Dual Role of Hepatic Macrophages in the Establishment of the Echinococcus multilocularis Metacestode in Mice.

Echinococcus multilocularis larvae, predominantly located in the liver, cause a tumor-like parasitic disease, alveolar echinococcosis (AE), that is characterized by increased infiltration of various immune cells, including macrophages, around the lesion that produces an "immunosuppressive" microenvironment, favoring its persistent infection. However, the role of hepatic macrophages in the host defense against E. multilocularis infection remains poorly defined. Using human liver tissues from patients with AE and a hepatic experimental mouse model of E. multilocularis, we investigated the phenotype and function of hepatic macrophages during the parasite infection. In the present study, we found that a large number of CD68+ macrophages accumulated around the metacestode lesion in the liver of human AE samples and that both S100A9+ proinflammatory (M1 phenotype) and CD163+ anti-inflammatory (M2 phenotype) macrophages were significantly higher in close liver tissue (CLT) than in distant liver tissue (DLT), whereas M2 macrophages represent the dominant macrophage population. Furthermore, E. multilocularis-infected mice exhibited a massive increase in macrophage (F4/80+) infiltration in the liver as early as day 5, and the infiltrated macrophages were mainly monocyte-derived macrophages (CD11bhi F4/80int MoMFs) that preferentially differentiated into the M1 phenotype (iNOS+) at the early stage of E. multilocularis infection and then polarized to anti-inflammatory macrophages of the M2 phenotype (CD206+) at the chronic stage of infection. We further showed that elimination of macrophages by treatment of mice with clodronate-liposomes before E. multilocularis infection impaired worm expulsion and was accompanied by a reduction in liver fibrosis, yielding a high parasite burden. These results suggest that hepatic macrophages may play a dual role in the establishment and development of E. multilocularis metacestodes in which early larvae clearance is promoted by M1 macrophages while persistent metacestode infection is favored by M2 macrophages.

Thioredoxin peroxidase secreted by Echinococcus granulosus (sensu stricto) promotes the alternative activation of macrophages via PI3K/AKT/mTOR pathway.

BACKGROUND: Larvae of Echinococcus granulosus (sensu lato) dwell in host organs for a long time but elicit only a mild inflammatory response, which indicates that the resolution of host inflammation is necessary for parasite survival. The recruitment of alternatively activated macrophages (AAMs) has been observed in a variety of helminth infections, and emerging evidence indicates that AAMs are critical for the resolution of inflammation. However, whether AAMs can be induced by E. granulosus (s.l.) infection or thioredoxin peroxidase (TPx), one of the important molecules secreted by the parasite, remains unclear. METHODS: The activation status of peritoneal macrophages (PMs) derived from mice infected with E. granulosus (sensu stricto) was analyzed by evaluating the expression of phenotypic markers. PMs were then treated in vivo and in vitro with recombinant EgTPx (rEgTPx) and its variant (rvEgTPx) in combination with parasite excretory-secretory (ES) products, and the resulting activation of the PMs was evaluated by flow cytometry and real-time PCR. The phosphorylation levels of various molecules in the PI3K/AKT/mTOR pathway after parasite infection and antigen stimulation were also detected. RESULTS: The expression of AAM-related genes in PMs was preferentially induced after E. granulosus (s.s.) infection, and phenotypic differences in cell morphology were detected between PMs isolated from E. granulosus (s.s.)-infected mice and control mice. The administration of parasite ES products or rEgTPx induced the recruitment of AAMs to the peritoneum and a notable skewing of the ratio of PM subsets, and these effects are consistent with those obtained after E. granulosus (s.s.) infection. ES products or rEgTPx also induced PMs toward an AAM phenotype in vitro. Interestingly, this immunomodulatory property of rEgTPx was dependent on its antioxidant activity. In addition, the PI3K/AKT/mTOR pathway was activated after parasite infection and antigen stimulation, and the activation of this pathway was suppressed by pre-treatment with an AKT/mTOR inhibitor. CONCLUSIONS: This study demonstrates that E. granulosus (s.s.) infection and ES products, including EgTPx, can induce PM recruitment and alternative activation, at least in part, via the PI3K/AKT/mTOR pathway. These results suggest that EgTPx-induced AAMs might play a key role in the resolution of inflammation and thereby favour the establishment of hydatid cysts in the host.

Evaluation of the diagnostic value of the immunoblotting and ELISA tests using recombinant Em18 antigen in human alveolar echinococcosis from Xingjiang China.

Alveolar echinococcosis (AE) is a prevalent epidemic in the northern hemisphere, especially in central Europe and western China. Serum diagnosis is important for patients with AE, especially during the first screening. The present study purified the recombinant Em18-GST (rEm18-GST), and detected its diagnostic performance in human alveolar echinococcosis patients of Xinjiang, China with immunoblotting (IB) and enzyme-linked immunosorbent assay (ELISA). Serum samples were collected from 50 patients with AE, 222 patients with cystic echinococcosis (CE), 158 patients with other unrelated infections and 106 healthy individuals. The IB results showed that serum samples of 47 patients with AE and 12 patients with CE were rEm18-positive. However, only one sample from patients with cancer showed a cross-reaction with rEm18 in IB. The overall sensitivity was 94%, and the total specificity was 96.58%. For the rEm18 results using ELISA, the sera of 46 patients with AE were positive, and the overall sensitivity was 92%. In conclusion, compared with imaging tools, including computed tomography, magnetic resonance imaging and positron emission tomography, rEm18 has considerable advantages for AE serodiagnosis.

Plasma IL-23 and IL-5 as surrogate markers of lesion metabolic activity in patients with hepatic alveolar echinococcosis.

Fluorodeoxyglucose (FDG) uptake by alveolar echinococcosis (AE) liver lesions is a signal of their metabolic activity and of disease progression. In order to find a surrogate marker for this status, we investigated whether parameters of the peripheral and/or periparasitic immune responses were associated with metabolic activity in a prospective case-control study of 30 AE patients and 22 healthy controls. Levels of 18 cytokines and chemokines, representative of innate and adaptive immune responses, were assessed in plasma and peripheral cells of two groups of patients with (MAAE) and without (MIAE) metabolically active lesions, and in the liver of MAAE patients. Mixed cytokine profile was observed in the peripheral blood of AE patients, with a predominance of Th2, Th17 and Treg responses. Among the detected markers only plasma IL-5 and IL-23, more elevated in MAAE patients, were found discriminant. Discrimination between MAAE and MIAE patients obtained by using IL-23 was improved when IL-5 was used in combination. The combination of elevated levels of IL-5 and IL-23 is significantly associated with FDG uptake at PET scan. It offers a new tool for the follow-up of AE patients which could substitute to FDG-PET whenever non-available to assess disease progression.

Expression of toll-like receptor 2, 4 and related cytokines in intraperitoneally inoculated Balb/C mice with Echinococcus multilocularis.

Immune response pattern between host and Echinococcus multilocularis (E. multilocularis) is considered as a crucial point in development of alveolar echinococcosis (AE). In this study, we are aiming to study the expression patterns of TLR2 and TLR4 with related cytokines and transcription factors in secondary E. multilocularis infected murine model. The murine model of AE was developed by using intraperitoneal inoculation of E. multilocularis protoscolexes and albendazole (E. m+ABZ group) or carboxy methyle cellulose (CMC; E. m+CMC group) administration via gastric tube was initiated in the third month and continued for one month. Mice with CMC administration served as negative controls (C+CMC group). The splenic cells and peritoneal exudates cells (PECs) were prepared and the levels of IFN-γ, IL-10, and IL-5 in splenic cells and PECs culture supernatants were detected using enzyme linked immune-sorbent assay (ELISA). Besides, the mRNA expression levels of TLR2, 4, transcription factors and cytokines were detected by using real-time fluorescent quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The concentration levels of IFN-γ, IL-10, and IL-5 in PECs and splenic cell supernatants were extremely lower, however, significantly elevated after stimulated with Concanavalin A (ConA) for 36 h with higher concentrations in E. m+CMC group comparing to both E. m+ABZ and C+CMC group. The mRNA levels of TLR2, 4 and GATA3, IFN-γ, IL-10 in splenic cells were significantly increased in E. m+CMC group comparing with other groups. Simultaneously, T-bet mRNA expressions were elevated in E. m+ABZ and C+CMC group compared to E. m+CMC group. In addition, T-bet/GATA3 ratios was higher in E. m+ABZ group compared to E. m+CMC group and were higher in C+CMC group than those in E. m+CMC group. TLR2 mRNA expression in splenic cells showed a positive correlation with IL-10 concentration levels in splenic cell culture supernatants. The present study provides evidence on the possible role of TLR2 in the process of immune tolerance during E. multilocularis infection and suggests albendazole treatment might reverse the immune tolerance situation and improve parasite clearance process.

Parasitic infection as a potential therapeutic tool against rheumatoid arthritis.

Parasites, which are a recently discovered yet ancient dweller in human hosts, remain a great public health burden in underdeveloped countries, despite preventative efforts. Rheumatoid arthritis is a predominantly cosmopolitan health problem with drastic morbidity rates, although encouraging progress has been achieved regarding treatment. However, although various types of methods and agents have been applied clinically, their broad usage has been limited by their adverse effects and/or high costs. Sustained efforts have been exerted on the 'hygiene hypothesis' since the 1870s. The immunosuppressive nature of parasitic infections may offer potential insight into therapeutic strategies for rheumatoid arthritis, in which the immune system is overactivated. An increasing number of published papers are focusing on the preventive and/or curative effect of various parasitic infection on rheumatoid arthritis from experimental studies to large-scale epidemiological studies and clinical trials. Therefore, the present review aimed to provide a general literature review on the possible beneficial role of parasitic infection on rheumatoid arthritis.

[Changes of Toll-like Receptor mRNA and Related Cytokines in Patients with Hepatic Alveolar Echinococcosis].

Objective: To investigate the expression of Toll-like receptor 2（TLR2） and TLR4 mRNA in peripheral blood mononuclear cells （PBMC） and in the liver of patients with hepatic alveolar echinococcosis (HAE), and their correlations with related cytokines in plasma. Methods: Twenty-eight HAE patients hospitalized in the First Affiliated Hospital of Xinjiang Medical University during January 2012 and June 2015 and 28 healthy volunteers as a control were enrolled in this study. Plasma levels of interferon-γ （IFN-γ）, interleukin-5 （IL-5）, IL-23, and IL-10 were measured by ELISA. qRT-PCR was performed to detect TLR2 and TLR4 mRNA levels in PBMCs and hepatic tissues. The percentage of peripheral blood eosinophil （Eo%） was determined by a hematology analyzer. The correlations of TLR2 and TLR4 mRNA levels in PBMCs with levels of related cytokines and Eo% were analyzed with the Spearman Correlation method. Results: ELISA results showed that the plasma levels of IFN-γ, IL-5, IL-23, and IL-10 in the HAE group were （301.100±47.290）, （43.420±11.380）, （86.580±31.990） and （8.766±7.568） pg/ml respectively, which were higher than those in the control［（301.100±67.790）, （40.970±6.310）, （46.770±15.490） and （6.272±10.360） pg/mlï¼½ with a statistical significance for IL-23 （P<0.01）. Results of qRT-PCR showed that the expression level of TLR2 in the HAE group （0.100±0.084） was significantly higher than that in the control （0.055±0.040） （P<0.05）, while the expression level of TLR4 in the HAE group （0.004±0.003） was comparable to that in the control（0.003±0.002）（P>0.05）. The expression of TLR2 and TLR4 mRNA in HAE lesions in the HAE group（29.680±25.650 and 21.340±16.640, respectively） were both significantly higher than that in para-lesion regions（2.308±4.140 and 5.541±9.233） and that in tissues of the control （1.112±1.431 and 1.100±1.734）（P<0.01）. There was also a significant difference in Eo% between the HAE（0.448±0.240） and the control（0.110±0.100） groups. Spearman correlation coefficients revealed a positive correlation of TLR2 mRNA in PBMCs with plasma IL-23 level and peripheral blood Eo% in HAE subjects（r=0.368, r=0.382, respectively）. Conclusion: There are increases in TLR2 and TLR4 mNRA expression in PBMCs and in HAE lesions in HAE patients. The TLR2 mNRA expression in PBMCs positively correlates with plasma IL-23 level and peripheral Eo%.

Expression of Toll-Like Receptors 2 and 4 and Related Cytokines in Patients with Hepatic Cystic and Alveolar Echinococcosis.

Several studies have demonstrated the important role of Toll-like receptors in various parasitic infections. This study aims to explore expression of Toll-like receptors (TLRs) and related cytokines in patients with human cystic echinococcosis (CE) and alveolar echinococcosis (AE). 78 subjects including AE group (N = 28), CE group (N = 22), and healthy controls (HC, N = 28) were enrolled in this study. The mRNA expression levels of TLR2 and TLR4 in blood and hepatic tissue and plasma levels related cytokines were detected by using ELISA. Median levels of TLR2 mRNA in AE and CE groups were significantly elevated as compared with that in healthy control group. Median levels of TLR4 expression were increased in AE and CE. Plasma concentration levels of IL-5, IL-6, and IL-10 were slightly increased in AE and CE groups compared with those in HC group with no statistical differences (p > 0.05). The IL-23 concentration levels were significantly higher in AE and CE groups than that in HC subjects with statistical significance. The increased expression of TLR2 and IL-23 might play a potential role in modulating tissue infiltrative growth of the parasite and its persistence in the human host.

The Potential Role of Th9 Cell Related Cytokine and Transcription Factors in Patients with Hepatic Alveolar Echinococcosis.

Human alveolar echinococcosis (AE) is a lethal parasitic infectious disease which may lead to liver failure if left untreated. It is caused by the larval stage of the fox tapeworm Echinococcus multilocularis and usually develops a substantial infiltrative occupation in solid organs. During the infection, T helper subsets are known to play crucial role in crosstalk between the parasite and human host. Th9 cells, a new member of CD4(+) T cell family which is characterized by its specific cytokine IL-9 and transcription factors PU.1 and IRF-4, have been known recently to have a critical role in allergic diseases, and cancers as well as the parasitic infection. To assess the potential role of Th9 cells during the infection, the mRNA levels of IL-9, PU.1, and IRF-4 both in peripheral blood mononuclear cells and in liver tissues were, respectively, detected by using real-time PCR. The plasma concentration levels of IL-9 were detected by using enzyme linked immunosorbent assay (ELISA). Th9 related cytokine IL-9 and transcription factors PU.1 and IRF-4 mRNA levels elevated both in PBMCs, and in hepatic lesion and paralesion tissues in AE patients. This may facilitate the infiltrative growth of the parasite and its persistence in human host.

[Research Achievements and Challenges for Echinococcosis Control].

Echinococcosis is an important communicable disease that has remarkable impacts on the global health. The disease is highly endemic in western China. In the last decades, achievements were obtained for the surgery and drug therapies for echinococcosis, as well as for studies on genomics, signaling pathways, and liver proliferation and injury of the intermediate hosts. Although steps have entered vaccine development, challenges remainin immunodiagnosis and drug treatment for intermediate hosts, and in vaccine development for definitive hosts. This paper gives an overview on the current achievements and challenges for echinococcosis control.

TNFRSF11B polymorphisms are associated with metabolic traits in Uyghur and Han ethnic groups.

UNLABELLED: Abstract Introduction: To test whether genetic variants of osteoprotegerin gene (TNFRSF11B) affect metabolic traits (body mass index [BMI], glucose, triglyceride, total cholesterol) and bone mass traits. METHODS: We conducted a population based association study to investigate associations of eight tagging single nucleotide polymorphisms (tSNPs) of the TNFRSF11B gene with the aforementioned traits in a Chinese Han population and an ethnic group admixed with Caucasians and Asians - Uyghur. The associations between the tSNPs and bone mass density (BMD) were also tested in Han population. RESULTS: We found that SNP rs3102727, located in the first intron of the TNFRSF11B gene, was significantly associated with triglyceride levels in Uyghur population and Han population simultaneously. T allele of the rs3102727 variant was associated with a 0.10 mmol/L and 0.09 mmol/L lower level of triglyceride than C allele in Uyghur (p = 0.019) and Han subjects (p = 0.037), respectively. In addition, the T allele is also associated with a lower level of hip BMD (p = 0.025) and total BMD (p = 0.048). Further, we found significant associations between SNP rs11573869 and BMI in Uyghur subjects and SNP rs3134062 with hip BMD in Han sbujects. Rs11573869-T allele was associated with a 0.81 kg/m(2) lower level of BMI than C allele (p = 0.002) and the hip BMD decreases with the copy of rs3134062-T allele increases (p = 0.002). CONCLUSION: We detected novel associations between TNFRSF11B polymorphisms and metabolic traits in Uyghur and Han populations. In addition, we found associations between TNFRSF11B polymorphisms and bone mass traits in Han population.

Dynamic changes of DC and T cell subsets in mice during Echinococcus multilocularis infection.

The aim of this study was to determine the dynamic changes of dendritic cell (DC) pheno-types and T cell in response to Echinococcus multilocularis (Em) infection in BALB/c mice. Mice comprised the control and Em-infected group. At day 0, 2, 7, 30, 60, 90 and 120 after infection, the size of larval cysts, the phenotype of DC and Th in splenocytes and the expression of CD40, CD86, TLR2 and TLR4, on DCs sulfur were examined. The results show that after 60 days' infection, larval cysts grow on the surface of liver, and they become larger over time. Compared with the control mice, MHC I and MHC II expressions on DC were significantly increased at day 7 (p < 0.05). At the same time, CD40, CD86, TLR2 and TLR4 increased rapidly, but after that they decreased gradually. At day 120, those markers were lower than in the control group. The ratio of CD4/CD8 was normal during 90 days of infection, while at day 120, a decline in CD4 T cell and increase in CD8 were foundleading to the inversion of the CD4/CD8 ratio. Our findings suggest that within the 120 days of Em infection, the major function of DC is to present antigens. Immune response is provided predominantly by Th1 cells, inducing host immune response against Em. However, after 120 days, DC matured and the function was suppressed. Furthermore, inversion of the CD4/CD8 ratio is beneficial to the growth of Em, thus favoring its immune evasion.

[Investigation on role of p38α mitogen-activated protein kinases in human esophageal squamous cell carcinoma cell line Eca109].

OBJECTIVE: To investigate the role of p38α mitogen-activated protein kinases (MAPK) in human esophageal squamous cell carcinoma cell line Eca109. METHODS: Specific short hairpin (shRNA) vector as well as eukaryotic expression vector harbouring full length cDNA of human p38α MAPK were transfected into Eca109 cells. Cell proliferation after transfection was detected by MTT, cell cycle and apoptosis were assayed by flow cytometry. The variation of migration and invasion after transfection was determined using wound healing assay and Transwell assay, respectively. RESULTS: The proliferation of Eca109 cells after knock-down for 48 h (0.951 ± 0.086) was significantly increased (t = 3.20, P < 0.05) compared with control (0.811 ± 0.012), Sphase was increased but not significantly. Cell apoptosis rate after knock down for 48 h (17.400 ± 5.495) was significantly increased (t = 40.06, P < 0.01) compared with control(1.000 ± 0.721) . Migration after knock down for 72 h (0.034 ± 0.031) were enhanced pronouncedly (t = -5.79, P < 0.01) compared with control (0.278 ± 0.021) and invasive ability also increased; whereas the proliferation of Eca109 cells after over-expression for 48 h (0.472 ± 0.089) was inhibited significantly (t = -7.50, P < 0.01) compared with control(0.811 ± 0.012), cells arrested at G1 phase (t = 4.80, P < 0.01). Cell apoptosis rate (32.233 ± 1.457) were decreased significantly (t = 17.20, P < 0.01) compared with control (1.000 ± 0.721) mm, migration after overexpression for 72 h ((0.770 ± 0.054) mm) was suppressed pronouncedly compared with control groups of (0.278 ± 0.021) mm(t = 11.00, P < 0.01).Invasion after overexpression was inhibited. CONCLUSIONS: p38α MAPK plays an anti-oncogenic role in the pathogenesis of esophageal squamous cell carcinoma cell line Eca109.

[Echinococcus multilocularis infection leads to increased expression of proliferation and anti-apoptosis factors in liver cells in BALB/c mice].

OBJECTIVE: To investigate the effects of Echinococcus multilocularis on host liver cell proliferation in vivo using a BALB/c mouse alveolar hydatid infection model. METHODS: Sixty-five 8-10-week-old female BALB/c mice were randomly divided into an experimental group (n = 40) and a control group (n = 25) and administered an abdominal injection into the left liver lobe of E. multilocularis protoscolices in saline solution or saline solution alone, respectively. At post-injection day 2, 8, 30, 60, and 90, liver samples were collected for analysis of lesions and lesion-adjacent tissue by hematoxylin-eosin staining and differential expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin A, and cyclin B1 by immunohistochemical staining. The significance of intergroup differences was assessed by Student's t-test. RESULTS: The control group showed normal liver histology at all time points. The experimental group developed E. multilocularis lesions that showed increased severity of pathological features, such as inflammatory cell invasion, steatosis and fibrous connective tissue hyperplasia, over time. At post-injection days 2 and 8, enlarged, binuclear and apocyte hepatocytes were observed close to the lesions. At post-injection days 30, 60, and 90, the number of hepatocytes expressing PCNA progressively increased in the experimental group, and the numbers were significantly higher than in the control group (7.01 +/- 1.89 vs. 1.03 +/- 0.52, 8.41 +/- 2.80 vs. 0.93 +/- 0.31, and 13.4 +/- 4.43 vs. 1.07 +/- 0.94; all P < 0.05). The same progressively increasing trend was seen in the number of hepatocytes expressing CyclinD1, but was only significantly different from controls at post-injection days 30 and 60 (6.73 +/- 2.52 vs. 0.48 +/- 0.43 and 8.22 +/- 3.09 vs. 0.55 +/- 0.34; both P < 0.05). In contrast, the number of hepatocytes expressing cyclin A was significantly increased at post-injection day 30 and then showed a decreasing trend at days 60 and 90, although the numbers of expressing cells remained significantly higher than control levels at all time points (7.75 +/- 3.05 vs. 0.69 +/- 0.36, 3.42 +/- 1.80 vs. 1.14 +/- 0.42, and 3.03 +/- 1.50 vs. 0.69 +/- 0.31; all P < 0.05). The number of hepatocytes expressing CyclinB1 in the experimental group was less robust than the other cyclins (with a general temporal trend of increase followed by decrease), but the differential expression was not significantly different from the control levels at any time point. CONCLUSION: E. multilocularis infection may promote the expression of host factors related to proliferation and anti-apoptosis in liver. This pathogen-mediated modulation of host cell-survival mechanisms may provide a rationale explanation for the clinical observations of hepatomegaly and the unexpected survival of alveolar echinococcosis patients following major hepatic resection.

A 6-week randomized controlled trial with 4-week follow-up of acupuncture combined with paroxetine in patients with major depressive disorder.

Acupuncture possesses the antidepressant potential. In this 6-week randomized controlled trial with 4-week follow-up, 160 patients with major depressive disorder (MDD) were randomly assigned to paroxetine (PRX) alone (n = 48) or combined with 18 sessions of manual acupuncture (MA, n = 54) or electrical acupuncture (EA, n = 58). Treatment outcomes were measured mainly using the 17-item Hamilton Depression Rating Scale (HAMD-17), Self-rating Depression Scale (SDS), clinical response and remission rates. Average PRX dose taken and proportion of patients who required an increased PRX dose due to symptom aggravation were also obtained. Both additional MA and EA produced a significantly greater reduction from baseline in score on HAMD-17 and SDS at most measure points from week 1 through week 6 compared to PRX alone. The clinical response was markedly greater in MA (69.8%) and EA (69.6%) groups than the group treated with PRX alone (41.7%, P = 0.004). The proportion of patients who required an increase dose of PRX due to symptom aggravation was significantly lower with MA (5.7%) and EA (8.9%) than PRX alone (22.9%, P = 0.019). At 4 weeks follow-up after completion of acupuncture treatment, patients with EA, but not MA, continued to show significantly greater clinical improvement. Incidence of adverse events was not different in the three groups. Our study indicates that acupuncture can accelerate the clinical response to selective serotonin reuptake inhibitors (SSRIs) and prevent the aggravation of depression. Electrical acupuncture may have a long-lasting enhancement of the antidepressant effects (Trial Registration: ChiCTR-TRC-08000278).

[Different Echinococcus granulosus antigens induced indoleamine 2,3-dioxygenase expression in dendritic cells].

OBJECTIVE: To observe the expression of indoleamine 2, 3-dioxygenase (IDO) in dendritic cells (DCs) via different Echinococcus granulosus antigens in vitro. METHODS: Bone Marrow DCs generated from bone marrow precursor cells of C57BL/6 mice and cultured in the presence of recombinant mouse GM-CSF (rmGM-CSF). Then, DCs were induced with 15 microg/ml recombinant antigen B (rAgB), 5 mg/ml mouse hydatid fluid (MHF), 1,000 U/ml IFN-gamma (as positive control), and RPMI 1640 complete medium (as negative control), respectively. Meanwhile, the treated DCs and cell supernatants were collected at 18, 24 and 48 h after induction. The positive expressions of D40, CD80, CD86 and I- A/I-E on DCs were determined by flow cytometry. By real-time fluorescent quantitative reverse-transcription polymerase chain reaction (FQ-RT-PCR), the expression level of IDO mRNA in DCs was measured. Concentrations of tryptophan (Try) were tested by high-performance liquid chromatography (HPLC) assay in cell supernatant. RESULTS: The data from flow cytometry showed that the positive expressions of CD40, CD80, CD86, I-A/I-E were decreased after stimulated by rAgB and MHF. At 24 h after induction, there was significant difference in the level of CD40, CD86 and I-A/I-E among rAgB-treated group [(22.60 +/- 2.69)%, (35.50 +/- 4.38)%, (57.30 +/- 4.38)%], MHF-treated group [(38.00 +/- 3.54)%, (53.00 +/- 3.39)%, (77.10 +/- 1.70)%] and negative control [(37.95 +/- 3.61)%, (19.55 +/- 1.06)% and (85.45 +/-1.63)%] (P < 0.05). At 18, 24 and 48 h after induction, the levels of IDO mRNA in rAgB-treated group [(9.20 +/- 0.01), (29.44 +/- 0.02), (16.48 +/- 0.04)] and MHF-treated group [(9.67 +/- 0.02), (17.52 +/- 0.01), (16.81 +/- 0.01)] was higher than that of negative control group [(2.46 +/- 0.01), (7.77 +/- 0.01), and (10.56 +/- 0.01)] (P < 0.01). And significant difference was found between rAgB-treated group and MHF-treated group (P < 0.05). At 18, 24 and 48 h after induction, the concentrations of Try were lowest in rAgB-treated group [(23.65 +/- 0.64), (13.95 +/- +1.06), (19.0 +/- 00.64) micro.mol/L]. At 24h after induction, Try concentration in negative control group (22.9 +/- 0.14) was higher than that of MHF-treated group (20.65 +/- 0.34) ( P < 0.05). CONCLUSION: Under in vitro condition, rAgB and MHF can up-regulate IDO expression. The ability of rAgB to up-regulate IDO activity was stronger than that of MHF at 24 h after induction.

[Effect of Echinococcus granulosus cyst fluid on the proliferation of HepG2 cells].

OBJECTIVE: To investigate whether Echinococcus granulosus cyst fluid-infected host liver cells had differential expression of mitogen-activated protein kinases (MAPKs) or differential cell cycle activity. METHODS: Human liver cells cultured with different concentrations of hydatid cyst fluid (HCF) were tested by the MTT method to determine effects on proliferation. The cell cycle was assessed by flow cytometry. Western blotting was used to detect changes in protein expressions of p-ERK, PCNA, cyclin-A, cyclin-B1, cyclin-D1, and cyclin-E. RESULTS: Forty-eight, 72 and 96 h of HCF at 15%, 30% and 60% concentrations in the cell media significantly promoted cell proliferation (F=67.845, P less than 0.01) and compared to controls (P less than 0.05). Cells exposed to 15% HCF for 48 h showed significantly induced expression of p-ERK (F=1.916, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 24 h showed significantly induced expression of cyclin-Dl (F=3.901, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 48 h or 30% HCF for 72 h showed significantly induced expression of PCNA (F=91.140, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 15% HCF for 48 h or 30% HCF for 72 h shed significantly induced expression of cyclin-A (F=18.587, P=0.002), higher than controls (P less than 0.01). Cells exposed to 15% HCF for either 48 h or 72 h showed significantly induced expression of cyclin-B1 (F=2.064, P less than 0.01), higher than controls (P less than 0.01). Cells exposed to 30% HCF for 96 h showed significantly induced expression of cyclin-E (F=1.068, P less than 0.01), higher than controls (P less than 0.01). CONCLUSION: Hydatid cyst fluid exerts no inhibitory effect on primary cultured host liver cells, but may promote cellular proliferation.

Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs of the host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage. Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK. METHODS: DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence. RESULTS: We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices. CONCLUSIONS: We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and protoscolex. These findings will be useful in further study of the biological functions of the gene in the growth and development of Echinococcus and will contribute to research on novel anti-echinococcosis drug targets.

[Echinococcus granulosus recombinant antigen B induced IDO expression in mouse bone marrow-derived dendritic cells in vitro].

OBJECTIVE: To observe the expression of indoleamine 2,3-dioxygenase (IDO) in mouse bone marrow-derived dendritic cells (DCs) after adding Echinococcus granulosus recombinant antigen B (rAgB) in vitro. METHODS: CD11c+ DCs generated from bone marrow precursor cells of C57BL/6 mice and cultured in the presence of recombinant mouse GM-CSF (rmGM-CSF). The morphology of DCs was observed by inverted microscope and scanning electronic microscope. The level of I-A/I-E, CD40, CD80, and CD86 on DCs were determined by flow cytometry. T cell proliferation induced by DCs were evaluated by using mixed lymphocyte reaction (MLR) assay. At day 6 post culture, the immature DCs were collected, and part of the immature DCs stimulated with lipopolysaccharide (LPS) for 24 h were examined by flow cytometry. Immature DCs were divided into 3 groups: negative control group, positive control group (rmIFN-gamma, 1000 U/ml) and rAgB group. Immature DCs of positive control group and rAgB group were induced with 1000 U/ml rmIFN-gamma and 15 microg/ml rAgB, respectively. IDO expression in DCs was examined 24 h after induction using immunohistochemical method and Western blotting. RESULTS: More than 80% CD11c+ DCs were harvested. The typical DCs were observed under inverted microscope and scanning electronic microscope. The level of CD40, CD80, and IA/IE (MHC II) in mature DCs group was significantly higher than that of immature DCs group (P < 0.05). In MLR, mitomycin-treated DCs can stimulate T lymphocytes proliferation activity. There were significantly differences in IDO expression in the negative control group [(4.544 +/- 1.752)%], positive control group [(20.464 +/- 4.452)%] and rAgB group [(11.148 +/- 1.966)%] (P < 0.05). Western blotting result indicated that the ratio of IDO/GAPDH in rAgB group (0.573 +/- 0.129) was significantly higher than that of negative group (0.229 +/- 0.085) (P < 0.05), and there were no significant difference in the ratio of IDO/GAPDH between IFN-gamma group (0.794 +/- 0.114) and rAgB group (P > 0.05). CONCLUSION: rAgB can induce IDO expression in bone marrow-derived dendritic cells in vitro.

[Expression of ERK1/2 MAPK signaling transduction pathway in esophageal cancers in Kazakh patients].

OBJECTIVE: To investigate the expression variation and significance of ERK1/2 MAPK signaling transduction pathway in the pathogenesis of esophageal squamous cell carcinoma (ESCC) in Kazakh patients. METHODS: The expression level of p-ERK1/2 after serum starvation and treatment with U0126 inhibitor was detected in esophageal cancer cell line EC9706 by Western blot assay. The mRNA level of total ERK1/2 (t-ERK1/2) and expression level of t-ERK1/2 and p-ERK1/2 proteins of 25 pairs of ESCC and adjacent normal esophageal mucosal tissues of Kazakh patients were examined and identified by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The expression of p-ERK1/2 protein was verified by immunohistochemistry in 126 paraffin-embeded specimens, including 19 normal esophageal mucosa, 55 esophageal carcinomas in situ and 52 invasive carcinomas. RESULTS: ERK1/2 MAPK signaling transduction pathway was in an active status in the EC9706 cells. The expression level of p-ERK1/2 in Ec9706 cells reached a peak at 10 min after transient serum stimulation, and p-ERK1/2 expression was totally restrained after the treatment with 50 µmol/L U0126. In the 25 pairs of ESCC and adjacent normal mucosa, the t-ERK1 mRNA level was 1.92 ± 3.49 in the ESCC tissues and 3.67 ± 7.47 in the adjacent normal mucosa. The t-ERK1 mRNA level in ESCC tissues was significantly lower than that in adjacent normal mucosa (P < 0.05), whereas there was no significant difference of t-ERK2 mRNA level between them(P > 0.05). The expression levels of p-ERK1 and p-ERK2 proteins were 0.87 ± 0.14 and 0.79 ± 0.10 in the ESCC tissues, and 1.10 ± 0.13 and 1.32 ± 0.12 in the adjacent normal mucosae. p-ERK1/2 protein in the ESCC tissues was significantly lower than that in the adjacent normal tissue (P < 0.01). However, there was no significant difference between their t-ERK1/2 protein levels (P > 0.05). In the 126 cases of paraffin-embeded specimens, positive expressions of both p-ERK1 and p-ERK2 in esophageal cancer tissues were 7.7% (4/52), significantly lower than those in adjacent normal mucosa (31.6%, 6/19) and carcinoma in situ (85.5%, 47/55, P < 0.05). CONCLUSIONS: ERK1/2 MAPK signaling pathway is in an active status in esophageal cancer and adjacent normal mucosa. Our results imply that the activation of p-ERK1/2 MAPK signaling transduction pathway plays a role in the early pathogenesis of ESCC in Kazakh patients.