Helminth-induced impairment of humoral immunity differently contribute to their anti-arthritic effects in mice: Comparison of Schistosoma mansoni and Trichinella spiralis.

AIMS: We have previously reported reduction of anti-type II collagen (IIC) IgG levels in collagen-induced arthritis (CIA) by Schistosoma mansoni (Sm) and Trichinella spiralis (Ts). To clarify the contribution of the impairment of humoral immunity to their anti-arthritic activities, we herein investigated the relationship between anti-IIC IgG levels and arthritic swelling in Sm- or Ts-infected mice. METHODS AND RESULTS: Male DBA/1J mice were infected with Sm cercariae or Ts muscle larvae prior to the IIC immunization. In the Sm-infected mice, paw swelling and anti-IIC IgG levels were continuously lower than those of non-infected control group. In contrast, arthritic swelling in the Ts-infected mice only decreased in the early phase of CIA progression, despite the continued impairment of anti-IIC IgG production throughout the experimental period. Correlation coefficients between residual paw swelling and anti-IIC IgG titers were similar or higher in the Sm group than in the control group, but were similar or lower in the Ts group than in the control group. CONCLUSION: The down-modulations of anti-IIC IgG levels by the two parasitic infections and the correlation analyses suggest that the anti-arthritic activity of Sm was primarily attributed to the modulation of IgG-independent arthritogenic mechanisms and secondarily to the impairment of anti-IIC IgG production. In contrast, Ts could alleviate CIA mainly via the impairment of antibody production.

The neuroprotective effects of FG-4592, a hypoxia-inducible factor-prolyl hydroxylase inhibitor, against oxidative stress induced by alpha-synuclein in N2a cells.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The pathological hallmark of PD is the appearance of intraneuronal cytoplasmic α-synuclein (α-Syn) aggregation, called Lewy bodies. α-Syn aggregation is deeply involved in the pathogenesis of PD. Oxidative stress is also associated with the progression of PD. In the present study, to investigate whether a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitor, FG-4592 (also called roxadustat), has neuroprotective effects against α-Syn-induced neurotoxicity, we employed a novel α-Syn stably expressing cell line (named α-Syn-N2a cells) utilizing a piggyBac transposon system. In α-Syn-N2a cells, oxidative stress and cell death were induced by α-Syn, and FG-4592 showed significant protection against this neurotoxicity. However, FG-4592 did not affect α-Syn protein levels. FG-4592 triggered heme oxygenase-1 (HO-1) expression downstream of HIF-1α in a concentration-dependent manner. In addition, FG-4592 decreased the production of reactive oxygen species possibly via the activation of HO-1 and subsequently suppressed α-Syn-induced neurotoxicity. Moreover, FG-4592 regulated mitochondrial biogenesis and respiration via the induction of the peroxisome proliferator-activated receptor-γ coactivator-1α. As FG-4592 has various neuroprotective effects against α-Syn and is involved in drug repositioning, it may have novel therapeutic potential for PD.

Trichinella pseudospiralis-secreted 53 kDa protein ameliorates imiquimod-induced psoriasis by inhibiting the IL-23/IL-17 axis in mice.

Trichinella infection can experimentally ameliorate many autoimmune diseases. However, the immune mechanism of the amelioration and the identification of corresponding Trichinella-derived molecule(s) are still not fully elucidated. Fifty-three kDa excretory-secretory (ES) protein from Trichinella pseudospiralis (Tpp53) is a molecule like TsP53 reported as a protein exerting immune-inhibitory effect in T. spiralis. In this study, we investigated the immunomodulatory effect of Tpp53 using imiquimod (IMQ)-induced psoriasis-like dermatitis model, which is a mouse model of autoimmune disease with the pathogenic interleukin 17 (IL-17) producing CD4+ T cells (Th17) via IL-23/IL17 axis. Administrating the recombinant Tpp53 (rTpp53) mixed with IMQ cream on the skin of mice ameliorated psoriatic lesions, as revealed by the improvement of erythema, scaling, skin thickening, epidermis hyperplasia and parakeratosis, thickening of acanthosis cell layer, epidermal extension of dermis, less infiltration of inflammatory cells, and decreased expression of inflammatory marker. The increased expression of the factors related to the IL-23/IL-17 axis, including IL-17A, IL-6, Il17F and Il23a, in the skins of IMQ-treated mice was inhibited by rTpp53 treatment. Moreover, the expression of activated keratinocyte-produced cytokines, chemokines, and antimicrobial peptides in the skin was also down-regulated in rTpp53-treated IMQ-treated mice. Co-culture of splenocytes with rTpp53 inhibited IL-17A and treatment of macrophages with rTpp53 reduced IL-6 production. Overall, our study revealed that the Trichinella-secreted 53 kDa ES protein could ameliorate IMQ-induced psoriasis by inhibiting the IL-23/IL-17 axis, suggesting that Tpp53 might involve in regulating host Th17 for immune evasion and have an alternative potential for psoriasis therapy.

AFF3, a susceptibility factor for autoimmune diseases, is a molecular facilitator of immunoglobulin class switch recombination.

Immunoglobulin class switch recombination (CSR) plays critical roles in controlling infections and inflammatory tissue injuries. Here, we show that AFF3, a candidate gene for both rheumatoid arthritis and type 1 diabetes, is a molecular facilitator of CSR with an isotype preference. Aff3-deficient mice exhibit low serum levels of immunoglobulins, predominantly immunoglobulin G2c (IgG2c) followed by IgG1 and IgG3 but not IgM. Furthermore, Aff3-deficient mice show weak resistance to Plasmodium yoelii infection, confirming that Aff3 modulates immunity to this pathogen. Mechanistically, the AFF3 protein binds to the IgM and IgG1 switch regions via a C-terminal domain, and Aff3 deficiency reduces the binding of AID to the switch regions less efficiently. One AFF3 risk allele for rheumatoid arthritis is associated with high mRNA expression of AFF3, IGHG2, and IGHA2 in human B cells. These findings demonstrate that AFF3 directly regulates CSR by facilitating the recruitment of AID to the switch regions.

TRAF6 signaling pathway in T cells regulates anti-tumor immunity through the activation of tumor specific Th9 cells and CTLs.

CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T (Th) cells play a critical role in protective immune responses to tumor cells. Particularly, Th9 cells exert anti-tumor activity by producing IL-9. TNF receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein that mediates the signals from both the TNFR superfamily and Toll-like receptors (TLRs). We have previously reported that T cell-specific TRAF6-deficent (TRAF6ΔT) mice spontaneously developed systemic inflammatory diseases. However, the physiological role of TRAF6 in T cells in controlling anti-tumor immune responses remains largely unclear. Here, we found that tumor formation of syngeneic colon cancer cells inoculated in TRAF6ΔT mice was accelerated compared to that in control mice. Although TRAF6-deficient naïve T cells showed enhanced differentiation of Th9 cells in vitro, these T cells produced lower amounts of IL-9 in response to a specific antigen. Moreover, CD4+ tumor-infiltrating lymphocytes (TILs) in tumor-bearing TRAF6ΔT mice expressed lower levels of IL-9 than those in WT mice. Importantly, administration of recombinant IL-9 (rIL-9) strongly suppressed tumor progression in TRAF6ΔT mice. Furthermore, expression levels of the T-box transcription factor Eomesodermin (Eomes) and its target molecules IFN-γ, granzyme B and perforin, as well as cytotoxic activity, were reduced in TRAF6-deficient CD8+ T cells in vitro. TRAF6-deficient T cells were found to express significantly increased levels of immune checkpoint molecules, CTLA-4 and PD-1 on the cell surface. These results demonstrate that the TRAF6 signaling pathway in T cells regulates anti-tumor immunity through the activation of tumor specific Th9 cells and CTLs in a tumor microenvironment.

Glucose transporter Glut1 controls diffuse invasion phenotype with perineuronal satellitosis in diffuse glioma microenvironment.

BACKGROUND: Gliomas typically escape surgical resection and recur due to their "diffuse invasion" phenotype, enabling them to infiltrate diffusely into the normal brain parenchyma. Over the past 80 years, studies have revealed 2 key features of the "diffuse invasion" phenotype, designated the Scherer's secondary structure, and include perineuronal satellitosis (PS) and perivascular satellitosis (PVS). However, the mechanisms are still unknown. METHODS: We established a mouse glioma cell line (IG27) by manipulating the histone H3K27M mutation, frequently harboring in diffuse intrinsic pontine gliomas, that reproduced the diffuse invasion phenotype, PS and PVS, following intracranial transplantation in the mouse brain. Further, to broadly apply the results in this mouse model to human gliomas, we analyzed data from 66 glioma patients. RESULTS: Increased H3K27 acetylation in IG27 cells activated glucose transporter 1 (Glut1) expression and induced aerobic glycolysis and TCA cycle activation, leading to lactate, acetyl-CoA, and oncometabolite production irrespective of oxygen and glucose levels. Gain- and loss-of-function in vivo experiments demonstrated that Glut1 controls the PS of glioma cells, that is, attachment to and contact with neurons. GLUT1 is also associated with early progression in glioma patients. CONCLUSIONS: Targeting the transporter Glut1 suppresses the unique phenotype, "diffuse invasion" in the diffuse glioma mouse model. This work leads to promising therapeutic and potential useful imaging targets for anti-invasion in human gliomas widely.

How does spaceflight affect the acquired immune system?

The impact of spaceflight on the immune system has been investigated extensively during spaceflight missions and in model experiments conducted on Earth. Data suggest that the spaceflight environment may affect the development of acquired immunity, and immune responses. Herein we summarize and discuss the influence of the spaceflight environment on acquired immunity. Bone marrow and the thymus, two major primary lymphoid organs, are evidently affected by gravitational change during spaceflight. Changes in the microenvironments of these organs impair lymphopoiesis, and thereby may indirectly impinge on acquired immunity. Acquired immune responses may also be disturbed by gravitational fluctuation, stressors, and space radiation both directly and in a stress hormone-dependent manner. These changes may affect acquired immune responses to pathogens, allergens, and tumors.

Th2 signals are not essential for the anti-arthritic effects of Trichinella spiralis in mice.

AIMS: Many parasitic helminths are known to alter host immune responses and consequently affect the progression of autoimmune and allergic diseases. The parasitic nematode Trichinella sp has been reported to suppress several experimental diseases in rodents, including experimental autoimmune encephalomyelitis, type 1 diabetes, colitis, airway inflammation and autoimmune arthritis. We tried to clarify requirement of Th2 cytokines in the anti-arthritic effects of Trichinella spiralis (Ts) against collagen-induced arthritis (CIA). METHODS AND RESULTS: We infected Ts and then induced CIA in STAT6KO DBA/1 mice, comparing the disease progression with that in wild-type (WT) DBA/1 mice, Ts significantly mitigated arthritis in WT mice, in addition to the impairment of anti-type II collagen (IIC) IgG production in a subclass-independent manner. The genetic absence of STAT6 in the mice did not abrogate the anti-arthritic effects of Ts. Alteration of splenic cytokines was not related to the anti-arthritic effects of the parasite. Moreover, lack of IL-10 did not abrogate the anti-arthritic effects of Ts. CONCLUSION: Our results suggest that the anti-arthritic effects of Ts do not require host Th2 signals.

Neutrophil Elastase Inhibition Ameliorates Endotoxin-induced Myocardial Injury Accompanying Degradation of Cardiac Capillary Glycocalyx.

Myocardial injury in sepsis may be caused by a burst of several inflammatory mediators, leading to vascular endothelial injuries. However, the contribution of neutrophil elastase (NE) to myocardial injury in sepsis is still unknown. We aimed to evaluate whether endotoxemia-induced myocardial injury is associated with NE. Lipopolysaccharide (LPS) was injected intraperitoneally at a dose of 20 mg/kg into granulocyte-colony-stimulating-factor knockout mice (G-CSF-KO), which have few neutrophils, and littermate control mice. The survival rate of G-CSF-KO mice 48 hours after LPS injection was significantly greater than that of control mice. The serum level of troponin I in G-CSF-KO mice was significantly lower than that in control mice. In addition, the concentration of inflammatory cytokine interleukin-6 (IL-6) was significantly decreased 6 and 12 hours after LPS administration compared with that in control mice. Ultrastructural analysis revealed that vascular endothelial structures and the endothelial glycocalyx in G-CSF-KO mice were clearly preserved. Next, mice were injected with 0.2 mg/kg sivelestat (an NE inhibitor) after LPS administration. The survival rate was significantly higher and the serum level of troponin I was lower in sivelestat-injected mice than in control mice, respectively. Furthermore, IL-6 levels were significantly decreased 6 and 12 hours after LPS administration compared with those in control mice. Vascular endothelial structures and the endothelial glycocalyx in sivelestat-treated mice were clearly preserved at the ultrastructural level. In conclusion, NE is significantly associated with myocardial injury in endotoxemia. Inhibition of NE may be a useful tool for the management of endotoxemia.

Controlling the Phenotype of Tumor-Infiltrating Macrophages via the PHD-HIF Axis Inhibits Tumor Growth in a Mouse Model.

The tumor microenvironment (TME) polarizes tumor-infiltrating macrophages toward tumor support. Macrophage-abundant tumors are highly malignant and are the cause of poor prognosis and therapeutic resistance. In this study, we show that the prolyl hydroxylase (PHD) inhibitor FG-4592 (FG) inhibits tumor growth of macrophage-abundant tumors and prolongs mouse survival. FG not only normalizes tumor vessels and improves tumor oxygenation but also directly affects macrophages and activates phagocytosis through the PHD-hypoxia-inducible factor (HIF) axis. Remarkably, FG can promote phagocytic ability of the Ly6Clo subset of tumor-infiltrating macrophages, leading to tumor growth inhibition. Moreover, Ly6Cneg macrophages contributed to blood vessel normalization. Using a malignant tumor mouse model, we characterized macrophage function and subsets. Altogether, our findings suggest that the PHD inhibitor can promote the anti-tumor potential of macrophages to improve cancer therapy.

Regulation of membrane phospholipid asymmetry by Notch-mediated flippase expression controls the number of intraepithelial TCRαß+CD8αα+ T cells.

Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αß T cells (TCRαß+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαß+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (Rbpj) or Notch1 and Notch2 in T cells. Rbpj-deficient TCRαß+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. Rbpj-deficient TCRαß+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαß+CD8αα+ IELs in Rbpj-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in Rbpj-deficient TCRαß+CD8αα+ IELs acts as an "eat-me" signal. Together, these results revealed that Notch-Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαß+CD8αα+ IELs.

Neutrophil Elastase Damages the Pulmonary Endothelial Glycocalyx in Lipopolysaccharide-Induced Experimental Endotoxemia.

Neutrophil elastase (NE) is necessary for effective sterilization of phagocytosed bacterial and fungal pathogens; however, NE increases alveolocapillary permeability and induces proinflammatory cytokine production in sepsis-induced acute respiratory distress syndrome. Under septic conditions, the pulmonary endothelial glycocalyx covering on the healthy endothelium surface is injured, but the contribution of NE to this injury remains unknown. Our aim was to examine whether NE-induced pulmonary endothelial injury is associated with endotoxemia. Lipopolysaccharide (LPS; 20 mg/kg) was injected intraperitoneally into 9- to 12-week-old granulocyte colony-stimulating factor knockout (G-CSFKO) mice, which harbor few neutrophils, and littermate control mice; in a second assay, mice were injected with the NE-inhibitor sivelestat (0.2 mg/kg) at 3, 6, 9, and 12 hours after LPS administration. Subsequently, vascular endothelial injury was evaluated through ultrastructural analysis. At 48 hours after LPS injection, survival rate was more than threefold higher among G-CSFKO than control mice, and degradation of both thrombomodulin and syndecan-1 was markedly attenuated in G-CSFKO compared with control mice. Ultrastructural analysis revealed attenuated vascular endothelial injury and clear preservation of the endothelial glycocalyx in G-CSFKO mice. Moreover, after LPS exposure, survival rate was approximately ninefold higher among sivelestat-injected mice than control mice, and sivelestat treatment potently preserved vascular endothelial structures and the endothelial glycocalyx. In conclusion, NE is associated with pulmonary endothelial injury under LPS-induced endotoxemic conditions.

A New Species of Lagochondria (Crustacea: Copepoda) Parasitic on Callionymid Fish: First Record of Precopulatory Mate Guarding in Fish-Parasitic Cyclopoid Copepods, and of the Occurrence of the Genus in the North Pacific.

A new species of the genus Lagochondria of the parasitic cyclopoid family Chondracanthidae is described from the gill cavity of the callionymid Repomucenus virgis (Jordan and Fowler, 1903) collected from off Kii Peninsula, Japan. This is the first record of the occurrence of the genus from the North Pacific, and is only the second species in the genus. The female of the new species is easily distinguished from that of the Australian type species L. nana Ho and Dojiri, 1988 by having a squared trunk lacking paired posterior processes, and by the very short neck. In the male, the new species can be differentiated from the type species by having three inner setae on the caudal ramus, and by the distal segment of the antennule having a setal formula of 4, 3, 7+ ae. An adult female was accompanied by an attached adult male, whereas fourth and fifth copepodid females each carried a fifth and a fourth attached copepodid male, respectively. This is the first record of precopulatory mate guarding in a cyclopoid family parasitic on fish hosts, and of mate guarding between late copepodids of both sexes. The zoogeography of the genus and its relatives with an atrophied tip on the antenna is also discussed.

The circulating immunoglobulins negatively impact on the parasite clearance in the liver of Leishmania donovani-infected mice via dampening ROS activity.

Visceral leishmaniasis, the most severe form of leishmaniasis, is caused by Leishmania donovani and L. infantum. Immunity to Leishmania infection has been shown to depend on the development of Th1 cells; however, the roles of B cells and antibodies during infection remain unclear. In the present study, we showed that AID and µs double-deficient mice (DKO), which have B cells but not circulating immunoglobulins (cIgs), became resistant to L. donovani infection, whereas µs or AID single-deficient mice did not. This resistance in DKO mice occurred in the liver from an early stage of the infection. The depletion of IFN-γ did not affect the rapid reduction of parasite burden, whereas NADPH oxidases was up-regulated in the livers of infected DKO mice. The inhibition of the reactive oxygen species pathway in vivo by apocynin, a NADPH oxidase inhibitor, resulted in a significant increase in the parasite burden in DKO mice. These results indicate that a circulating Ig deficiency induces a protective response against L. donovani infection by elevating IFN-γ-independent NADPH oxidase activity, and also that cIgs play a regulatory role in controlling L. donovani infection in mice.

Tumour blood vessel normalisation by prolyl hydroxylase inhibitor repaired sensitivity to chemotherapy in a tumour mouse model.

Blood vessels are important tissue structures that deliver oxygen and nutrition. In tumour tissue, abnormal blood vessels, which are hyperpermeable and immature, are often formed; these tissues also have irregular vascularisation and intravasation. This situation leads to hypoperfusion in tumour tissue along with low oxygen and nutrition depletion; this is also called the tumour microenvironment and is characterised by hypoxia, depleted nutrition, low pH and high interstitial pressure. This environment induces resistance to anticancer drugs, which causes an increase in anticancer drug doses, leading to increased side effects. We hypothesised that normalised tumour blood vessels would improve tumour tissue perfusion, resupply nutrition and re-oxygenate the tumour tissue. Chemotherapy would then be more effective and cause a decrease in anticancer drug doses. Here we report a neovascularisation-inducing drug that improved tumour vascular abnormalities, such as low blood flow, blood leakage and abnormal vessel structure. These results could lead to not only an increased chemo-sensitivity and tissue-drug distribution but also an up-regulated efficiency for cancer chemotherapy. This suggests that tumour blood vessel normalisation therapy accompanied by angiogenesis may be a novel strategy for cancer therapy.

Nonencapsulated Trichinella pseudospiralis Infection Impairs Follicular Helper T Cell Differentiation with Subclass-Selective Decreases in Antibody Responses.

Infectious microorganisms often modify host immunity to escape from immune elimination. Trichinella is a unique nematode of the helminth family, whose members parasitize the muscle cells inside the host without robust eliminative reactions. There are several species of Trichinella; some develop in muscle cells that become encapsulated (e.g., Trichinella spiralis) and others in cells that do not encapsulate (e.g., Trichinella pseudospiralis). It has already been established that Trichinella infection affects host immune responses in several experimental immune diseases in animal models; however, most of those studies were done using T. spiralis infection. As host immune responses to T. spiralis and T. pseudospiralis infections have been reported to be different, it is necessary to clarify how T. pseudospiralis infection influences the host immune responses. In this study, we investigated the influence on host humoral immunity in T. pseudospiralis-infected mice. We demonstrated that T. pseudospiralis infection decreased antigen-specific IgG2a and IgG2b antibody (Ab) production in mice immunized with a model antigen. This selective decrease in gamma interferon (IFN-γ)-dependent Ab production was not due to a decrease in IFN-γ production, and we instead found impaired follicular helper T (Tfh) cell differentiation. The affinity maturation of antigen-specific Ab tended to be delayed but was not significant in T. pseudospiralis-infected mice. We also observed that CD11b+ spleen cells in T. pseudospiralis-infected mice expressed CD206 and PD-L2, the phenotype of which was M2 macrophages with weak production of interleukin-6 (IL-6), possibly resulting in impaired Tfh differentiation. Taken together, our results indicate that nonencapsulated Trichinella infection induces selective dampening in humoral immunity with the suppression of Tfh differentiation.

Transmission of survival signals through Delta-like 1 on activated CD4+ T cells.

Notch expressed on CD4+ T cells transduces signals that mediate their effector functions and survival. Although Notch signaling is known to be cis-inhibited by Notch ligands expressed on the same cells, the role of Notch ligands on T cells remains unclear. In this report we demonstrate that the CD4+ T cell Notch ligand Dll1 transduces signals required for their survival. Co-transfer of CD4+ T cells from Dll1-/- and control mice into recipient mice followed by immunization revealed a rapid decline of CD4+ T cells from Dll1-/- mice compared with control cells. Dll1-/- mice exhibited lower clinical scores of experimental autoimmune encephalitis than control mice. The expression of Notch target genes in CD4+ T cells from Dll1-/- mice was not affected, suggesting that Dll1 deficiency in T cells does not affect cis Notch signaling. Overexpression of the intracellular domain of Dll1 in Dll1-deficient CD4+ T cells partially rescued impaired survival. Our data demonstrate that Dll1 is an independent regulator of Notch-signaling important for the survival of activated CD4+ T cells, and provide new insight into the physiological roles of Notch ligands as well as a regulatory mechanism important for maintaining adaptive immune responses.

Practical methods for collecting Trichinella parasites and their excretory-secretory products.

Trichinella is a unique nematode. Its developmental stages include adult worms, newborn larvae, and muscle larvae. Besides humans, the parasite also infects many kinds of animals, including mice. Mice are widely used as an animal model in the research fields of immunology, cell biology, and host-parasite relationships of trichinellosis. The different developmental stages of Trichinella share similar, but unique characteristics. Therefore, it is important to collect different sources of Trichinella-derived materials for research with appropriate methods. In the present study, we introduce methods to collect Trichinella at different stages as well as their ES products. By optimizing the concentration of artificial gastric juice, volume of medium, and time of incubation for ES collection in vitro, muscle larvae, adult worms, and newborn larvae were collected with less contamination by host materials, and the ES products collected were confirmed to be originally antigenic and biologically active. The DNA, RNA, and proteins isolated from the parasites collected were confirmed to be applicable to analyses, including PCR, real-time PCR, Western blotting, and stimulators of cell cultures (macrophages, splenocytes, and tumor cells). The present study compiled protocols to collect materials from Trichinella and provides a reference for research on Trichinella.

Significance of S100P as a biomarker in diagnosis, prognosis and therapy of opisthorchiasis-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignancy of bile duct with the difficulty in early diagnosis, poor prognosis and less alternation in therapy. S100P is a member of S100 family proteins and plays important roles in cancers. We investigated the S100P expression and its correlation with clinicopathology in 78 cases of opisthorchiasis-associated CCA, and the effects of S100P knockdown with shRNA interference on the proliferation, cell cycle, migration, apoptosis and sensitivity to anti-cancer drug. Extremely high expression of S100P mRNA was detected in the CCA tumor tissues. The increased S100P protein expression was immunohistochemically confirmed and localized in the CCA cytoplasm and/or nuclei as well as in the hyperneoplasia and dysplasia bile ducts, but not in normal bile ducts. The intensity of immunostaining was correlated with survival, tumor stage and metastasis, and the high expression could be an independent prognostic factor. High levels of S100P were detected in the serum and bile fluid of CCA patients. The shRNA-mediated knockdown of S100P expression inhibited the proliferation in vitro and in vivo, and migration of CCA cells, arrested cell cycle with the up-regulated expression of cell cycle arrest related factors, p21, p27, GADD45A, and 14-3-3 zeta. S100P knockdown also promoted CCA cell apoptosis by up-regulating expression of apoptosis related factors, DR5, TRADD, caspase 3 and BAX, and increased the sensitivity of CCA cells to the chemotherapeutic agents sunitinib and apigenin. Taken together, this study indicates that S100P might be a promising biomarker for the diagnosis, prognosis and therapy of CCA.