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Simple Summary Despite the rapid advancement in immunotherapy and targeted agents, many patients diagnosed with cancer have poor prognosis with dismal overall survival. One of the key hallmarks of cancer is the ability of cancer cells to reprogram their energy metabolism. O-GlcNAcylation is an emerging potential mechanism for cancer cells to induce proliferation and progression of tumor cells and resistance to chemotherapy. This review summarizes the mechanism behind O-GlcNAcylation and discusses the role of O-GlcNAcylation, including its function with receptor tyrosine kinase and chemo-resistance in cancer, and immune response to cancer and as a prognostic factor. Further pre-clinical studies on O-GlcNAcylation are warranted to assess the clinical efficacy of agents targeting O-GlcNAcylation. Abstract Cancer cells are able to reprogram their glucose metabolism and retain energy via glycolysis even under aerobic conditions. They activate the hexosamine biosynthetic pathway (HBP), and the complex interplay of O-linked N-acetylglucosaminylation (O-GlcNAcylation) via deprivation of nutrients or increase in cellular stress results in the proliferation, progression, and metastasis of cancer cells. Notably, cancer is one of the emerging diseases associated with O-GlcNAcylation. In this review, we summarize studies that delineate the role of O-GlcNAcylation in cancer, including its modulation in metastasis, function with receptor tyrosine kinases, and resistance to chemotherapeutic agents, such as cisplatin. In addition, we discuss the function of O-GlcNAcylation in eliciting immune responses associated with immune surveillance in the tumor microenvironment. O-GlcNAcylation is increasingly accepted as one of the key players involved in the activation and differentiation of T cells and macrophages. Finally, we discuss the prognostic role of O-GlcNAcylation and potential therapeutic agents such as O-linked β-N-acetylglucosamine-transferase inhibitors, which may help overcome the resistance mechanism associated with the reprogramming of glucose metabolism.

Simple Summary Patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer can have central nervous system (CNS) metastases during their disease course. A high unmet medical need exists especially for patients with T790M-positive NSCLC whose disease progressed after first-line EGFR-TKI. Osimertinib is a third-generation EGFR-TKI with selective activity for both sensitizing and EGFR T790M mutations and has improved CNS activity over first- and second-generation EGFR TKIs and chemotherapies. This study confirmed the clinical activity and CNS efficacy of osimertinib in an unselected real-world population. Abstract Up to 40% of patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) may develop central nervous system (CNS) metastases throughout their disease. Moreover, the first- and second-generation EGFR-tyrosine kinase inhibitors have limited efficacy because of their poor blood–brain barrier permeability. Therefore, we conducted preplanned analyses of ASTRIS, a clinical study of the third-generation EGFR-TKI osimertinib to demonstrate its potential role in intracranial response efficacies. We retrospectively examined 89 NSCLC patients with brain evaluation who were not amenable to curative surgery or radiotherapy and received osimertinib upon confirmation of the presence of the T790M mutation. We collected the information regarding patients’ baseline characteristics, baseline intracranial status, including leptomeningeal metastases (LM), and intracranial responses measured by Response Evaluation Criteria in Solid Tumors version 1.1, using independent central review. The median age was 60 years, and 69.7% of the patients were female. Sixty-five patients (73.0%) had brain metastases (BM) at baseline and nineteen patients (23.5%) had additional LM. Among patients with brain metastases, 24 (36.9%) had ≥1 measurable brain metastases and 16 were evaluated for the objective response. In the CNS evaluable for response set, the intracranial objective response rate (cORR) and disease control rate (cDCR) were 62.5% (95% confidence interval (CI), 38.3–82.6%) and 93.8% (95% CI, 74.3–99.3%), respectively. The median intracranial progression-free survival (cPFS) was 13.0 (95% CI, 7.21–18.8) months, including patients with measurable and non-measurable BM or LM. Our cORR, cDCR, and cPFS were comparable to those observed in previous clinical trials. The outcome of this study helps to demonstrate the potential role of intracranial efficacies of osimertinib 80 mg administration in T790M-positive advanced NSCLC with/without BM or LM.

Objectives The treatment for stage III non-small cell lung cancer (NSCLC) is quite variable because stage III NSCLC is a heterogenous disease. Programmed death ligand 1 (PD-L1) and CD8+ tumor infiltrating lymphocytes (TILs) are thought to be related to treatment outcome in many tumors. To improve treatment outcome in stage III NSCLC, it is necessary to obtain data on PD-L1 expression and CD8+ TIL counts following CCRT and their relationship to treatment outcome. Materials and methods We retrospectively enrolled 43 patients with stage III NSCLC treated with neoadjuvant CCRT followed by surgery at Yonsei Cancer Center Severance Hospital in Korea between June 2008 and October 2010. PD-L1 level and CD8+ TIL numbers in tumors following CCRT were analyzed by immunohistochemistry, and their association with patient survival was evaluated with Kaplan-Meier method. Results More than half patients (52%) showed up- or downregulation of PD-L1 expression, and most patients (81%) showed change in CD8+ TIL counts by CCRT. Patients with PD-L1 expression following CCRT tended to have shorter recurrence free survival (RFS) (P = 0.182) or overall survival (OS) (P = 0.215) compared to the ones without PD-L1 expression. In the survival analysis with pre-CCRT specimens, neither RFS nor OS showed statistically significant differences. Patients with increased CD8+ TIL counts following CCRT regardless of pathological response strongly showed longer OS (median: not reached vs. 14.2 months for others; P = 0.017). Conclusions CCRT dynamically alters PD-L1 expression and CD8+ TIL numbers in stage III NSCLC. Our data provide a rationale for combining CCRT and immunotherapy for the treatment of potentially resectable NSCLC.

Background Immune checkpoint blockade with Programmed cell death 1 (PD-1)/PD-L1 inhibitors has been effective in various malignancies and is considered as a standard treatment modality for patients with non-small-cell lung cancer (NSCLC). However, emerging evidence show that PD-1/PD-L1 blockade can lead to hyperprogressive disease (HPD), a flair-up of tumor growth linked to dismal prognosis. This study aimed to evaluate the incidence of HPD and identify the determinants associated with HPD in patients with NSCLC treated with PD-1/PD-L1 blockade. Patients and methods We enrolled patients with recurrent and/or metastatic NSCLC treated with PD-1/PD-L1 inhibitors between April 2014 and November 2018. Clinicopathologic variables, dynamics of tumor growth, and treatment outcomes were analyzed in patients with NSCLC who received PD-1/PD-L1 blockade. HPD was defined according to tumor growth kinetics (TGK), tumor growth rate (TGR), and time to treatment failure (TTF). Immunophenotyping of peripheral blood CD8+ T lymphocytes was conducted to explore the potential predictive biomarkers of HPD. Results A total of 263 patients were analyzed. HPD was observed in 55 (20.9%), 54 (20.5%), and 98 (37.3%) patients according to the TGK, TGR, and TTF. HPD meeting both TGK and TGR criteria was associated with worse progression-free survival [hazard ratio (HR) 4.619; 95% confidence interval (CI) 2.868–7.440] and overall survival (HR, 5.079; 95% CI, 3.136–8.226) than progressive disease without HPD. There were no clinicopathologic variables specific for HPD. In the exploratory biomarker analysis with peripheral blood CD8+ T lymphocytes, a lower frequency of effector/memory subsets (CCR7−CD45RA− T cells among the total CD8+ T cells) and a higher frequency of severely exhausted populations (TIGIT+ T cells among PD-1+CD8+ T cells) were associated with HPD and inferior survival rate. Conclusion HPD is common in NSCLC patients treated with PD-1/PD-L1 inhibitors. Biomarkers derived from rationally designed analysis may successfully predict HPD and worse outcomes, meriting further investigation of HPD.

Clinical benefit of ALK tyrosine kinase inhibitors (ALK‐TKIs) in ALK‐rearranged lung cancer has been limited by the inevitable development of acquired resistance, and bypass‐molecular resistance mechanisms remain poorly understood. We investigated a novel therapeutic target through screening FDA‐approved drugs in ALK‐TKI‐resistant models. Cerivastatin, the rate‐limiting enzyme inhibitor of the mevalonate pathway, showed anti‐cancer activity against ALK‐TKI resistance in vitro/in vivo, accompanied by cytoplasmic retention and subsequent inactivation of transcriptional co‐regulator YAP. The marked induction of YAP‐targeted oncogenes (EGFR, AXL, CYR61, and TGFβR2) in resistant cells was abolished by cerivastatin. YAP silencing suppressed tumor growth in resistant cells, patient‐derived xenografts, and EML4‐ALK transgenic mice, whereas YAP overexpression decreased the responsiveness of parental cells to ALK inhibitor. In matched patient samples before/after ALK inhibitor treatment, nuclear accumulation of YAP was mainly detected in post‐treatment samples. High expression of YAP in pretreatment samples was correlated with poor response to ALK‐TKIs. Our findings highlight a crucial role of YAP in ALK‐TKI resistance and provide a rationale for targeting YAP as a potential treatment option for ALK‐rearranged patients with acquired resistance to ALK inhibitors., Acquired resistance to targeted therapy is a major challenge in treatment for ALK‐rearranged NSCLC. The transcriptional activation of YAP induces multiple resistance factors, conferring acquired resistance to ALK‐TKIs. Statins represent proxy inhibitors to overcome YAP‐mediated drug resistance.

The development of cancer therapies using monoclonal antibodies has been successful during the last 30 years. Recently much progress was achieved with technologies involving bispecific and multi-specific antibodies. Bispecific antibodies (BsAbs) are antibodies that bind two distinct epitopes, and a large number of potential clinical applications of BsAbs have been described. Here we review mechanism of action, clinical development and future challenges of BsAbs which could be a serve as a valuable arsenal in cancer patients.

Objectives: Patients with epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) ultimately acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) during treatment. In 5–22% of these patients, resistance is mediated by aberrant mesenchymal epithelial transition factor (MET) gene amplification. Here, we evaluated the emergence of MET amplification after EGFR-TKI treatment failure based on clinical parameters. Materials and Methods: We retrospectively analyzed 186 patients with advanced EGFR-mutant NSCLC for MET amplification status by in situ hybridization (ISH) assay after EGFR-TKI failure. We collected information including baseline patient characteristics, metastatic locations and generation, line, and progression-free survival (PFS) of EGFR-TKI used before MET evaluation. Multivariate logistic regression analysis was conducted to evaluate associations between MET amplification status and clinical variables. Results: Regarding baseline EGFR mutations, exon 19 deletion was predominant (57.5%), followed by L858R mutation (37.1%). The proportions of MET ISH assays performed after first/second-generation and third-generation TKI failure were 66.7% and 33.1%, respectively. The median PFS for the most recent EGFR-TKI treatment was shorter in MET amplification-positive patients than in MET amplification-negative patients (median PFS 7.0 vs. 10.4 months, p = 0.004). Multivariate logistic regression demonstrated that a history of smoking, short PFS on the most recent TKI, and less intracranial progression were associated with a high probability of MET amplification (all p &lt, 0.05). Conclusions: Our results demonstrated the distinct clinical characteristics of patients with MET amplification-positive NSCLC after EGFR-TKI therapy. Our clinical prediction can aid physicians in selecting patients eligible for MET amplification screening and therapeutic targeting.

An anti-malarial candidate, 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251), was studied to characterize its potential as a novel anti-Toxoplasma gondii drug. In the present study, IC50 and LC50 of N-251 on host cells and T. gondii were compared to those of artemisinin and sulfadiazine. The IC50 on Huh-7 cells was 10.19μg/ml, 67.69μg/ml and 310.17μg/ml for N-251, artemisinin, and sulfadiazine, respectively. The LC50 for anti-T. gondii effect was shown to be 1.11μg/ml, 5.79μg/ml, and 5.45μg/ml for N-251, artemisinin and sulfadiazine, respectively. N-251 concentration causing complete parasiticidal effect with minimal cytotoxicity on host cells was determined to be 5μg/ml. Additionally, the anti-T. gondii effect of N-251 was confirmed by ultrastructural changes, loss of organelles, degenerated morphology and the increase of amylopectin as detected by transmission electron microscope (TEM). Accordingly, the present study suggests that the anti-malarial synthetic endoperoxide, N-251, is an emerging drug candidate more effective than artemisinin and sulfadiazine.

// Kyoung-Ho Pyo 1, 4 , You-Won Lee 1 , Sun Min Lim 2 , Eun-Hee Shin 1, 3 1 Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine and Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, Republic of Korea 2 Division of Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Republic of Korea 3 Seoul National University Bundang Hospital, Seongnam, Republic of Korea 4 Current address: JE-UK Laboratory of Molecular Cancer Therapeutics, Yonsei Cancer Institute, Yonsei University College of Medicine, Seoul, Republic of Korea Correspondence to: Eun-Hee Shin, email: ehshin@snu.ac.kr Keywords: Toxoplasma gondii, Toll-like receptor, profilin, antitumor, vaccine Received: July 15, 2016 Accepted: September 21, 2016 Published: September 28, 2016 ABSTRACT Profilin-like protein in Toxoplasma gondii (TgPLP) is a Toll-like receptor (TLR) agonist. In this study, we investigated whether TgPLP has an adjuvant effect on immune function in autologous whole-tumor-cell vaccine (AWV) treatment. Mice vaccinated with AWV together with recombinant TgPLP protein had smaller CT26 tumors and increased survival. TgPLP treatment strongly increased the production of IL-12 through MyD88 signaling and several chemokines, including CCL5, CCL12, and XCL1, in bone marrow-derived macrophages (BMMs). In addition, TgPLP increased the phagocytosis of tumor cells by BMMs and promoted immune cell mobility on a tumor-matrigel scaffold. TgPLP triggered immune responses as demonstrated by increased expression of antigen presenting cell markers (MHC class I and II, B7.1, and B7.2) in BMMs and increased IL-12 and IFN-γ expression in mice. Mice vaccinated with AWV and TgPLP had more immune cells (CD4 + and CD8 + T cells, natural killer cells, and macrophages) in the spleen and higher total IgG and IgG2a concentrations in the blood than mice vaccinated with AWV alone. These findings suggest that TgPLP is a TLR-based vaccine adjuvant that enhances antitumor immune responses during vaccination with AWV.

To better understand the immune profile within the tumor microenvironment (TME) and the potential immune-evasion capacity that may arise from administration of immune checkpoint inhibitors, we investigated immunological phenotypes and their correlations with genomic alterations in order to provide predictive/prognostic implications in lung adenocarcinoma (LAC).Surgical sections with corresponding peripheral blood mononuclear cells were collected from 76 patients with pathologically confirmed Ia-IIIa. The histological contents expressing immune checkpoint molecules in the tumor nest and stromal area were imaged and quantified using a multi-spectral imaging system. Distribution of tumor mutation burden (TMB) and non-synonymous somatic mutations were analyzed in whole exome sequencing (WES) data.Total 76 patients consisted of EGFR-mutant (n=37) and EGFR-wild type (n=39) tumors. As is well known, the number of tumor-infiltrating cytotoxic T cells (174.7 ± 24.4 cells/mm2) and TMB (3.8 ± 0.4) in EGFR-mutant tumors was significantly lower than those of EGFR wild type tumors (394.2 ± 82.3 cells/mm2 and 6.7 ± 1.2) (p=0.0146 and p=0.0254, respectively), elucidating EGFR mutation-mediated immune evasion. The subjects carrying EGFR wild-type were classified into two subgroups by the density of tumor-infiltrating cytotoxic T cells, defined as the high TIL (n=20) and low TIL (n=19). There was great difference of mean density of cytotoxic T cells between low TIL group (129.6 ± 16.8 cells/mm2) and high TIL group (672.8±143.5 cells/mm2). Tumor-infiltrating T cells were granzyme B-positive, suggesting most cells were tumor-specific. Interestingly, no significant difference in TMB was found between the two subgroups (7.5 ± 1.9 in low TIL group vs. 5.8±1.3 in high TIL group; p=0.4642). Tumor proportion score (TPS) of PD-L1 was remarkably higher in high TIL group than low TIL group (29.7 ± 6.5% vs. 13.9 ± 2.8%, p=0.0008, respectively), but no significant differences of tumoral CD73 and IDO between the two groups (p=0.643 and p=0.923, respectively). Even there were no significant differences in counts of tumor-infiltrating macrophages in all types of macrophage, significantly higher counts of PD-L1+ M1-type macrophages (CD68+ CD163low) in the stromal area were discovered in the high TIL group compared to those of the low TIL group (396.2 ± 93.6 vs. 139.8 ± 30.1, p=0.0114, respectively), indicating that most of macrophages resided in the stromal area. Interestingly, the low TIL group exhibited higher counts of CD73+ M2-type macrophages (CD68+ CD163high) in the stromal area than the high TIL group (644.5 ± 110.9 vs. 387.9 ± 73.2, p=0.0639, respectively). Cell count ratio (M1/M2) of macrophages was significantly higher in the high TIL group (p=0.0042), suggesting that the TME with a smaller number of TILs tends to contain more M2-type macrophages. The tumor recurrence was more frequent in the low TIL group compared to the high TIL group with no statistical significance (p=0.127). In surgically resected lung adenocarcinoma patients, the subgroup with a low density of tumor-infiltrating cytotoxic T cells had a large number of CD73+ M2-type macrophages, which may contribute to poor prognosis. Acknowledgement: This study was sponsored by Ono Pharma Co., Ltd Citation Format: Jae-Hwan Kim, Tae-Min Kim, Sung-Gu Her, Kyoung-Ho Pyo, Jeong- Oh Kim, Beung-Chul Ahn, Byoung Chul Cho, Jin Hyoung Kang. Comprehensive analysis of immune phenotypes identifies CD73+ CD163high macrophages as a prognostic biomarker in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4981.

RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, in RET-rearranged LADC has not been reported. The aims of the study are to explore antitumor effects and mechanisms of acquired resistance of dovitinib in RET-rearranged LADC. Using structural modeling and in vitro analysis, we demonstrated that dovitinib induced cell-cycle arrest at G0–G1 phase and apoptosis by selective inhibition of RET kinase activity and ERK1/2 signaling in RET-rearranged LC-2/ad cells. Strong antitumor effect of dovitinib was observed in an LC-2/ad tumor xenograft model. To identify the acquired resistance mechanisms to dovitinib, LC-2/ad cells were exposed to increasing concentrations of dovitinib to generate LC-2/ad DR cells. Gene-set enrichment analysis of gene expression and phosphor-kinase revealed that Src, a central gene in focal adhesion, was activated in LC-2/ad DR cells. Saracatinib, an src kinase inhibitor, suppressed ERK1/2 phosphorylation and growth of LC-2/ad DR cells. Taken together, these findings suggest that dovitinib can be a potential therapeutic option for RET-rearranged LADC, in which acquired resistance to dovitinib can be overcome by targeting Src. Mol Cancer Ther; 14(10); 2238–48. ©2015 AACR.

Toxoplasma gondii is an intracellular protozoan parasite that causes a Th1 cellular immunity. Our previous study showed that T. gondii lysate antigen (TLA) treatment in S180 tumor-bearing mice resulted in tumor reduction by suppressing CD31 expression, a marker of angiogenesis. In the present study, to investigate tumor suppressive effect of TLA under the absence of T lymphocytes, athymic nude mice were compared with euthymic mice in the anti-tumorigenic effect triggered by TLA in CT26 tumors. According to the results, intratumorally injected TLA reduced tumor growth and TIMP-1 level, a metastatic marker, in both euthymic and athymic mice. TLA treatment led to a sharp increase in IL-12 expression in serum cytokine profiling of athymic mice, and increased MyD88 signals in macrophages derived from the bone marrow, implying the activation of innate immunity. The selective induction of IL-12 by TLA treatment had an anti-tumorigenic effect.

Anaplastic lymphoma kinase (ALK) inhibitors are highly effective in patients with ALK fusion-positive lung cancer, but acquired resistance invariably emerges. Identification of secondary mutations has received considerable attention, but most cases cannot be explained by genetic causes alone, raising the possibility of epigenetic mechanisms in acquired drug resistance. Here, we investigated the dynamic changes in the transcriptome and enhancer landscape during development of acquired resistance to ALK inhibitors. Histone H3 lysine 27 acetylation (H3K27ac) was profoundly altered during acquisition of resistance, and enhancer remodeling induced expression changes in both miRNAs and mRNAs. Decreased H3K27ac levels and reduced miR-34a expression associated with the activation of target genes such as AXL. Panobinostat, a pan-histone deacetylase inhibitor, altered the H3K27ac profile and activated tumor-suppressor miRNAs such as miR-449, another member of the miR-34 family, and synergistically induced antiproliferative effects with ALK inhibitors on resistant cells, xenografts, and EML4-ALK transgenic mice. Paired analysis of patient samples before and after treatment with ALK inhibitors revealed that repression of miR-34a or miR-449a and activation of AXL were mutually exclusive of secondary mutations in ALK. Our findings indicate that enhancer remodeling and altered expression of miRNAs play key roles in cancer drug resistance and suggest that strategies targeting epigenetic pathways represent a potentially effective method for overcoming acquired resistance to cancer therapy.Significance: Epigenetic deregulation drives acquired resistance to ALK inhibitors in ALK-positive lung cancer. Cancer Res; 78(12); 3350-62. ©2018 AACR.

Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as an immune check point molecule is expressed in lung cancer cells. The homophilic interaction of CEACAM1 inhibits killing effect of T cells by co-inhibitory signal transduction. Thus, up-regulated CEACAM1 in the tumor microenvironment is considered as a promising therapeutic target for cancer. In this study, anti-cancer effect of anti-CEACAM1 monoclonal antibody (MG1124, from GC Pharma, Yongin, South Korea) was investigated with lung cancer-xenograft humanized mice. Materials & Methods: Multispectral image analysis using Vectra polaris system was performed in 49 of human lung cancers (36 adenocarcinoma; 13 squamous cell carcinoma) to evaluate the expressions of CEACAM1, PD-L1 and CD8. Four tumors (YHIM-01, YHIM-02, YHIM-03 and YHIM-04) with high CEACAM1 expression were subcutaneously implanted into humanized mice (hu-CD34 NSG) matched with HLA-A. Efficacy of MG1124 was performed by the single mouse trial format. Humanized mice were treated with MG1124 (q2w, 20 mpk, i.p. injection) with or without pembrolizumab (q2w, 10 mpk, i.p. injection). Findings: Among 49 lung cancers evaluated, proportion of tumor cells expressing CEACAM1 and PD-L1 with any intensity ranged 0.1- 89.2% (median, 15.4%) and 0.2% - 95.6% (median, 6.8%) respectively, and TIL counts ranged 0 - 1,200 cells/mm2 (median, 129 cells/mm2). Four tumors with high CEACAM1(14.5% in YHIM-01, 81.2% in YHIM-02, 23.8% in YHIM-03 and 36.7% in YHIM-04) were selected to establish humanized mouse models. MG1124 as a monotherapy demonstrated antitumor activity and also was superior to pembrolizumab in 1 (YHIM-02, TGI=63.4±1.7% for combination vs. TGI=58.7±9.1% for pembrolizumab alone) out of 4 humanized mouse models. Combination of MG1124 with pembrolizumab also demonstrated synergic anti-tumor effect in 3 models (YHIM-01, TGI= 39.6±19.7% vs. 0.6±11.4% for pembrolizumab alone, vs. 16.5±33.3% for MG1124 alone; YHIM-02, TGI=58.7±9.1% vs. 38.2±14.9% for pembrolizumab, vs. 63.4±1.7% for MG1124 alone; YHIM-03, TGI= 35.6±13.2% vs. TGI=0.0±13.2% for pembrolizumab alone, vs. TGI=3.1±13.3% for MG1124 alone). PD-L1 expression was not associated with MG1124 alone or in combination with pembrolizumab (PD-L1, 2.5% in YHIM-01, 0.5% in YHIM-02 and 14.0% in YHIM-03 respectively). Interestingly, however, combinational effects of MG1124 and pembrolizumab were shown only in 3 models showing high CD8+ T cell infiltration in the tumors (209, 432, 362 vs. 46 in a model without combination synergy). Conclusion: CEACAM1-targeting monoclonal antibody, MG1124, exhibits promising anti-cancer effects in CEACAM1-expressing lung cancers. Tumoral CEACAM1 expression and high intratumoral CD8+ T cell infiltration could serve as a predictive biomarker to MG1124 monotherapy or in combination with pembrolizumab and need to be further investigated. Citation Format: Jae-Hwan Kim, Kyoung-Ho Pyo, Min-Jee Jung, Minkyu Hur, Jonghwa Won, Byoung Chul Cho. Efficacy of CEACAM1-targeting immunoglobulin in combination with pembrolizumab in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2266.

Artificial digestive solution based on pepsin is essential for collecting metacercariae from fish. To promote the enzymatic reactivity of pepsin, the pH of the solution has to be adjusted to pH 1.0–2.0. Hydrochloride (HCl) is usually used for this purpose, but the use of HCl raises safety concerns. The aim of this work was to address the usefulness of citric acid as an alternative for HCl for the acidification of pepsin solution, and to examine its potential to damage metacercariae during in vitro digestion as compared with HCl. Changes in pH after adding 1–9% of citric acid (m/v) to pepsin solution were compared to a 1% HCl (v/v) addition. Digestion of fish muscle was evaluated by measuring released protein concentrations by spectrophotometry. In addition, survival rates of metacercariae in pepsin solution were determined at different citric acid concentrations and were compared that of with 1% HCl. The present study shows that addition of citric acid reduced the pH of pepsin solutions to the required level. Addition of more than 5% of citric acid resulted in the effective digestion of fish muscle over 3h in vitro, and 5% citric acid was less lethal to metacercariae than 1% HCl in pepsin solution. Pepsin solution containing 5% citric acid had digestive capacity superior to pepsin solution containing 1% HCl after 3h incubation with released protein concentrations of 12.0ng/ml for 5% citric acid and 9.6ng/ml for 1% HCl. Accordingly, the present study suggests that the addition of 5% citric acid to pepsin solution is a good alternative to 1% HCl in infection studies because citric acid is a stable at room temperature and has a good safety profile. In addition, we suggest that the use of citric acid enables the preparation of commercial digestive solutions for the detection of microorganisms in fish and other vertebrate muscle tissue.

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.

Aberrant fibroblast growth factor receptor (FGFR) activation/expression is a common feature in lung cancer (LC). In this study, we evaluated the antitumor activity of and the mechanisms underlying acquired resistance to two potent selective FGFR inhibitors, AZD4547 and BAY116387, in LC cell lines. The antitumor activity of AZD4547 and BAY1163877 was screened in 24 LC cell lines, including 5 with FGFR1 amplification. Two cell lines containing FGFR1 amplifications, H1581 and DMS114, were sensitive to FGFR inhibitors (IC50m). Clones of FGFR1-amplified H1581 cells resistant to AZD4547 or BAY116387 (H1581AR and H1581BR cells, respectively) were established. Receptor tyrosine kinase (RTK) array and immunoblotting analyses showed strong overexpression and activation of Met in H1581AR/BR cells, compared with that in the parental cells. Gene set enrichment analysis against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that cytokine–cytokine receptor interaction pathways were significantly enriched in H1581AR/BR cells, with Met contributing significantly to the core enrichment. Genomic DNA quantitative PCR and fluorescent in situ hybridization analyses showed MET amplification in H1581AR, but not in H1581BR, cells. Met amplification drives acquired resistance to AZD4547 in H1581AR cells by activating ErbB3. Combination treatment with FGFR inhibitors and an anaplastic lymphoma kinase (ALK)/Met inhibitor, crizotinib, or Met-specific short interfering RNA (siRNA) synergistically inhibited cell proliferation in both H1581AR and H1581BR cells. Conversely, ectopic expression of Met in H1581 cells conferred resistance to AZD4547 and BAY1163877. Acquired resistance to FGFR inhibitors not only altered cellular morphology, but also promoted migration and invasion of resistant clones, in part by inducing epithelial-to-mesenchymal transition. Taken together, our data suggest that Met activation is sufficient to bypass dependency on FGFR signaling. Concurrent inhibition of the Met and FGFR pathways may have synergistic clinical benefits when targeting FGFR-dependent LC.

To investigate the expression of programmed death-ligand 1 (PD-L1) and immune checkpoints and their prognostic value for resected head and neck squamous cell cancer (HNSCC). PD-L1 expression on tumor cells (TC) and tumor-infiltrating immune cells (IC), abundance of tumor-infiltrating lymphocytes (TILs), and expression of the immune checkpoints were investigated in 402 HNSCC patients. PD-L1 expression on TC and IC was categorized into four groups according to the percentage of PD-L1-positive cells. PD-L1 positivity was defined as ≥5% of cells based on immunohistochemistry. High PD-L1 expression on IC, but not TC, was an independent favorable prognostic factor for RFS and OS adjusted for age, gender, smoking, stage, and HPV. High frequencies of CD3+ or CD8+ TILs, Foxp3+ Tregs, and PD-1+ TILs were strongly associated with favorable prognosis. PD-L1 was exclusively expressed on either TC or IC. Transcriptome analysis demonstrated that IC3 expressed higher levels of the effector T cell markers than TC3, suggesting that PD-L1 expression is regulated via an adaptive IFNγ-mediated mechanism. High PD-L1 expression on IC, but not TC, and high abundance of PD-1+ T cells and Foxp3+ Tregs are favorable prognostic factors for resected HNSCC. This study highlights the importance of comprehensive assessment of both TC and IC.

The influence of temperature on the development and embryonation of Ascaris suum eggs was studied using coarse sand medium in an environmental chamber with 50% humidity. The time required for development and embryo- nation of eggs was examined under 3 different temperature conditions, 5°C, 25°C, and 35°C. A. suum eggs did not de- velop over 1 month at the temperature of 5°C. However, other temperature conditions, 25°C and 35°C, induced egg de- velopment to the 8-cell-stage at days 5-6 after incubation. All eggs examined developed to the 8-cell stage at day 6 after incubation in the sand medium at 25°C. The higher temperature, 35°C, slightly accelerated the A. suum egg development compared to 25°C, and the development to the 8-cell stage occurred within day 5 after incubation. The formation of lar- vae in A. suum eggs at temperatures of 35°C and 25°C appeared at days 17 and 19 after incubation, respectively. These findings show that 35°C condition shortens the time for the development of A. suum eggs to the 8-cell-stage in compari- son to 25°C, and suggest the possibility of accelerated transmission of this parasite, resulting from global warming and ecosystem changes.

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1 × 10(5) tachyzoites) or day 11 PI (with 1 × 10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4(+) and CD8(+) T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-γ and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-α was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-γ, TNF-α, and IL-10, in response to T. gondii infection.

Objectives The epidermal growth factor receptor (EGFR) abnormalities including amplification, mutation, and overexpression are frequent in non-small cell lung cancer (NSCLC). We investigated in vitro and in vivo antitumor activity of ER2, a novel human anti-EGFR monoclonal antibody, in NSCLC. Methods A panel of NSCLC cell lines (A549, H460, H322, H358, H1299, HCC827, PC9, H1975, and PC9-GR) was used to evaluate in vitro antitumor activity of ER2 and cetuximab. The inhibitory effects of ER2 and cetuximab on downstream signaling were assessed by western blot. Secreted VEGF was measured by Human VEGF Quantikine ELISA kit. Antitumor effects of ER2 and cetuximab as single agents and in combination with cisplatin were evaluated in H322, HCC827 and A549 xenograft models. Results ER2 efficiently inhibits EGFR and its downstream signaling molecules including Akt and Erk1/2 in NSCLC cell lines with wild-type or mutant EGFR. ER2 inhibited cell viability of H322, HCC827 and A549 cells in a dose-dependent manner by inducing cell cycle arrest and apoptosis. Also, ER2 suppressed EGF-stimulated VEGF production as efficiently as cetuximab in H322, HCC827 and A549 cells. Moreover, ER2 alone and in combination with cisplatin showed a significant anti-tumor efficacy in xenograft mouse models. Conclusion Taken together, ER2 has significant anti-tumor activity in in vitro and in vivo NSCLC models, suggesting a rationale for clinical development of ER2 in NSCLC.

The authors describe a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method targeting the 18S rRNA gene for the species detection of medically important trematode infecting fish and oysters, and suggest that this PCR-RFLP method based on a specific Tre-18 primer and the restriction enzymes, Acc1, Ava2, Msp1, and Hinf1, is useful for the detection of parasites in aquatic food.

Background:Treatment with ALK tyrosine kinase inhibitors elicits profound initial antitumor responses in ALK fusion-positive patients with lung adenocarcinoma. However, patients invariably develop acquired resistance to ALK inhibitors. Although the recent functional genetic studies characterized the mechanism of resistance to ALK inhibition, epigenetic mechanism of acquired resistance is poorly understood. Method: We established in vivo and in vitro models of acquired resistance to ceritinib and crizotinib using H3122 and H2228 cells. For in vivo model, mice with established H3122-derived tumors were treated with ceritinib to derive ceritinib-resistant tumors. To investigate the epigenetic mechanisms of resistance, we performed methylated DNA binding domain sequencing (MBD-seq) for DNA methylation and chromatin immunoprecipitation-sequencing (ChIP-seq) for histone modifications associated with enhancers (H3K4me1 and H3K27ac) of H3122 and H3122 ceritinib-resistant (LR) cells. We also performed in situ hybridization of miR-34 and miR-449 in patient samples. Results: Epithelial-to-mesenchymal transition with AXL activation was found in multiple in vitro and in vivo ALK-rearranged lung cancer models with acquired resistance to ceritinib. Genome and epigenome-wide analysis identified that miR-34a and miR-449a are decreased with loss of H3K27ac, while AXL and mesenchymal genes are increased in the process of acquired resistance. We confirmed that miR-34a and miR-449 were involved in AXL-dependent epithelial-mesenchymal transition (EMT) by treating mimics and inhibitors of miR-34a and miR-449a. In addition, pharmacological inhibition of AXL or ectopic expression of miR-34a or miR-449a restored sensitivity to ceritinib in H3122-LR cells. Histone deacetylase (HDAC) inhibitor, panobinostat, synergistically induced anti-proliferative effects with ceritinib in resistant cells. Treatment with panobinostat led to increased expression of miR-449a with increased H3K28ac signal in H3122 LR cells. Panobinostat also reversed EMT phenotype by downregulating N-cadherin and upregulating E-cadherin expression. Additionally, combination of panobinostat and ceritinib induced enhanced antitumor efficacy in acquired resistant xenograft models. To clinically validate our preclinical findings, we measured the expression of miR-34a and miR-449 by in situ hybridization in 5 matched ALK-rearranged lung cancers obtained from individuals both before and after ALK inhibitor treatment. We detected decreased expression of miR-34a and miR-449 in 3 of 5 samples (60%). Conclusion: Our findings demonstrate that H3K27ac remodeling is a crucial event in ceritinib resistance and inhibition of both ALK and HDAC could prevent or overcome acquired resistance to ALK inhibitors in individuals with ALK-rearranged lung cancer. Citation Format: Sun Min Lim, Mi Ran Yun, Kyoung Ho Pyo, Seong Keun Kim, Seon-Kyu Kim, Yong Sung Kim, Mirang Kim, Byoung Chul Cho. Epigenetic downregulation of miRNA-34a and miRNA-449 induce epithelial-mesenchymal transition and acquired resistance to ceritinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3346. doi:10.1158/1538-7445.AM2017-3346

The phylogenetic relationships of the 3 Neodiplostomum spp. (Digenea: Neodiplostomidae) occurring in Korea (N. seoulense, N. leei, and N. boryongense) were analyzed using the partial mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. The adult flukes were recovered from Sprague-Dawley rats (N. seoulense) and newborn chicks (N. leei and N. boryongense) experimentally infected with the neodiplostomula from the grass snake, Rhabdophis tigrinus tigrinus. The genomic DNA was amplified using specific primers, and the sequence of CO1 was obtained. According to the results, the pairwise similarity was 96.1% between N. boryongense and N. seoulense, but was 95.0% between N. boryongense and N. leei and 94.2% between N. leei and N. seoulense. The results demonstrated a closer phylogenetic relationship between N. seoulense and N. boryongense. This high relationship of N. seoulense and N. boryongense may be related to their similar morphologic features including the limited distribution of vitellaria and the presence of a genital cone. N. leei is distinct on the other hand with an extensive distribution of vitellaria and the absence of a genital cone.

The activation of macrophages by trypsin-digested silk peptides was investigated by considering CD11b and CD40 expression in the RAW264.7 cell, a murine macrophage. Silk protein hydrolysates were digested with trypsin, following by centrifugal purification using the Centriprep 30k concentrator. Trypsin-digested total silk peptides and its centrifugal fractions were tested for macrophage activation in vitro. The functional peptide of fractionated silk peptides was examined by LC/MS/MS analysis. Trypsin-digested and fractionated silk peptides of more than 30 kDa induced an increase in the activation markers CD11b and CD40 in RAW264.7 cells. These results are supported by morphological changes reflecting an increase in the number of dendrites in activated cells. The fractionated silk peptides examined by LC/MS/MS contained partial peptides of Bombyx mori fibroin. These results suggest that the activation of RAW264.7 macrophages may be induced not by sericin-derived peptides but by fibroin-derived ones.

Background: EML4-ALK is a fusion-type protein typrosin kinase that is generated in lung cancer. We have established transgenic mouse line. The surfactant protein C gene (SPC) is only expressed in alveolar epithelial cells. We applied SPC promoter on encoded human-EML4-ALK gene. Normally, EML4-ALK gene is silenced when LoxP-PGK-LoxP gene is turned on. When Cre recombinase is present with the specific inducer, tamoxifen, EML4-ALK gene is expressed in alveolar epithelial cell, and converses to lung cancer. Materials and Methods: For generation of EML4-ALK fusion transgenic mouse, a cDNA fragment encoding FLAG tagged EML4-ALK (variant 1) was ligated to end of the CMV-LoxP-PGK-LoxP. The expression cassette was injected into pronuclear-stage embryos of B6 mouse to generate Tg mouse. Transgenic mouse was prepared for interbreed with KI mouse (SPC-CreER2T-trTA-NeoR). The KI-Tg fusion mouse was confirmed by genotyping, then the mouse was injected with tamoxifen (cre recombinase inducer) intraperitoneally. The lung tumors were observed by MRI or CT. The lung pathology was analyzed by immunohistochemistry and hematoxylin & eosin staining. For confirmation of EML4-ALK protein expression, western blot was performed in different organs. EML4-ALK tumor-bearing mouse was treated with ALK tyrosine kinase inhibitor (TKI) for 2 weeks and tumor shrinkage was measured. Results: EML4-ALK tumor was observed after one week. Tumor nodules were widely observed in all lung lobes. Pathologically, the histologic type was adenocarcinoma and had homology to patient tissue on H&E stain. We confirmed EML4-ALK expression in different organs including lung, heart, liver, stomach, kidney, colon and brain. EML4-ALK (120kDa) was strongly expressed in lung, but not in other organs. Indeed, ALK and Flag expression were observed in tumor region by immunohistochemistry. The EML4-ALK tumor bearing mice were treated with ALK TKI for 2 weeks. The ALK TKI-treated mice showed dramatic shrinkage of tumors and showed gain in body weight. Conclusions: EML4-ALK Tg mouse was successfully established. The benefits of the model compared to a previous conditioned Tg mouse model are (1) no need to treat Cre inducer daily, and (2) short duration (approximately 1 week) of tumorigenesis. We confirmed dramatic tumor shrinkage after treatment with ALK TKI. This model provides a strong preclinical model for testing ALK TKIs in lung cancer. Citation Format: Kyoung-Ho Pyo, Hye Ryun Kim, Sun Min Lim, Mi Ran Yun, Sung-Moo Kim, Hwan Kim, Han Na Kang, Ji Min Lee, Sang Gyun Kim, Byoung Chul Cho. EML4-ALK fusion gene expressing transgenic mouse for lung cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 677.

Background: We conducted a co-clinical trial with PDX models in conjunction with a phase II clinical trial with dovitinib in patients with fibroblast growth factor receptor 1 (FGFR1) amplified LSCC in order to identify a predictive biomarker for dovitinib, a FGFR inhibitor. Methods: PDX models were established using tumor samples obtained from LSCC patients enrolled in the trial. We conducted an in vivo efficacy test with dovitinib in PDX models and comprehensive molecular profiling by whole exome sequencing (WES), array comparative genomic hybridization (aCGH), and microarray-based gene expression. Results: The histological and genomic characteristics of xenograft tumors (F2) resembled case-matched original tumors (F0). Dovitinib treatment resulted in tumor shrinkage in PDXL01, but not in PDXL02-04, which corresponded to responses in LSCC patients enrolled in the clinical trial. The patient, from whom PDXL01 was derived, showed a partial response to dovitinib with progression-free survival of 6 months, whereas the patient, from whom PDXL04 was derived had rapid disease progression within 2 months of treatment. Mutation profiles of F0 and F2 were largely concordant with each other in at least 70% of somatic mutations. The genome-wide copy number alterations were also largely concordant between F0 and F2, suggesting that PDX tumors can successfully represent the genomic features of the tumors from LSCC patients. Notably, mutation in FGFR 1-3 genes was not observed. FGFR1 amplification largely due to arm-level 8p gain was consistently observed, regardless of sensitivity to dovitinib. This finding suggests that FGFR1 amplification may not be a predictor for dovitinib sensitivity. Finally, we compared gene expression between a responder and non-responders. In a Heatmap analysis of the top 50 significantly up- and down-regulated genes, FGFR gene signature including FGF3 and FGF19 was significantly up-regulated in the responder. Gene set enrichment analysis (GSEA) identified that gene sets, such as FGFR ligand binding and activation and SHC-mediated cascade pathway, were significantly enriched in the responder, highlighting FGFR pathway activation as a key molecular determinant for sensitivity to dovitinib. To identify predictive gene signatures from independent datasets, we further performed GSEA in dovitinib-sensitive lung cancer cell lines compared to resistant ones as available in the CCLE (Cancer Cell Line Encyclopedia) database. Among the four functional modules (FGFR signaling, Immune, Cell cycle, and RNA) that we identified, the FGFR signaling module performed well in the prediction of dovitinib sensitivity of PDXL01-04 Conclusions: FGFR gene expression signatures are predictors for response to dovitinib in LSCC. Citation Format: Hye Ryun Kim, Han Na Kang, Sung Moo Kim, Hwan Kim, Kyoung-Ho Pyo, Myung-Ju Ahn, Tae Min Kim, Byoung Chul Cho. A Mouse-Human co-clinical trial with patient-derived xenograft (PDX) models demonstrates a predictive signature for dovitinib (TKI258), an FGFR and VEGFR inhibitor,in lung squamous cell carcinomalung cancer (LSCC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5194.

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8(+) T-cells and CD4(+) T-cells. However, in mice immunized using a gene gun, significantly higher (P0.05) proportions of CD8(+) cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.

The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression.

Immunosuppression is a characteristic feature of Toxoplasma gondii-infected murine hosts. The present study aimed to determine the effect of the immunosuppression induced by T. gondii infection on the pathogenesis and progression of Alzheimer's disease (AD) in Tg2576 AD mice. Mice were infected with a cyst-forming strain (ME49) of T. gondii, and levels of inflammatory mediators (IFN-γ and nitric oxide), anti-inflammatory cytokines (IL-10 and TGF-β), neuronal damage, and β-amyloid plaque deposition were examined in brain tissues and/or in BV-2 microglial cells. In addition, behavioral tests, including the water maze and Y-maze tests, were performed on T. gondii-infected and uninfected Tg2576 mice. Results revealed that whereas the level of IFN-γ was unchanged, the levels of anti-inflammatory cytokines were significantly higher in T. gondii-infected mice than in uninfected mice, and in BV-2 cells treated with T. gondii lysate antigen. Furthermore, nitrite production from primary cultured brain microglial cells and BV-2 cells was reduced by the addition of T. gondii lysate antigen (TLA), and β-amyloid plaque deposition in the cortex and hippocampus of Tg2576 mouse brains was remarkably lower in T. gondii-infected AD mice than in uninfected controls. In addition, water maze and Y-maze test results revealed retarded cognitive capacities in uninfected mice as compared with infected mice. These findings demonstrate the favorable effects of the immunosuppression induced by T. gondii infection on the pathogenesis and progression of AD in Tg2576 mice.

To examine the infection status of freshwater fish with Gnathostoma spp. larvae in Myanmar, we purchased 15 snakeheads, Channa striatus, from a local market in a suburban area of Naypyidaw, the new capital city. Two larval gnathostomes were collected using an artificial digestion technique, and observed by a light microscope and a scanning electron microscope. The size of an intact larva was 2.65 mm long and 0.32 mm wide. The characteristic morphology of the larvae included the presence of a long esophagus (0.80 mm long), 2 pairs of cervical sacs (0.43 mm long), and a characteristic head bulb with 4 rows of hooklets. The number of hooklets in the 1st, 2nd, 3rd, and 4th row was 45, 48, 50, and 52, respectively. Based on these morphological characters, the larvae were identified as the advanced 3rd-stage larvae of Gnathostoma spinigerum. This is the first report of detection of G. spinigerum 3rd-stage larvae in the central part of Myanmar. Our study suggests that intake of raw meat of snakehead fish in Myanmar may result in human gnathostomiasis.