Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

Objective:To compare the applicability of the SYBK Grcen-Ⅰ assay with the standard schizont maturalion assay,for determination of sensitivity of Plasmodium vivax(P.vivax) to chloroquine and a new antifolale WR 99210.Methods:The study was conducted at Mae Tao Clinic for migrant workers,Tak Province during April 2009 to July 2010.A total of 64 blood samples(1 mL blood collected into sodium heparinized plastic tube) were collected from patients with monoinfection with P.vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P.vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-Ⅰ assays.Results:A total of 30 out of 64 blood samples collected from patients with P.vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays.The failure rates of the schizont maturation inhibition assay(50%) and the SYBR Green-I assay(54%) were similar(P=0.51).The median IC_(10)s,IC_(50)s and IC_(90)s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P.vivax tested.Based on the cut-off of 100 nM,the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates,respectively.The strength of agreement between the two methods was very poor for both chloroquine and WR992I0.Conclusions:On the basis of this condition and its superior sensitivity,the microscopic method appears better than the SYUK Green-I Green assay for assessing in vitro sensitivity of fresh P.vivax isolates to antimalarial drugs.

In vitro inhibitory effects of plumbagin,the promising antimalarial candidate,on human cytochrome P450 enzymes

Objective:To investigate the propensity of plumbagin to inhibit the three isoforms of human cytochrome P450(CYP),ie.,CYP1A2,CYP2C19,and CYP3A4 using human liver microsomes in ritro.Methods:Inhibitory effects of plumbagin on the three human CYP isoformswere investigated using pooled human liver microsomes.Phenacetin O-deethylation,omeprazole hydroxylation and nifedipine oxidation were used as selective substrates for CYP1A2,CYP2C19 and CYP3A4 activities,respectively.Concentrations of paracetamol,5-hydroxyomeprazole,and oxidized nifedipine were determined in microsomal incubation mixture using high performance liquid chromatography.Results:Plumbagin showed significantinhibitory effects on all CYP isoforms.but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation.The IC50(concentration that inhibits enzyme activity by 50%) values of plumbagin and nootkatone(selective inhibitor) for CYP2C19 were(0.78±0.01) and(27.31±0.66) μM,respectively.The inhibitory activities on CYP1 A2-mediated phenacetin O-deethylation and CYP3A4-mediated nifedipine oxidation were moderate.The IC_(50) values of plumbagin and-naphthoflavone(selective inhibitor) for CYP1A2 were(1.39±0.01) and(0.02±.0.36) μM,respectively.The corresponding IC_(50) values of plumbagin and ketoconazole(selective inhibitor) for CYP3A4 were(2.37+0.10) and(0.18±0.06) μM,respectively.Conclusions:Clinical relevance of the interference of human drug metabolizing enzymes should be aware of for further development scheme of plumbagin as antimalarial drug when used in combination with other antimalarial drugs which are metabolized by these CYP isoforms.

Possible role of PGD_2 in malaria infections

Objective:To preliminarily investigate the possible role of prostaglandin D_2(PGD_2) 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).PGD_2 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 PGD_2 and malaria infections is clearly demonstrated with PGD_2 concentration in urine.The urinary PGD_2 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 PGD_2 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.

Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model

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 concanavilin A(Con A)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LCMS/MS. Results: Among the 24 Con A-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 x3(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 NSFL1 C glycoproteins isolated by Con A-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1 C 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.

Electromagnetic Stimulation Combined with Aloe vera Increases Collagen Reorganization in Burn Repair

Objectives： Burns are shown as a clinical problem for their severity and multiple complications due to the time required to heal. Therapies that improve their healing are of great importance, especially for being minimally invasive, of low cost and best performance, all related to the speed and quality of healing. This study investigated the effects of the magnetic electro stimulator Haihuá CD9 isolated or in association with Aloe vera in rats skin burns. Methods： Experimental groups （n = 30/group） were： （C） Carbopol gel; （F） A. vera/Carbopol gel; （H） Haihuá＋Carbopol gel; （H＋F） Haihuá＋A. vera/Carbopol gel. Samples were collected on the 7th, 14th, and 21st experimental days for structural and morphometric analysis, hydroxyproline and glycosaminoglycans quantification, zymography for MMP-2 and MMP-9 and Western Blotting for TGF-β1, VEGF, Collagen I and III. Key findings： The expression of TGF-β1 in H＋F was increased on the 7th day and of MMP-9 on the 7th and 14th days. The expression of VEGF increased in the first experimental periods and decreased in the last for the treated groups. There was an increase in the fibroblasts and birefringent collagen fibers in groups treated with Haihuá isolated or in association with A. vera in all periods. The quantification of collagen I increased, while collagen III decreased in H＋F. The higher amount of GAGs and MMP-2 active isoform was detected in H and H＋F during all periods. Conclusions： Considering the results of the present study, electromagnetic stimulation in association with the A. vera extract promoted an increase in the number of fibroblasts, GAGs content, MMP-2 activity, the deposition and organization of collagen fibers, favoring the repair of injuries to second degree burns, and may also present therapeutic potential in this injury type.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94

Objective:To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3)induced by pyrimethamine,and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis(2-DE).Methods:Proteins were solubilized and separated according to their charges and sizes.The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software.Results:Differentially expressed protein patterns(up—or down-regulation)were separated from the extracts from the two clones.A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3)clones,respectively.Marked reduction in density of protein expression was observed with the extract from the mutant(resistant)clone compared with the parent(sensitive)clone.A total of 25 protein spots showed at least two-fold difference in density,some of which exhibited as high as ten-fold difference.Conclusions:These proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine.Further study to identify the chemical structures of these proteins by mass spectrometry is required.

Preliminary investigation on the prevalence of malaria and HIV co-infection in Mae Sot District, Tak Province of Thailand

Objective: To preliminarily investigate the prevalence of HIV co-infection in patients with malaria in Mae Sot District, Tak Province of Thailand.Methods: The study was a retrospective study on blood samples collected from a total of 256 patients with malaria(all species and severity) who attended Mae Tao clinic for migrant workers, Tak Province during 2005-2007(148 samples) and 2010-2012(108 samples). Malaria diagnosis was performed based on microscopic examination of patients' blood smears. Chemiluminescent microparticle immunoassay and gel particle passive agglutination were employed for the detection of HIV antigen in patients' plasma. Results: Plasmodium falciparum(P. falciparum) and Plasmodium vivax(P. vivax) are the two predominant malaria species with the ratio of about 1: 1 to 1.5:1. Most of the P. falciparum cases were presented with acute uncomplicated signs and symptoms with highest parasitemia of 1 045 000 asexual parasites/μL bloods. The prevalence of malaria and HIV co-infection during 2005-2007 was 1.35%(2/148 cases, 1 each for P. falciparum and P. vivax co-infection), but was increased to 2.78%(3/108 cases, 2 and 1 for P. falciparum and P. vivax co-infection, respectively) during 2010-2012.Conclusions: The increasing trend of prevalence of malaria and HIV co-infection in Mae Sot, Tak province was of a great concern on either pharmacodynamics or pharmacokinetics aspect. The study in a larger numbers of malaria patients in different endemic areas throughout the country with different time periods is underway.

Lasertherapy as A Strategy for Treatment Healing under Caloric Restriction – Study in Rats

Complications in the healing process are challenging, especially in clinical situations of caloric restriction （CR）. The lasertherapy becomes an important strategy that aids the repair, especially in CR. Thus, it is important to investigate the InGaAlP-660 nm laser as an strategy to repair cutaneous wounds in rats submitted to 30% of CR and to understand the tissue repair in clinical situations of CR. Thirty-six male Wistar rats were used, of which half were fed with 30% less ration, and half with ad libitum diet, for 21 days. Then, punch lesions of 1.5 cm in diameter were made on the animals backs, which were divided into： NR （no-restricted）, R （restricted）-both before lesion; C （control）, RC （restricted-control）, L （laser）, RL （restricted-laser）-after lesion. Samples of the skin/lesion/scar were collected on the 2nd, 7th and 14th days post-injury for histological, biochemical and molecular analyses. The R group showed reduction of body mass, epidermal/dermal thickness, inflammation, angiogenesis, fibroplasia and collagenesis. The RL group showed control of inflammation, oxidative damage and increase of antioxidants than RC, which probably favored angiogenesis, collagenesis and reepithelialization, similar to C and L. Thus, 30% of CR impaired the skin （before lesion）. In the lesion, lasertherapy has shown to be effective in tissue repair mainly in CR status, being thus, the laser clinically important strategy to tissue repair in critical situations of caloric restriction.

Polymorphisms of the oxidant enzymes glutathione S-transferase and glutathione reductase and their association with resistance of Plasmodium falciparum isolates to antimalarial drugs

Objective:To investigate the association between amplification of the two regulatory genes controlling glutathione(GSH) levels,glutathione reductase(PfGR) and glutathione S-transferase (PfGST) genes and sensitivity of Plasmodium falciparum(P.falciparum) isolates collected from different malaria endemic areas of Thailand to standard antimalarial drugs.Methods:A total of 70 P.falciparum isolates were collected from endemic areas of multi-drug resistance (Tak,Chantaburi and Ranong Provinces) during the year 2008-2009.The in vitro assessment of antimalarial activity of P.falciparum clones(K1- and Dd2 chloroquine resistant and 3D7- chloroquine sensitive) and isolates to chloroquine,quinine,mefloquine and arteusnate was performed based on SYBR Green modified assay.Results:68(97.14%),11(15.71%) and 28(40%) isolates respectively were classified as chloroquine-,quinine- and mefloquine-resistant isolates. With this limited number of P.falciparum isolates included in the analysis,no significant association between amplification of PfGST gene and sensitivity of the parasite to chloroquine, quinine,mefloquine and quinine was found.Based on PCR analysis,Dd2,Kl and 3D7 clones all contained only one copy of the PfGST gene.All isolates(70) also carried only one copy number of PfGST gene.There appears to be an association between amplification of PfGR gene and chloroquine resistance.The 3D7 and Dd2 clones were found to carry only one PfGR gene copy, whereas the K1 clone carried two gene copies.Conclusions:Chloroquine resistance is likely to be a consequence of multi-factors and enzymes in the GSH system may be partly involved. Larger number of parasite isolates are required to increase power of the hypothesis testing in order to confirm the involvement of both genes as well as other genes implicated in glutathione metabolism in conferring chloroquine resistance.

Exogenous phosphatidic acid reduces acetaminophen-induced liver injury in mice by activating hepatic interleukin-6 signaling through inter-organ crosstalk

We previously demonstrated that endogenous phosphatidic acid(PA)promotes liver regeneration after acetaminophen(APAP)hepatotoxicity.Here,we hypothesized that exogenous PA is also beneficial.To test that,we treated mice with a toxic APAP dose at 0 h,followed by PA or vehicle(Veh)posttreatment.We then collected blood and liver at 6,24,and 52 h.Post-treatment with PA 2 h after APAP protected against liver injury at 6 h,and the combination of PA and N-acetyl-L-cysteine(NAC)reduced injury more than NAC alone.Interestingly,PA did not affect canonical mechanisms of APAP toxicity.Instead,transcriptomics revealed that PA activated interleukin-6(IL-6)signaling in the liver.Consistent with that,serum IL-6 and hepatic signal transducer and activator of transcription 3(Stat3)phosphorylation increased in PA-treated mice.Furthermore,PA failed to protect against APAP in IL-6-deficient animals.Interestingly,IL-6 expression increased 18-fold in adipose tissue after PA,indicating that adipose is a source of PA-induced circulating IL-6.Surprisingly,however,exogenous PA did not alter regeneration,despite the importance of endogenous PA in liver repair,possibly due to its short half-life.These data demonstrate that exogenous PA is also beneficial in APAP toxicity and reinforce the protective effects of IL-6 in this model.

Comparison of the systemic action of electroacupuncture and laserpuncture in experimental injuries on the back skin of Wistar rats(Rattus novergicus)

OBJECTIVE:To evaluate the systemic action of electroacupuncture and laserpuncture in the repair of excisional cutaneous injuries on the back of adult female Wistar rats.METHODS:Ninety animals were divided into three experimental groups:C-control;E-treated with electroacupuncture[acupoints:Feishu(BL13),Geshu(BL17),Zusanli(ST36)];L-treated with laserpuncture(same acupoints)and euthanized on the 7 th,14 th and 21 st days for tissue removal and preparation for histomorphometric and biochemical(dosages,zymography and Western blotting)analysis.RESULTS:The number of fibroblasts in the E and L groups presented higher values than the C on the14 th and 21 st days.The number of granulocytes was lower than C on the 21 st day in groups E and L.The total number of newly formed vessels increased on the 21 st day and was higher in both treatments when compared to C.The birefringent collagen fibers detected on the 21 st day was lower in groups E and L than group C.The glycosaminoglycans and hydroxyproline amount was similar among groups throughout the experimental period.The active isoform of matrixmetallo proteinase(MMP)-2 and the latent isoform of MMP-9 did not show any differences among all groups and experimental periods.The amount of collagenⅠpresented higher values in the L group on the 14 th day and in the E and L groups on the 21 st day.For typeⅢcollagen,groups E and L presented values lower than C in all experimental periods.The amount of transforming growth factor-β1 in the E and L groups showed higher amounts than C on the 7 th day and lower on the 14 th and 21 st days.For vascular endothelial growth factor,E and L groups presented similar and lower values than C on the 7 th and 14 th days,respectively,and similar on the 21 st day.CONCLUSION:The therapies evaluated in this article stimulated fibroblast proliferation,neoangiogenesis and altered the reorganization of collagen fibers in the repair of excisional injuries in female adult rats.

Pre-treatment twice with liposomal clodronate protects against acetaminophen hepatotoxicity through a pre-conditioning effect

Background and aim:Acetaminophen(APAP)overdose is a major cause of acute liver injury,but the role of macrophages in the propagation of the hepatotoxicity is controversial.Early research revealed that macrophage inhibitors protect against APAP injury.However,later work demonstrated that macrophage ablation by acute pre-treatment with liposomal clodronate(LC)exacerbates the toxicity.To our surprise,during other studies,we observed that pre-treatment twice with LC seemed to protect against APAP hepatotoxicity,in contrast to acute pre-treatment.The aim of this study was to confirm that observation and to explore the mechanisms.Methods:We treated mice with empty liposomes(LE)or LC twice per week for 1 week before APAP overdose and collected blood and liver tissue at 0,2,and 6 h post-APAP.We then measured liver injury(serum alanine aminotransferase activity,histology),APAP bioactivation(total glutathione,APAP-protein adducts),oxidative stress(oxidized glutathione(GSSG)),glutamate-cysteine ligase subunit c(Gclc)mRNA,and nuclear factor erythroid 2-related factor(Nrf2)immunofluorescence.We also confirmed the ablation of macrophages by F4/80 immunohistochemistry.Results:Pre-treatment twice with LC dramatically reduced F4/80 staining,protected against liver injury,and reduced oxidative stress at 6 h post-APAP,without affecting APAP bioactivation.Importantly,Gclc mRNA was higher in the LC group at 0 h and total glutathione was higher at 2 h,indicating accelerated glutathione re-synthesis after APAP overdose due to greater basal glutamate-cysteine ligase.Oxidative stress was lower in the LC groups at both time points.Finally,total Nrf2 immunofluorescence was higher in the LC group.Conclusions:We conclude that multiple pre-treatments with LC protect against APAP by accelerating glutathione re-synthesis through glutamate-cysteine ligase.Investigators using twice or possibly more LC pre-treatments to deplete macrophages,including peritoneal macrophages,should be aware of this possible confounder.