The effects of CCR7 and related signaling pathways on Leishmania major -infected human dendritic cells.

OBJECTIVES: In our research, we aimed to investigate the roles of CC-chemokine receptor 7 (CCR7) and relevant signaling pathways in Leishmania major-infected human dendritic cells (DCs). METHODS: Differentially expressed genes (DEGs) in L. major-infected human DCs were selected out and visualized using R program. Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted for investigation of significantly enriched signaling pathways and Gene Ontology enrichment analysis was carried out for the unveiling of enriched Molecular Functions and Biological Processes in L. major-infected human DCs. Besides, Hub gene was screened out using weighted gene coexpression network analysis and Cytoscape. In addition, enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction were used for detection of relative expression of CCR7, interleukin-12 (IL-12), and interferon-γ (IFN-γ) in L. major-infected human DCs and western blot analysis was used for detection of relative expression of CCR7 and other proteins in JAK-STAT signaling pathway in L. major-infected human DCs. RESULTS: CCR7 was upregulated and both chemokine and JAK-STAT signaling pathway were activated in L. major-infected human DCs. During the L. major infection, total number of L. major-infected human DCs were increased, as well as the relative expression levels of CCR7, IL-12, and IFN-γ and proteins in the JAK-STAT signaling pathway. Overexpression of CCR7 not only increased expression levels of IL-12 and IFN-γ but also activated the JAK-STAT signaling pathway to affect the leishmaniasis progression. CONCLUSION: L. major infection-induced activation of CCR7, as well as JAK2 and STAT1, might well upregulate the expression of BAX yet suppress the expression of both Bcl2 and c-Jun to affect leishmaniasis progression.

In vitro efficacies of solubility-improved mebendazole derivatives against Echinococcus multilocularis.

Recently, we introduced an epoxy group to mebendazole by a reaction with epichlorohydrin and obtained two isoforms, mebendazole C1 (M-C1) and mebendazole C2 (M-C2). The in vitro effects of mebendazole derivatives at different concentrations on Echinococcus multilocularis protoscoleces and metacestodes as well as cytotoxicity in rat hepatoma (RH) cells were examined. The results demonstrated that the solubility of the two derivatives was greatly improved compared to mebendazole. The mortality of protoscoleces in vitro reached to 70-80% after 7 days of exposure to mebendazole or M-C2, and M-C2 showed higher parasiticidal effects than mebendazole (P > 0.05). The parasiticidal effect of M-C1 was low, even at a concentration of 30 µm. The percentage of damaged metacestodes that were treated with mebendazole and M-C2 in vitro at different concentrations were similar, and M-C1 exhibited insignificant effects on metacestodes. Significant morphological changes on protoscoleces and metacestodes were observed after treatment with mebendazole and M-C2. In addition, the introduction of an epoxy group to mebendazole also reduced its cytotoxicity in RH cells. Our results demonstrate that the introduction of an epoxy group not only improved the solubility of mebendazole, but also increased its parasiticidal effects on E. multilocularis and reduced its cytotoxicity in RH cells.

The MAPK Signaling Pathways as a Novel Way in Regulation and Treatment of Parasitic Diseases.

Few major advances in fighting parasitic diseases have been made in China since the development of new methods for prevention, control, and elimination. However, the proportion of immunocompromised individuals has increased due to the growth of chronic diseases, population aging, and more frequent cases of patients with AIDS and cancer. All these problems can promote development of parasitic infections, which is commonly associated with manipulation of host signaling pathways and the innate immune system. Mitogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved in metazoan organisms, which play critical roles in the cell cycle, gene expression, growth, differentiation, apoptosis, and parasite⁻host interactions. Recent discoveries of the MAPK components involved in activation, regulation, and signal transduction appeared to be promising for the diagnosis, prevention, and treatment of parasitic diseases in the future. This review summarizes the involvement and critical role of the MAPK family in parasitic disease development and maintenance in the host. Moreover, it highlights recent studies concerning the mechanisms and novel drug development for inhibition and regulation of MAPK pathways in order to prevent parasitic disease. In addition, we discuss some antigenic proteins as prospective inhibitory molecules or vaccines for the regulation and control of MAPK signaling involved in parasite physiological activity.

In Vitro and in Vivo Effect of MAPK Signal Transduction Pathway Inhibitors on Echinococcus multilocularis.

To evaluate the effect of mitogen-activated protein kinase (MAPK) signal transduction pathway inhibitors against alveolar echinococcosis in vitro and in vivo, Echinococcus multilocularis metacestode cysts and protoscolices were obtained from infected mice. Protein chip technology was utilized to screen for key highly expressed target proteins in the MAPK pathway in this parasite and their corresponding inhibitors. Four-week-old Balb/c female mice used for the in vivo experiment underwent inoculation of E. multilocularis by intraperitoneal injection, as well as intragastric administration of MAPK inhibitors for 6 wk. We included 6 groups of mice: a phosphate-buffered saline (PBS) group (negative control); an albendazole-treated group (positive group); and 4 experimental groups treated with TRx0237 mesylate, GDC-0994, pifithrin-ß hydrobromide, or Selonsertib. Echinococcus multilocularis protoscolices were collected and cultured in 1066 medium with penicillin/streptomycin and 10% fetal bovine serum. The in vitro experiment included a PBS group (negative control), a dimethyl sulfoxide-treated group (solvent group), and 4 inhibitor-treated groups as in the in vivo experiment (experimental groups). Each inhibitor group received 4 drug concentrations (5, 30, 55, and 80 µM), and the experiment was performed in triplicate per sample. Fluorescence microscopy was used to evaluate the survival rate of the protoscolices every 48 hr beginning from the first 24 hr. The same grouping was used to evaluate cytotoxicity on E. multilocularis germinal cells and L02 cells. The average weights of E. multilocularis metacestode cyst tissue from each group of the in vivo experiment were 873 mg (PBS), 335 mg (albendazole), 323 mg (TRx0237 mesylate), 420 mg (GDC-0994), 340 mg (pifithrin-ß hydrobromide), and 642 mg (Selonsertib). Results showed albendazole, TRx0237 mesylate, and pifithrin-ß hydrobromide had significant inhibitory effects on inhibition of E. multilocularis. We found a positive correlation between drug concentrations and the inhibitory effects seen in the in vitro experiment, with the differences in contrast with the control group becoming statistically significant after 72 hr of treatment ( P < 0.05). The inhibition rates of TRx0237 mesylate to germinal cells by drug concentration were 23.73, 46.59, 74.71, and 77.44%. Other drugs had no effect on germinal cells. All the inhibitors had low toxicity on L02 cells. Inhibitors of the MAPK signal transduction pathway showed significant inhibitory effects on E. multilocularis, suggesting these may be potential candidates for the treatment of alveolar echinococcosis.

Inhibitory Effect of Ionizing Radiation on Echinococcus granulosus Hydatid Cyst.

BACKGROUND: Heavy ion radiation has more advantages than traditional radiation therapy in the treatment of cancer, mainly because of its superior biological effects. However, there is currently no reliable evidence that heavy ion radiation can induce cell death in hydatid cysts at the cellular and molecular level. In addition, we believe heavy ion therapy could be a potential alternative approach for the treatment of hydatid cysts. METHODOLOGY/PRINCIPAL FINDING: The hydatid cysts and protoscolices were obtained from an experimentally infected KunMing mice. LD50 was used to evaluate the death of the protoscolex. The cellular and ultrastructure of the parasites were observed under light and electron microscopes, the damage and copy numbers of mitochondrial DNA (mtDNA) were decided by QPCR. The apoptosis was evaluated by the expression and activity of caspase3. Dose-dependent ionizing radiation induced damage to the initial mtDNA. Echinococcosis cyst after ionizing radiation showed sparse cytoplasm, disorganized and clumped organelles, huge vacuoles, and villus deletions. The kinetic of DNA repair activity after X-ray irradiation was faster than those after carbon-ion irradiation. High doses of carbon ion radiation caused irreversible attenuation of mitochondrial DNA. Cysts showed obvious reduction in size after radiation. Carbon ion radiation was more effective than X-ray radiation in inhibiting hydatid cysts. CONCLUSIONS: These studies provide evidence that heavy-ion radiation can cause the extinction of hydatid cysts in vitro. The carbon-ion radiation is more advantageous than X-ray radiation in suppress hydatid cyst.

In vitro and in vivo efficacies of carbazole aminoalcohols in the treatment of alveolar echinococcosis.

Benzimidazoles, including albendazole and mebendazole, are the major drugs for clinical chemotherapy of echinococcosis. They mainly exert parasitostatic effects depending on high dosages for long-term. Previous studies have identified carbazole aminoalcohols as novel anti-CE (cystic echinococcosis) agents. However, it is still to be confirmed whether it is effective on alveolar echinococcosis (AE) or not. In the present study, efficacies of novel carbazole aminoalcohols, propylamine, R-propylamine and S-propylamine were evaluated under in vitro and in vivo conditions. Carbazole aminoalcohols were tested against Echinococcus multilocularis (E. multilocularis) protoscoleces (PSC) in vitro. The effects of propylamine and R-propylamine exhibited a time-dependent manner at different concentrations, while the effect of S-propylamine was very poor. At a concentration of 20 µM, the mortality of PSC achieved to 100% on the 11th day after exposure to R-propylamine. The treatment of carbazole aminoalcohols to infected mice resulted in statistically significant reductions in the cyst weights compared with those obtained from negative control mice (p < 0.05), and no significant differences were found between albendazole and carbazole aminoalcohols (p > 0.05). The cytotoxicity examination in rat hepatoma (RH) cells indicated that propylamine and R/S-propylamine were lower that of albendazole at a low concentration (5 µM). In addition, histopathological observation of organs (liver, spleen and kidney) for experimental mice showed mild inflammatory changes in the liver and spleen. This study reveals the potential of carbazole aminoalcohols as a class of novel anti-AE agents.