Pro-Angiogenic Activity of Monocytic-Type Myeloid-Derived Suppressor Cells from Balb/C Mice Infected with Echinococcus Granulosus and the Regulatory Role of miRNAs.

BACKGROUND/AIMS: This study aims to predict the pro-angiogenic functions of monocytic-type myeloid-derived suppressor cells (M-MDSCs) derived from mice infected with Echinococcus granulosus. METHODS: M-MDSCs were collected from Balb/c mice infected with E. granulosus and normal mice (control) and cultured in vitro. Human umbilical vein endothelial cells (HUVECs) were stimulated with the cell supernatant, and angiogenesis was investigated and analysed by the Angiogenesis module of the software NIH Image J. RNA was extracted from fresh isolated M-MDSCs and analysed with miRNA microarray; differentially expressed miRNAs and their potential functions were analysed through several bioinformatics tools. Finally, quantitative PCR was used to confirm the results of microarray analysis. RESULTS: M-MDSCs from mice infected with E. granulosus could promote the formation of tubes from HUVECs in vitro. Moreover, vascular endothelial growth factor (VEGF) showed significantly high expression, whereas soluble fms-like tyrosine kinase-1 (sFlt-1) showed low expression at the transcriptional level in M-MDSCs from mice infected with E. granulosus. Microarray analysis of miRNAs showed that 28 miRNAs were differentially expressed in M-MDSCs from the two experimental mice groups, and 272 target genes were predicted using the microRNA databases TargetScan, PITA and microRNAorg. These target genes were mainly involved in the biological processes of intracellular protein transport, protein targeting to the lysosome and protein transport, and mainly located in the cytoplasm, neuronal cell body and membrane. Moreover, they were mainly involved in the molecular functions of protein binding, metal ion binding and SH3 domain binding. Further, the differentially expressed miRNAs were mainly enriched in the endocytosis, Wnt and axon guidance pathways, as well as the MAPK, focal adhesion, PI3K-Akt, cAMP, mTOR and TGF-ß signalling pathways, which are linked to immunoregulation and angiogenesis based on the results of bioinformatics analysis with DIANA-miRPath 3.0. In addition, the expression of eight miRNAs was randomly verified by quantitative PCR independently in three mice infected with E. granulosus and three normal mice. CONCLUSION: M-MDSCs have a potential angiogenic role during E. granulosus infection, and miRNAs may play a role in the immune response and angiogenesis functions of M-MDSCs through regulation of the identified signalling pathways.

[In Vitro Effects of Aminoalcohol-carbazole Compound BTB3 against Echinococcus granulosus].

Objective: To evaluate the effects of a aminoalcohol-carbazole compound BTB3 against Echinococcus granulosus in vitro. Methods: The protoscoleces from sheep and germinal cells from secondary-infected mice were cultured and treated with 1, 2, 4, 8, 10 and 20 µg/ml BTB3 for 3 days. The viability of protoscoleces and cells was determined by the methylene blue exclusion method and CCK-8 assay. Meanwhile, the effect of 10 µg/ml BTB3 on germinal cells was assessed by scanning electron microscopy（SEM）. The metacestodes were treated with 1, 5 and 10 µg/ml BTB3 for two weeks, and the integrity of the metacestodes was evaluated by SEM. Results: The 10 µg/ml and 20 µg/ml BTB3 caused a death rate of （100.0±0.0）% and （85.2±7.2）% respectively for protoscoleces. As the concentration decreased, the death rate remained below 10%. Moreover, the activity inhibition rate on germinal cells was about 100% for 8, 10 and 20 µg/ml BTB3, and was reduced with the decrease of BTB3 concentration other than the afore-mentioned three. SEM revealed detachment, shrinkage, and cavitation of germinal cells after BTB3 treatment. And the metacestodes all showed the loss of turgidity after BTB3 treatment for 14 days, which indicated cell detachment and uneven distribution of cells in internal cyst of metacestodes. Conclusion: BTB3 has strong effects against Echinococcu granulosus protoscoleces, germinal cells and metacestodes in vitro, which make it a potential drug against hydatid diseases.

Inhibitory effects of 19 antiprotozoal drugs and antibiotics on Babesia microti infection in BALB/c mice.

INTRODUCTION: Different results have been achieved in the evaluation of antiparasitic drug activity in Mongolian jirds, hamsters, and BALB/c mice infected with Babesia microti. The aims of the present study were to find a preferable method for drug screening and to re-evaluate the activity of several drugs against B. microti. METHODOLOGY: The activity of 19 drugs on B. microti-infected BALB/c mice was evaluated. The study was built on Peters' four-day suppressive test, and the pathogenicity of the blood from the treated mice was also used as indicator. RESULTS: The results showed that 15 of the 19 drugs had little or no in vivo effect against B. microti. The inhibitory rates of atovaquone and azithromycin were high at all doses, but the microscopy-negative blood of recovered mice was still infectious. Similar to robenidine hydrochloride at 25 and 50 mg/kg, primaquine at 100 mg/kg had a 100% inhibitory rate. Robenidine hydrochloride achieved a 100% inhibitory rate at 100 mg/kg, and the blood of recovered mice did not result in parasitemia in subpassage experiments. Parasite-negative blood from mice treated with antimalarial drugs (clinically used for babesiosis) still caused parasitemia in subpassage experiments. This suggests that these drugs cannot eradicate the parasites. CONCLUSIONS: Peters' four-day suppressive test and the pathogenicity of the blood from the treated mice are suitable methods for preliminary investigating possible drugs against B. microti. Considering that robenidine hydrochloride achieved the best activity against B. microti in BALB/c mice in our study, further studies are needed.

An alternative mebendazole formulation for cystic echinococcosis: the treatment efficacy, pharmacokinetics and safety in mice.

BACKGROUND: Cystic echinococcosis is a serious zoonotic infection worldwide caused by metacestodes of Echinococcus gruanulosus. Mebendazole and albendazole are the only two drugs used in the treatment of this disease with cure rates only about 30% due to the poor oral absorption. Thus an alternative treatment for this disease is needed. METHODS: A mebendazole oily suspension (MBZ-OS) was prepared and orally administrated to mice infected with echinococcus cysts for 8 months at 12.5 mg/kg and 25 mg/kg for 14 consecutive days. Mebendazole suspended in 1% tragacanth (MBZ-1% tragacanth) served as treated control. In addition, liver and serum samples were collected from these treated mice (25 mg/kg) for histopathology examination and liver function test. For pharmacokinetic analysis, plasma, parasite (cyst wall and cyst fluid) and tissue samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16 and 24 h after orally administrating MBZ-OS and MBZ-1% tragacanth to E. granulosus-infected mice at 25 mg/kg. These samples were then processed and quantitatively analyzed by HPLC. RESULTS: The administration of MBZ-OS resulted in a treatment efficacy with the cyst weight reductions higher than 80%, significantly better than the corresponding MBZ-1% tragacanth groups. The better treatment efficacy of MBZ-OS was related to the higher drug concentration in plasma, parasites and tissues. It was also shown that the injury of the liver was not significantly altered by taking MBZ-OS compared to the untreated control. CONCLUSION: These findings demonstrate that MBZ-OS is a promising new formulation of MBZ for treatment of hydatid diseases without showing significantly liver toxicity.

Research gaps for three main tropical diseases in the People's Republic of China.

This scoping review analyzes the research gaps of three diseases: schistosomiasis japonica, malaria and echinococcosis. Based on available data in the P.R. China, we highlight the gaps between control capacity and prevalence levels, and between diagnostic/drug development and population need for treatment at different stages of the national control programme. After reviewing the literature from 848 original studies and consultations with experts in the field, the gaps were identified as follows. Firstly, the malaria research gaps include (i) deficiency of active testing in the public community and no appropriate technique to evaluate elimination, (ii) lack of sensitive diagnostic tools for asymptomatic patients, (iii) lack of safe drugs for mass administration. Secondly, gaps in research of schistosomiasis include (i) incongruent policy in the implementation of integrated control strategy for schistosomiasis, (ii) lack of effective tools for Oncomelania sp. snail control, (iii) lack of a more sensitive and cheaper diagnostic test for large population samples, (iv) lack of new drugs in addition to praziquantel. Thirdly, gaps in research of echinococcosis include (i) low capacity in field epidemiology studies, (ii) lack of sanitation improvement studies in epidemic areas, (iii) lack of a sensitivity test for early diagnosis, (iv) lack of more effective drugs for short-term treatment. We believe these three diseases can eventually be eliminated in mainland China if all the research gaps are abridged in a short period of time.

[In vitro metabolism of fenbendazole prodrug].

Synthesized fenbendazole prodrug N-methoxycarbonyl-N'-(2-nitro-4-phenylthiophenyl) thiourea (MPT) was analyzed in vitro in artificial gastric juice, intestinal juice and mouse liver homogenate model by using HPLC method, and metabolic curve was then generated. MPT was tested against Echinococcus granulosus protoscolices in vitro. The result showed that MPT could be metabolized in the three biological media, and to the active compound fenbendazole in liver homogenate, with a metabolic rate of 7.92%. Besides, the prodrug showed a weak activity against E. granulosus protoscolices with a mortality of 45.9%.

[Comparative observation on inhibition of hemozoin formation and their in vitro and in vivo anti-schistosome activity displayed by 7 antimalarial drugs].

OBJECTIVE: To observe and compare the inhibition of hemozoin formation and the in vitro as well as in vivo antischistosomal activity induced by seven antimalarial drugs. METHODS: Inhibition of hemozoin formation displayed by chloroquine phosphate, quinine hydrochloride, quinidine, mefloquine hydrochloride, pyronaridine phosphate and lumefantrine at 25 micromol/L, and artemether at 100 micromol/L was performed by assay of inhibition of beta-hematin formation in 1 mol/L sodium acetate buffers containing hematin with various pH of 4.0, 4.2, 4.4, 4.6, 4.8, and 5.0. In in vitro antischistosomal study, the medium of RPMI 1640 supplemented by 10% calf serum was used to maintain the adult Schistosoma japonicum, and the 50% and 95% lethal concentrations (LC50 and LC95) to kill the adult worms of each drug were then determined. Meanwhile, the interaction of quinine, pyronaridine and chloroquine combined with hemin against adult schistosomes was also undertaken. As to in vivo test, the efficacy of seven antimalarial drugs administered orally or intraperitoneally to mice infected with adult schistosomes was observed. RESULTS: In the acidic acetate-hematin solution, 25 micromol/L pyronaridine showed significant inhibition of beta-hematin formation at pH 4.4-5.0 with inhibition rates of 81.3%-97.0%. At pH 4.6, the inhibition rates of beta-hematin formation in acetate-hematin solution induced by mefloquine, chloroquine or quinine at concentration of 25 beta mol/L were 79.7%, 72.8% or 65.8%, respectively, and the beta-hematin formation was continually inhibited by these 3 antimalarial drugs at pH 4.8 and 5.0 with inhibition rates of 83.1%-90.6%, 41.9%-49.0% or 53.2-62.0%. The inhibition rates of beta-hematin formation at pH 4.6 and 4.8-5.0 induced by lumefantrine 25 micromol/L were 74.3% and 40.4%-40.5%, respectively. While under the same concentration of quinidine, 53.4% and 50.9% inhibition rates of beta-hematin formation were observed at pH 4.8 and 5.0. As to artemether, higher concentration of 100 micromol/L only showed light inhibition of beta-hematin formation at pH 4.4-4.8 with inhibition rates of 16.6%-25.0%. As regard to in vitro test, the LC50 and LC95 of mefloquine, pyronaridine, quinine and quinidine were 4.93 and 6.123 microg/ml, 37.278 and 75.703 microg/ml, 93.688 and 134.578 microg/ml, as well as 101.534 and 129.957 microg/ml, respectively. When adult schistosomes were exposed to the medium containing chloroquine, lumefantrine or artemether at higher concentrations of 100 or 120 microg/ml for 72 h, no or only individual worms died. Hence the LC50 and LC95 of these 3 drugs could not be determined. In other in vitro test, adult schistosomes exposed to quinine 50 micromol/L (20 microg/ml) in combination with 153.4 micromol/L (100 microg/ml) hemin, all worms died within 72 h post incubation. While the worms exposed to 50 micromol/L (26 microg/ml) chloroquine combined with the same concentration of hemin, only 18.8%(3/16) of worm died at 72 h post exposure. Unexpectedly, in schistosomes exposed to pyronaridine at a toxic concentrations of 50 micromol/L (46 microg/ml) in combination with 153.4 mol/L (100 microg/ml) hemin for 72 h, all of the worms were protected from the toxic action induced by pyronaridine, which revealed in normal motor activity and appearance of morphology in majority of the worms. In in vivo test, mice infected with adult schistosomes were treated orally with chloroquine, pyronaridine or lumefantrine at a daily dose of 400 mg/kg for 3 days, or intraperitoneally with chloroquine or pyronaridine at a daily dose of 100 mg/kg for 2 or 3 days, no apparent efficacy was seen. When mefloquine, quinine, quinidine or artemether were administered orally to infected mice at a single dose of 400 mg/kg or 200 mg/kg (mefloquine), all groups of mice treated showed moderate or higher efficacy with worm burden reductions of 61.1%-98.1%. CONCLUSION: Among the seven antimalarial drugs tested, their inhibitions of hemozoin (beta-hematin) exhibit no definite correlation to their in vitro and in vivo antischistosomal activity. Quinine in combination with hemin shows synergistic effect against schistosomes in vitro. While antagonist effect is observed in pyronaridine combined with hemin.

[Effect of long-term use of albendazole on mice liver].

OBJECTIVE: To observe the change in serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), direct bilirubin (DBL), indirect bilirubin (IBIL), albumin (ALB) and globulin (GLB), and mouse liver ultrastructure during 1-16 weeks of albendazole treatment. METHODS: 180 female Kunming mice were divided randomly into albendazole treatment group and negative control group. Each mouse of albendazole treatment group was treated with 136.3 mg/(kg x d) albendazole. The mice in control group were given same amount of physiological saline. After 1, 2, 4, 6, 8, 10, 12, 14 and 16 weeks of treatment, 10 mice from each group were randomly selected, serum samples were collected and analyzed for the above seven liver function indices. Pathological changes of liver were observed by transmission electron microscopy. Linear regression analysis was conducted for the relationship between liver function indices(dependent variable) and pathological scores (independent variable). RESULTS: During 1-16 weeks of albendazole treatment, there was no significant difference in serum levels of DBL, IBIL, ALB and GLB between albendazole treatment group and control group. Compared with other treatment period, after 12 weeks of treatment the serum levels of ALT (55.2 +/- 23.7), AST(176.4 +/- 49.2) and ALP(141.1 +/- 19.4) in albendazole treatment group were higher than that of the control (35.5 +/- 8.6, 108.2 +/- 21.9, 84.0 +/- 24.8) (P < 0.05). After 2, 8, 10, 12 and 14 weeks of treatment, the pathological score of albendazole treatment group was 11.8 +/- 4.8, 10.6 +/- 4.8, 13.6 +/- 3.5, 29.8 +/- 10.7, and 5.6 +/- 2.5, respectively, which was higher than that of the control (0.8 +/- 0.4, 1.2 +/- 0.8, 2.4 +/- 2.0, 1.2 +/- 0.4, 1.4 +/- 1.1) (P < 0.05). Among the three liver function indices AST, ALT and ALP, AST was the best fit index for linear regression. The regression formula was Y = -17.616 + 0.188X. CONCLUSION: Long-term treatment with albendazole at a dosage of 136.3 mg/(kg x d) for mice can cause significant elevation of serum levels of ALT, AST and ALP, and result in mild pathological changes in the liver.

Enhanced bioavailability and cysticidal effect of three mebendazole-oil preparations in mice infected with secondary cysts of Echinococcus granulosus.

The aim of the present study is to explore the possibility to increase the efficacy of mebendazole (MBZ) against secondary cysts of Echinococcus granulosus harbored in mice by augmenting the solubility and bioavailability of the drug. Firstly, the saturated solubility of MBZ in nine kinds of oil was determined by high performance liquid chromatography (HPLC), and MBZ was found exhibiting the highest, secondary, and lowest solubility in oleic acid (OA), glycerol trioleate (GT), and soybean oil (SB), respectively. Secondly, MBZ-OA suspension, MBZ-GT suspension, MBZ-SB suspension, and MBZ suspended in 1 % tragacanth (MBZ-1 % tragacanth) were selected for further studies on pharmacokinetics and experimental therapy in mice. Four groups of mice were treated orally with one of aforementioned four MBZ preparations at a single dose of 25 mg/kg, and concentrations of MBZ in plasma obtained from each mouse at various intervals within 24 h postadministration were determined by HPLC. The major pharmacokinetic parameters calculated by MBZ plasma concentration-time curve demonstrated that the peak concentration of the drug (C (max) ) values obtained from three MBZ-oil preparation groups was 1.6-2.8 times higher than that of MBZ-1 % tragacanth group. The same was true that the area under the drug concentration-time curve (AUC(0-∞)) values of 19.8 (2.5)-28.2 (2.5) µg/ml × h revealed in the three MBZ-oil preparation groups was significantly higher than that of 11.6 (2.0) µg/ml × h in MBZ-1 % tragacanth group, and the bioavailability of the three MBZ-oil preparation groups was 71-143 % higher than that of MBZ-1 % tragacanth group. In mice infected with secondary cysts of E. granulosus for 8 months treated orally with MBZ-1 % tragacanth at a daily dose of 25 mg/kg for 14 consecutive days, the mean cyst weight was lower than that of untreated control, but the difference was not statistically significant with cyst weight reduction of 48 %. When the infected mice received three MBZ-oil preparations at the same oral dose schedule as aforementioned, the mean cyst weights were significantly lower than those in MBZ-1 % tragacanth group or control group with cyst weight reductions of 71.2-84.7 %. The results indicate that the solubility of MBZ in oils may increase to various degrees according to the kinds of oil used. Meanwhile, three MBZ-oil (OA, GT, and SB) preparations administered orally to mice not only improve the bioavailability of MBZ relative to that of MBZ suspended in 1 % tragacanth, but their effects against hydatid cysts also significantly enhance.

[Research progress on the efficacy, metabolism and bioavailability of mebendazole in hydatid disease].

Mebendazole is currently used in the treatment of hydatid disease. Its poor absorption from the gastrointestinal tract and low bioavailability aroused further research on new formulations of mebendazole to increase the bioavailability and improve the therapeutic efficacy. This review summarizes the recent research progress.