approximately 10-fold higher than that in the general population, and HBV Ae is the most common subgenotype among HIV infected individuals. Highly active antiretroviral therapy(HAART) is an effective treatment for HIV/Acquired Immune Deficiency Syndrome. Lamivudine, a component of HAART, is an effective treatment for HBV, HIV, and HBV/HIV co-infection; however, cost, emerging drug resistance, antiretroviral-associated liver toxicity and liver-related morbidity due to HCV progression are particular concerns. HCV/HIV co-infection may accelerate the clinical progression of both HCV and HIV. The high prevalence of HBV/HIV and HCV/HIV co-infections in Asia underscores the need to improve prevention and control measures, as fewer evidencebased prevention strategies are available(compared with Western countries). In this review, the most recent publications on the prevalence of HBV/HIV and HCV/HIV co-infections and related issues, such as therapy and problems in Asia, are updated and summarized.

Factors associated with stroke direct cost in francophone West Africa, Benin example

Background: In sub-Saharan Africa, stroke constitutes a public health issue. Few studies were conducted to assess the cost involved in its treatment. Objective: To determine the factors involved in direct cost of stroke in Cotonou-Benin. Method: It consists in a transversal and prospective research of economic type with analytical and descriptive aim. It was conducted from 20thFebruary 2011 to 30thSeptember 2011. The research dealt with 122 stroke patients. With regard to the economic approach, bottom-up was the data collection technique which was adopted. Cost was estimated not only based on the patient himself/herself but considering societal aspect. Cost estimation period was hospitalization period. Data analysis was conducted via software such as Epi info and SPSS. Results: Overall expenses in terms of direct cost varied from $144.9 to $9393.9;average expenses were $1030.1 ± $101.7. Patients aged 50 and above had higher stroke hospitalization cost ($1277.4) than those aged below 50 ($857.4) p = 0.001;male patients made more expenses than females (FCFA 1157.5 against $831.8) p = 0.01;direct cost of stroke was increased in proportion to neurological deficit (score NIHSS) p = 0.043. This cost was higher in cases of hemorrhagic stroke than ischemic stroke (FCFA $1375 against $1098) p = 0.002. Stroke direct cost was also increased in proportion to severance of disability level of patients. Stroke type (hemorrhagic) and RANKIN score were firmly correlated to stroke hospitalization cost. Conclusion: Stroke is very expensive for patientsin Benin and they constitute a burden for both patients and their family. There is a great need to increase awareness regarding risk factor control in order to reduce the cost involved in treating this malady.

Searching for the best agarose candidate from genus Gracilaria, Eucheuma,Gelidium and local brands

Objective: To explore the potential of local agar of genus Gracilaria, Eucheuma,Gelidium and local brands as an alternative for imported agarose for DNA electrophoresis, and to examine their ability related to separation and migration of DNA fragments in DNA electrophoresis.Methods: Their performance at various concentrations were compared via an experimental study with a specific brand of imported commercial agarose used in molecular biology research. The measured variables were separation and migration during electrophoresis of a DNA fragment.Results: The local agar genus Gracilaria gigas, Gelidium, brand “B” and brand “S”could separate DNA fragments at a concentration between 1% and 2%, with an optimum concentration of 2% w/v, as good as a specific brand of imported commercial agarose.Conclusions: Their performance were very close to that of commercial agarose and can still be improved by further agar purification as well as by p H and sulfur control.

HIV-Associated Polyneuropathy in Resource-Limited Settings: Genetic Predisposition and Vitamin Variations

Human immunodeficiency virus-related polyneuropathy remains a painful condition resulting from damaged nerve endings. HIV infection strongly associates with a predominantly polyneuropathy that is attributed to HIV infection itself, or a toxic neuropathy associated with combination antiretroviral therapy (CART). In non-HIV-infected individuals, both deficiency and high intake of vitamins have been associated with polyneuropathy. For that reason, clinicians recommend vitamin supplements before and during CART. Although some, but not all, HIV-related vitamin deficiencies may replete during treatment with CART, it is predictable that high vitamin supplement intakes may contribute to nerve disorders. In resource-limited settings where the diagnosis of polyneuropathy heavily relies on symptoms, data on risk factors for polyneuropathy including vitamin status, alcohol consumption, and co-infections are limited. In addition, studies on genetic influence on the concentration of micronutrients in the blood of long-term users of CART are scarce. Possible sources of high intakes of vitamins could arise from the fact that a number of HIV-infected persons self-medicate. In addition, since HIV-infected individuals have an increased lifespan, relying on symptoms alone to specifically diagnose HIV-associated neuropathies could be a barrier to effective treatment in recourse-poor settings. This paper reviews evidence on single nucleotide polymorphisms (SNPs) with the potential to influence bioavailability of vitamins in HIV-infected patients. Genome-wide association studies have reported SNPs in alkaline phosphatase, fucosyltransferase 2, cubilin, transcobalamin 1, and tumor necrosis factor as potential determinants of various blood levels of vitamin B-6, B-12 and E. As long term CART increasingly become, personalized, future research should focus on SNPs, which influence vitamin blood levels, and with potential to augment long-term treatment with CART.

Induced Monocytes-Derived HSCs (CD34+) with LPS Accelerated Homing Rat Bone Marrow-Mesenchymal Stem Cell (BM-MSCs, CD105) in Injured Pancreas

Investigating the function of combining induced rat monocytes-derived bone marrow-haemopoietic stem cell (rat BM-HSCs) with LPS and rat bone marrow-mesenchymal stem cell (rat BM-MSCs) was to analyze the acceleration of homing process mechanism in injured pancreas. Mononucleated stem cells were isolated from aspirated whole rat BM using ficoll and cultured in α-MEM complete growth medium in 10 cm petridish. After two days, adherent cells after washing twice in petridish were added α-MEM growth medium and then mesenchymal cells were characterized using CD105 marker in third passage and labeled PKH26. Then haemopoietic stem cells (HSCs) were isolated with magnetic beads CD34+ and differentiated in vitro, and then induced monocytes with LPS. Animal experiment used 28 male Wistar rats, and divided them into 4 groups. After transplantation combined, both cells between monocyte derived HSc (mHSCs) and rat BM-MSC were analyzed expression of pair box gen 4 (Pax4), pancreatic and duodenal homeobox (Pdx1), C-peptide using immunohistochemistry, then secretion of insulin and C-peptide analyzed using indirect ELISA. Results showed that the expressions of Pax4, Pdx1, C-peptide found in the surface membrane cell of pancreatic cell, and secreted C-peptide and insulin were shown significant (P < 0.05) in transplanted group 2, 3 and 4, but in group 3 were transplanted with combined cells more dominant than non-combined cells. Conclusions suggested that combining of induced monocytes-derived HSCs and rat BM-MSCs has accelerated homing MSCs into injured pancreatic tissue.

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.

New injectable two-step forming hydrogel for delivery of bioactive substances in tissue regeneration

A hydrogel based on chitosan,collagen,hydroxypropyl-c-cyclodextrin and polyethylene glycol was developed and characterized.The incorporation of nano-hydroxyapatite and pre-encapsulated hydrophobic/hydrophilic model drugs diminished the porosity of hydrogel from 81.6262.25%to 69.9863.07%.Interactions between components of hydrogel,demonstrated by FTIR spectroscopy and rheology,generated a network that was able to trap bioactive components and delay the burst delivery.The thixotropic behavior of hydrogel provided adaptability to facilitate its implantation in a minimally invasive way.Release profiles from microspheres included or not in hydrogel revealed a two-phase behavior with a burst-and a controlled-release period.The same release rate for microspheres included or not in the hydrogel in the controlled-release period demonstrated that mass transfer process was controlled by internal diffusion.Effective diffusion coefficients,Deff,that describe internal diffusion inside microspheres,and mass transfer coefficients,h,i.e.the contribution of hydrogel to mass transfer,were determined using‘genetic algorithms’,obtaining values between 2.64·10^(-15) and 6.67·10^(-15)m^(2)/s for Deff and 8.50·10^(-10) to 3.04·10^(-9) m/s for h.The proposed model fits experimental data,obtaining an R2-value ranged between 95.41 and 98.87%.In vitro culture of mesenchymal stem cells in hydrogel showed no manifestations of intolerance or toxicity,observing an intense proliferation of the cells after 7 days,being most of the scaffold surface occupied by living cells.