Inhibition of AMPK activation in Echinococcus granulosus sensu stricto limits the parasite's glucose metabolism and survival.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by larvae of the Echinococcus granulosus sensu lato (s.l.) cluster. There is an urgent need to develop new drug targets and drug molecules to treat CE. Adenosine monophosphate (AMP)-activated protein kinase (AMPK), a serine/threonine protein kinase consisting of α, ß, and γ subunits, plays a key role in the regulation of energy metabolism. However, the role of AMPK in regulating glucose metabolism in E. granulosus s.l. and its effects on parasite viability is unknown. In this study, we found that targeted knockdown of EgAMPKα or a small-molecule AMPK inhibitor inhibited the viability of E. granulosus sensu stricto (s.s.) and disrupted the ultrastructure. The results of in vivo experiments showed that the AMPK inhibitor had a significant therapeutic effect on E. granulosus s.s.-infected mice and resulted in the loss of cellular structures of the germinal layer. In addition, the inhibition of the EgAMPK/EgGLUT1 pathway limited glucose uptake and glucose metabolism functions in E. granulosus s.s.. Overall, our results suggest that EgAMPK can be a potential drug target for CE and that inhibition of EgAMPK activation is an effective strategy for the treatment of disease.

Involvement of TIGIT in Natural Killer Cell Exhaustion and Immune Escape in Patients and Mouse Model With Liver Echinococcus multilocularis Infection.

BACKGROUND AND AIMS: Alveolar echinococcosis (AE) is a lethal helminthic liver disease caused by persistent infection with Echinococcus multilocularis. Although more attention has been paid to the immunotolerance of T cells caused by E. multilocularis infection, the role of natural killer (NK) cell, a critical player in liver immunity, is seldom studied. APPROACH AND RESULTS: Here, we observed that NK cells from the blood and closed liver tissue (CLT) of AE patients expressed a higher level of inhibitory receptor TIGIT and were functionally exhausted with a lower expression of granzyme B, perforin, interferon-gamma (IFN-γ), and TNF-α. Addition of anti-TIGIT (T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) monoclonal antibody into AE patients' peripheral blood mononuclear cell culture significantly enhanced the synthesis of IFN-γ and TNF-α by NK cells, indicating the reversion of exhausted NK cells by TIGIT blockade. In the mouse model of E. multilocularis infection, liver and splenic TIGIT+ NK cells progressively increased dependent of infection dosage and timing and were less activated and less degranulated with lower cytokine secretion. Furthermore, TIGIT deficiency or blockade in vivo inhibited liver metacestode growth, reduced liver injury, and increased the level of IFN-γ produced by liver NK cells. Interestingly, NK cells from mice with persistent chronic infection expressed a higher level of TIGIT compared to self-healing mice. To look further into the mechanisms, more regulatory CD56bright and murine CD49a+ NK cells with higher TIGIT expression existed in livers of AE patients and mice infected with E. multilocularis, respectively. They coexpressed higher surface programmed death ligand 1 and secreted more IL-10, two strong inducers to mediate the functional exhaustion of NK cells. CONCLUSIONS: Our results indicate that inhibitory receptor TIGIT is involved in NK cell exhaustion and immune escape from E. multilocularis infection.

Roles of immune cells in the concurrence of Echinococcus granulosus sensu lato infection and hepatocellular carcinoma.

Immune cells are pivotal players in the immune responses against both parasitic infection and malignancies. Substantial evidence demonstrated that there may exist possible relationship between echinococcus granulus sensu lato (E. granulosus s.l.) infection and hepatocellular carcinoma (HCC) development. Thus, this study aimed to observe crucial roles of immune cells in the formation of subcutaneous lesions after transplanting HepG2 cell lines with or without E. granulosus s.l. protoscoleces (PSCs). HepG2 cell lines were subcutaneously injected into nude mice in the control group. In the co-transplantation group, HepG2 cells were subcutaneously co-injected with high dosage of E. granulosus s.l. PSCs. From the 25th day of transplantation, volume of subcutaneous lesions was measured every four days, which were removed at the 37th day for further studies. Basic pathological and functional changes were observed. Moreover, expression of Ki67, Bcl-2, Caspase3, α-smooth muscle actin (α-SMA), T cell markers (CD3, CD4, CD8), PD1/PD-L1, nature killer (NK) cell markers (CD16, CD56) were further detected by immunohistochemical staining and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Subcutaneous lesions were gradually increased in volume and there occurred pathologically heterogeneous tumor cells, which were more significant in the co-transplantation group. Compared to the control group, expression of proliferation markers Ki67 and Bcl-2 was at higher levels in the co-transplantation group. Reversely, apoptotic marker Caspase3 was highly detected in the control group, suggesting promoting effects of E. granulosus s.l. PSCs on HCC development. Interestingly, subcutaneous lesions of the co-transplantation group were more functional in synthesizing and storing glycogen. Collagen and α-SMA+ cells were also at higher levels in the co-transplantation group than those in the control group. Most importantly, co-transplantation of HepG2 cells with E. granulosus s.l. PSCs led to significant increase in the expression of T cell markers, PD1/PD-L1 and NK cells markers. E. granulosus s.l. may have promoting effects on HCC development, which was closely associated with the immune responses of T cells and NK cells.

Immune Exhaustion of T Cells in Alveolar Echinococcosis Patients and Its Reversal by Blocking Checkpoint Receptor TIGIT in a Murine Model.

BACKGROUND AND AIMS: The cestode Echinococcus multilocularis infection, a serious health problem worldwide, causes alveolar echinococcosis (AE), a tumor-like disease predominantly located in the liver and able to spread to any organs. Until now, there have been few studies that explore how T-cell exhaustion contributes to the parasite's escape from immune attack and how it might be reversed. APPROACH AND RESULTS: In this study, we found that liver T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) expression was significantly enhanced and positively correlated with lesion activity in AE patients. High TIGIT expression in both liver-infiltrating and blood T cells was associated with their functional exhaustion, and its ligand CD155 was highly expressed by hepatocytes surrounding the infiltrating lymphocytes. In co-culture experiments using human blood T cells and hepatic cell line HL-7702, CD155 induced functional impairment of TIGIT+ T cells, and in vitro blockade with TIGIT antibody restored the function of AE patients' T cells. Similar TIGIT-related functional exhaustion of hepatic T cells and an abundant CD155 expression on hepatocytes were observed in E. multilocularis-infected mice. Importantly, in vivo blocking TIGIT prevented T-cell exhaustion and inhibited disease progression in E. multilocularis-infected mice. Mechanistically, CD4+ T cells were totally and CD8+ T cells partially required for anti-TIGIT-induced regression of parasite growth in mice. CONCLUSIONS: This study demonstrates that E. multilocularis can induce T-cell exhaustion through inhibitory receptor TIGIT, and that blocking this checkpoint may reverse the functional impairment of T cells and represent a possible approach to immunotherapy against AE.

An improved experimental method for simultaneously isolating hepatocytes and hepatic stellate cells in mouse liver infected with Echinococcus multilocularis.

BACKGROUND: Alveolar echinococcosis (AE) is a zoonotic disease caused by the larval stage of Echinococcus multilocularis parasitizing in the human liver, causing local pathological changes in the liver and manifesting as hyperplasia, liver fibrosis, atrophy, degeneration, and necrosis. Here, we report a method that can simultaneously isolate hepatocytes and hepatic stellate cells (HSCs) from mice infected with Echinococcus multilocularis. METHODS: A mouse model of AE was established. Hepatocytes and HSCs were isolated from mouse liver using a two-step method combining in situ collagenase perfusion and gradient centrifugation. Expressions of Alb, Desmin, and α-SMA were detected with immunofluorescence to identify the isolated hepatocytes and HSCs. RESULTS: The viability and purity of hepatocytes and HSCs both reached 90% or above. For hepatocytes, clear cell boundaries were observed, and the nuclei were round or oval, with clear nucleoli. There was a homogeneous distribution of the hepatocyte marker Alb in the cytoplasm of hepatocytes. Lipid droplets and Desmin expression were observed in the cytoplasm of freshly isolated HSCs. During the activation of HSCs, the lipid droplets gradually decreased and disappeared with a high expression of α-SMA. CONCLUSION: Hepatocytes and HSCs are simultaneously isolated. This may provide a research tool to investigate the interaction between hepatocytes and HSCs and to investigate the mechanism of Echinococcus multilocularis infection-induced liver pathological changes.

Human CD96 Correlates to Natural Killer Cell Exhaustion and Predicts the Prognosis of Human Hepatocellular Carcinoma.

Immune checkpoint blockade has become a promising therapeutic approach to reverse immune cell exhaustion. Coinhibitory CD96 and T-cell immunoglobulin and ITIM domain (TIGIT), together with costimulatory CD226, bind to common ligand CD155. The balancing between three receptors fine-tunes immune responses against tumors. In this study, we investigated the expression of CD96, TIGIT, and CD226 in 55 fresh human hepatocellular carcinoma (HCC) samples, 236 paraffin-embedded HCC samples, and 20 normal human livers. The cumulative percentage, absolute count, and mean fluorescence intensity (MFI) of CD96+ NK cells are significantly increased in the intratumoral tissues of HCC and break the balance between three receptors. Human CD96+ NK cells are functionally exhausted with impaired interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production, high gene expression of interleukin (IL)-10 and transforming growth factor-beta 1 (TGF-ß1), and low gene expression of T-bet, IL-15, perforin, and granzyme B. In addition, blocking CD96-CD155 interaction specifically increases lysis of HepG2 cells by NK cells. HCC patients with a high level of CD96 or CD155 expression within tumor are strongly associated with deteriorating disease condition and shorter disease-free survival (DFS) and overall survival times. Patients with a higher cumulative percentage of CD96+ NK cells within tumor also exhibit shorter DFS. High plasma level of TGF-ß1 in HCC patients up-regulates CD96 expression and dynamically shifts the balance between CD96, TIGIT, and CD226 in NK cells. Blocking TGF-ß1 specifically restores normal CD96 expression and reverses the dysfunction of NK cells. Conclusion: These findings indicate that human intratumoral CD96+ NK cells are functionally exhausted and patients with higher intratumoral CD96 expression exhibit poorer clinical outcomes. Blocking CD96-CD155 interaction or TGF-ß1 restores NK cell immunity against tumors by reversing NK cell exhaustion, suggesting a possible therapeutic role of CD96 in fighting liver cancer.

Identification of Functional MKK3/6 and MEK1/2 Homologs from Echinococcus granulosus and Investigation of Protoscolecidal Activity of Mitogen-Activated Protein Kinase Signaling Pathway Inhibitors In Vitro and In Vivo.

Cystic echinococcosis is a zoonosis caused by the larval stage of Echinococcus granulosussensu lato There is an urgent need to develop new drugs for the treatment of this disease. In this study, we identified two new members of mitogen-activated protein kinase (MAPK) cascades, MKK3/6 and MEK1/2 homologs (termed EgMKK1 and EgMKK2, respectively), from E. granulosussensu stricto Both EgMKK1 and EgMKK2 were expressed at the larval stages. As shown by yeast two-hybrid and coimmunoprecipitation analyses, EgMKK1 interacted with the previously identified Egp38 protein but not with EgERK. EgMKK2, on the other hand, interacted with EgERK. In addition, EgMKK1 and EgMKK2 displayed kinase activity toward the substrate myelin basic protein. When sorafenib tosylate, PD184352, or U0126-ethanol (EtOH) was added to the medium for in vitro culture of E. granulosus protoscoleces (PSCs) or cysts, an inhibitory and cytolytic effect was observed via suppressed phosphorylation of EgMKKs and EgERK. Nonviability of PSCs treated with sorafenib tosylate or U0126-EtOH, and not with PD184352, was confirmed through bioassays, i.e., inoculation of treated and untreated protoscoleces into mice. In vivo treatment of E. granulosussensu stricto-infected mice with sorafenib tosylate or U0126-EtOH for 4 weeks demonstrated a reduction in parasite weight, but the results did not show a significant difference. In conclusion, the MAPK cascades were identified as new targets for drug development, and E. granulosus was efficiently inhibited by their inhibitors in vitro The translation of these findings into in vivo efficacy requires further adjustment of treatment regimens using sorafenib tosylate or, possibly, other kinase inhibitors.

Echinococcus multilocularis inoculation induces NK cell functional decrease through high expression of NKG2A in C57BL/6 mice.

BACKGROUND: Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis (E. multilocularis), and considered as public health issue. Parasite-host immune interaction is pivotal during infection. As a subset of innate lymphoid cells, NK cells are known to play an important role during virus, bacteria, intra/extracellular parasitic infections and tumor progression. However, the possible role of NK cells in E. multilocularis infection in both human and murine is little known. Herein, the functional alteration of hepatic NK cells and their related molecules in E. multilocularis infected mice were studied. METHODS: 2000 protoscoleces (PSCs) were injected to C57BL/6 mice via the portal vein to establish secondary E. multilocularis infection. NK cells population and their related molecules (CD69, Ly49D, Ly49G2, Ly49H, Ly49I, NKG2A, NKG2D, granzyme B, IFN-γ, TNF-α) were assessed by using fluorescence-activated cell sorter (FACS) techniques and qRT-PCR. NK cell depletion was performed for further understanding the possible function of NK cells during infection. RESULTS: The total frequencies of NK cells and NK-derived IFN-γ production were significantly reduced at designated time points (2, 4, 12 weeks). The liver resident (CD49a+DX5-) NK cells are decreased at 4 weeks after inoculation and which is significantly lower than in control mice. Moreover, in vivo antibody-mediated NK cell depletion increased parasitic load and decreased peri-parasitic fibrosis. Expression of the inhibitory receptor NKG2A was negatively related to NK- derived IFN-γ secretion. CONCLUSIONS: Our study showed down regulates of NK cells and upper regulates of NKG2A expression on NK cells during E. multilocularis infection. Reduction of NK cell frequencies and increased NKG2A might result in low cytotoxic activity through decreased IFN-γ secretion in E. multilocularis infection. This result might be helpful to restore NK cell related immunity against E. multilocularis infection to treat alveolar echinococcosis.

Foxp3+ T Regulatory Cells as a Potential Target for Immunotherapy against Primary Infection with Echinococcus multilocularis Eggs.

Alveolar echinococcosis (AE) is a lethal disease caused by infection with the metacestode stage of the helminth Echinococcus multilocularis, which develops into a tumorlike mass in susceptible intermediate hosts. The growth potential of this parasite stage is directly linked to the nature of the surrounding periparasitic immune-mediated processes. In a first step (experiment 1), mice were orally infected with E. multilocularis eggs, to be used for assessing the hepatic expression profiles of 15 selected cytokine and chemokine genes related to acquired immunity from 21 to 120 days postinfection. The early stage of infection in immunocompetent animals was marked by a mixed Th1/Th2 immune response, as characterized by the concomitant presence of gamma interferon (IFN-γ) and interleukin-4 (IL-4) and their related chemokines. At the late stage of AE, the profile extended to a combined tolerogenic mode including Foxp3, IL-10, and transforming growth factor beta (TGF-ß) as key components. In a second step (experiment 2), the effect of T regulatory cell (Treg) deficiency on metacestode growth was assessed in E. multilocularis-infected DEREG (depletion of regulatory T cells) mice upon induction of Treg deficiency with diphtheria toxin (DT). The parasite lesions were significantly smaller in the livers of treated mice than in corresponding control groups. Foxp3+ Tregs appear to be one of the key players in immune-regulatory processes favoring metacestode survival by affecting antigen presentation and suppressing Th1-type immune responses. For these reasons, we suggest that affecting Foxp3+ Tregs could offer an attractive target in the development of an immunotherapy against AE.

Larval Echinococcus multilocularis infection reduces dextran sulphate sodium-induced colitis in mice by attenuating T helper type 1/type 17-mediated immune reactions.

The tumour-like growth of larval Echinococcus multilocularis tissue (causing alveolar echinococcosis, AE) is directly linked to the nature/orientation of the periparasitic host immune-mediated processes. Parasite-mediated immune suppression is a hallmark triggering infection outcome in both chronic human and murine AE. So far, little is known about secondary systemic immune effects of this pathogen on other concomitant diseases, e.g. endogenous gut inflammation. We examined the influence of E. multilocularis infection on murine dextran sodium sulphate (DSS) -induced colitis. At 3 months after E. multilocularis infection (chronic stage), the mice were challenged with 3% DSS in the drinking water for 5 days plus subsequently with tap water (alone) for another 4 days. After necropsy, fixed tissues/organs were sectioned and stained with haematoxylin & eosin for assessing inflammatory reactions. Cytokine levels were measured by flow cytometry and quantitative RT-PCR. Colitis severity was assessed (by board-certified veterinary pathologists) regarding (i) colon length, (ii) weight loss and (iii) a semi-quantitative score of morphological changes. The histopathological analysis of the colon showed a significant reduction of DSS-induced gut inflammation by concomitant E. multilocularis infection, which correlated with down-regulation of T helper type 1 (Th1)/Th17 T-cell responses in the colon tissue. Echinococcus multilocularis infection markedly reduced the severity of DSS-induced gut inflammation upon down-regulation of Th1/Th17 cytokine expression and attenuation of CD11b+ cell activation. In conclusion, E. multilocularis infection remarkably reduces DSS-induced colitis in mice by attenuating Th1/Th17-mediated immune reactions.

Molecular Cloning and Characterization of a P38-Like Mitogen-Activated Protein Kinase from Echinococcus granulosus.

Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus. We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus. Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-ß1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus, as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-ß1.

[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.

[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.

Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus.

Smad family proteins are essential cellular mediators of the transforming growth factor-ß superfamily. In the present study, we identified two members of the Smad proteins, Smad8 and Smad4 homologues (termed as EgSmadE and EgSmadD, respectively), from Echinococcus granulosus, the causative agent of cystic echinococcosis (CE). Phylogenetic analysis placed EgSmadE in the Smad1, 5, and 8 subgroup of the R-Smad sub-family and EgSmadD in the Co-Smad family. Furthermore, EgSmadE and EgSmadD attained a high homology to EmSmadE and EmSmadD of E. multilocularis, respectively. Both EgSmadE and EgSmadD were co-expressed in the larval stages and exhibited the highest transcript levels in activated protoscoleces, and their encoded proteins were co-localized in the sub-tegumental and tegumental layer of the parasite. As shown by yeast two-hybrid and pull-down analysis, EgSmadE displayed a positive binding interaction with EgSmadD. In addition, EgSmadE localized in the nuclei of Mv1Lu cells (mink lung epithelial cells) upon treatment with human TGF-ß1 or human BMP2, indicating that EgSmadE is capable of being translocated into nucleus, in vitro. Our study suggests that EgSmadE and EgSmadD may take part in critical biological processes, including echinococcal growth, development, and parasite-host interaction.

Brain areas involved in the acupuncture treatment of AD model rats: a PET study.

BACKGROUND: Acupuncture may effectively treat certain symptoms of Alzheimer's disease (AD). Although several studies have used functional brain imaging to investigate the mechanisms of acupuncture treatment on AD, these mechanisms are still poorly understood. We therefore further explored the mechanism by which needling at ST36 may have a therapeutic effect in a rat AD model. METHODS: A total of 80 healthy Wistar rats were divided into healthy control (n = 15) and pre-model (n = 65) groups. After inducing AD-like disease, a total of 45 AD model rats were randomly divided into three groups: the model group (n = 15), the sham-point group (n = 15), and the ST36 group (n = 15). The above three groups underwent PET scanning. PET images were processed with SPM2. RESULTS: The brain areas that were activated in the sham-point group relative to the model group were primarily centred on the bilateral limbic system, the right frontal lobe, and the striatum, whereas the activated areas in the ST36 group were primarily centred on the bilateral limbic system (pyriform cortex), the bilateral temporal lobe (olfactory cortex), the right amygdala and the right hippocampus. Compared with the sham-point group, the ST36 group showed greater activation in the bilateral amygdalae and the left temporal lobe. CONCLUSION: We concluded that needling at a sham point or ST36 can increase blood perfusion and glycol metabolism in certain brain areas, and thus may have a positive influence on the cognition of AD patients.

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.

Numerical investigation of effect of geometric parameters on performance of rotational hydrodynamic cavitation reactor.

The objective of this paper is to discuss the influence of geometric parameters on the performance of the rotational hydrodynamic cavitation reactor (RHCR) using numerical method. The novel RHCR is implemented by modifying a centrifugal impeller into a new one using the annular slit constriction (ASC) with circumferentially distributed blind holes. The cavitation intensity and cavitation generation rate are selected to evaluate the cavitation performance, the head is used to assess conveying performance, and the entropy generation theory is used to evaluate the energy loss in the impeller. The effect of the axial width, radial length and radial position of the ASC on the cavitating flow of the RHCR is investigated by CFD method. The results indicate that three patterns of cavitation are induced in the RHCR, including separation cavitation, vortex cavitation and shear cavitation. The axial width, radial length and radial position of the ASC are the important geometric parameter that affect the performance of the RHCR. A small width is superior to a large width in terms of cavitation performance, although the conveying performance suffers as a result. The energy loss in the impeller initially increases and then decreases as the width decreases. Both a reduction in radial length and radial position leads to higher cavitation and conveying capacity, accompanying slight increase in energy loss. Compared to the original model, the RHCR with an axial width of 3 mm, a radial length of 17 mm, and a radial position of 0.541 achieves the highest performance.

Phenotype and function of MAIT cells in patients with alveolar echinococcosis.

Mucosal-associated invariant T (MAIT) cells are a subpopulation of unconventional T cells widely involved in chronic liver diseases. However, the potential role and regulating factors of MAIT cells in alveolar echinococcosis (AE), a zoonotic parasitic disease by Echinococcus multilocularis (E. multilocularis) larvae chronically parasitizing liver organs, has not yet been studied. Blood samples (n=29) and liver specimens (n=10) from AE patients were enrolled. The frequency, phenotype, and function of MAIT cells in peripheral blood and liver tissues of AE patients were detected by flow cytometry. The morphology and fibrosis of liver tissue were examined by histopathology and immunohistochemistry. The correlation between peripheral MAIT cell frequency and serologic markers was assessed by collecting clinicopathologic characteristics of AE patients. And the effect of in vitro stimulation with E. multilocularis antigen (Emp) on MAIT cells. In this study, MAIT cells are decreased in peripheral blood and increased in the close-to-lesion liver tissues, especially in areas of fibrosis. Circulating MAIT exhibited activation and exhaustion phenotypes, and intrahepatic MAIT cells showed increased activation phenotypes with increased IFN-γ and IL-17A, and high expression of CXCR5 chemokine receptor. Furthermore, the frequency of circulating MAIT cells was correlated with the size of the lesions and liver function in patients with AE. After excision of the lesion site, circulating MAIT cells returned to normal levels, and the serum cytokines IL-8, IL-12, and IL-18, associated with MAIT cell activation and apoptosis, were altered. Our results demonstrate the status of MAIT cell distribution, functional phenotype, and migration in peripheral blood and tissues of AE patients, highlighting their potential as biomarkers and therapeutic targets.