Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development.

Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3's biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.

The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.

Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

Ciliary signaling in stem cells in health and disease: Hedgehog pathway and beyond.

The primary cilium is a hair-like sensory compartment that protrudes from the cellular surface. The primary cilium is enriched in a variety of signaling molecules that regulate cellular activities. Stem cells have primary cilia. They reside in a specialized environment, called the stem cell niche. This niche contains a variety of secreted factors, and some of their receptors are localized in the primary cilia of stem cells. Here, we summarize the current understanding of the function of cilia in compartmentalized signaling in stem cells. We describe how ciliary signaling regulates stem cells and progenitor cells during development, tissue homeostasis and tumorigenesis. We summarize our understanding of cilia regulated signaling -primary involving the hedgehog pathway- in stem cells in diverse settings that include neuroepithelial cells, radial glia, cerebellar granule neuron precursors, hematopoietic stem cells, hair follicle stem cells, bone marrow mesenchymal stem cells and mammary gland stem cells. Overall, our review highlights a variety of roles that ciliary signaling plays in regulating stem cells throughout life.

Biallelic KARS pathogenic variants cause an early-onset progressive leukodystrophy.

The leukodystrophies cause severe neurodevelopmental defects from birth and follow an incurable and progressive course that often leads to premature death. It has recently been reported that abnormalities in aminoacyl t-RNA synthetase (ARS) genes are linked to various unique leukodystrophies and leukoencephalopathies. Aminoacyl t-RNA synthetase proteins are fundamentally known as the first enzymes of translation, catalysing the conjugation of amino acids to cognate tRNAs for protein synthesis. It is known that certain aminoacyl t-RNA synthetase have multiple non-canonical roles in both transcription and translation, and their disruption results in varied and complicated phenotypes. We clinically and genetically studied seven patients (six male and one female; aged 2 to 12 years) from five unrelated families who all showed the same phenotypes of severe developmental delay or arrest (7/7), hypotonia (6/7), deafness (7/7) and inability to speak (6/7). The subjects further developed intractable epilepsy (7/7) and nystagmus (6/6) with increasing age. They demonstrated characteristic laboratory data, including increased lactate and/or pyruvate levels (7/7), and imaging findings (7/7), including calcification and abnormal signals in the white matter and pathological involvement (2/2) of the corticospinal tracts. Through whole-exome sequencing, we discovered genetic abnormalities in lysyl-tRNA synthetase (KARS). All patients harboured the variant [c.1786C>T, p.Leu596Phe] KARS isoform 1 ([c.1702C>T, p.Leu568Phe] of KARS isoform 2) either in the homozygous state or compound heterozygous state with the following KARS variants, [c.879+1G>A; c.1786C>T, p.Glu252_Glu293del; p.Leu596Phe] ([c.795+1G>A; c.1702C>T, p.Glu224_Glu255del; p.Leu568Phe]) and [c.650G>A; c.1786C>T, p.Gly217Asp; p.Leu596Phe] ([c.566G>A; c.1702C>T, p.Gly189Asp; p.Leu568Phe]). Moreover, similarly disrupted lysyl-tRNA synthetase (LysRS) proteins showed reduced enzymatic activities and abnormal CNSs in Xenopus embryos. Additionally, LysRS acts as a non-canonical inducer of the immune response and has transcriptional activity. We speculated that the complex functions of the abnormal LysRS proteins led to the severe phenotypes in our patients. These KARS pathological variants are novel, including the variant [c.1786C>T; p.Leu596Phe] (c.1702C>T; p.Leu568Phe) shared by all patients in the homozygous or compound-heterozygous state. This common position may play an important role in the development of severe progressive leukodystrophy. Further research is warranted to further elucidate this relationship and to investigate how specific mutated LysRS proteins function to understand the broad spectrum of KARS-related diseases.

Verruciform xanthoma accompanying a cystic growth: A case report.

Verruciform xanthoma, an uncommon, benign lesion with characteristic histopathological features, usually develops on the oral mucosa or genital area. We present an unusual case of verruciform xanthoma observed on the inguinal skin of a 52-year-old healthy man along with an underlying cystic component. The superficial lesion was a pedunculated nodule with a fissured surface and an 18-mm mound-like pigmented tumor underneath it. The histopathologically deep lesion was continuously attached to the superficial lesion. It was composed of fistula or sinus-like spaces and covered with acanthotic epithelium. The epidermis and upper dermis of both lesions showed identical histopathological findings: varying degrees of acanthosis, elongation of rete ridges, eosinophilic parakeratotic layer extending toward the dermis, and densely infiltrating foam cells confined to the papillary layer of the dermis. This finding of a cystic component in the deep dermis expands the histopathologic features of verruciform xanthoma.

[WT1 Class I Peptide/WT1 Class II Peptide Pulsed Dendritic Cell Therapy Efficacy in 60 Patients with a Wide Range of Advanced Cancers].

We assessed the efficacy of WT1 class I peptide and WT1 class II peptide pulsed dendritic cell(DC)therapy for a wide range of advanced cancers. This retrospective study included 60 advanced cancer patients who were vaccinated 5times or more in this clinic between September 2013 and December 2015. The clinical response was examined. This treatment was approved by the ethics panel at this institution. Sixty patients were injected an average of 6.15times with dendritic cells(DCs) (2.6×10 / 7 cells/injection). Overall, 55of 60(92%)patients achieved a clinical benefit per the RECIST v1.1 criteria. The median survival time(MST)from diagnosis was 26.9 months, and the MST from the first admission at this institution was 12.2 months. Complete response(CR)was achieved in 5patients(9.1%), partial response(PR)in 12(22%), and stable disease(SD) in 21(38%), with confirmed progressive disease(PD)in 17(31%). In 11 cases of pancreatic cancer, the response(RR)and disease control rates(DCR)were 27%and 55%,respectively. In 8 cases of colorectal cancer, the rates were 25% and 75%; in 7 cases of lung cancer, the rates were 29% and 43%; in 7 cases of gastric cancer, the rates were 71% and 86%; in 22 other types, the rates were 23% and 74%. These results demonstrated the potential clinical efficacy of DC-based immunotherapy.

A potential molecular pathogenesis of cardiac/laterality defects in Oculo-Facio-Cardio-Dental syndrome.

Pitx2 is the last effector of the left-right (LR) cascade known to date and plays a crucial role in the patterning of LR asymmetry. In Xenopus embryos, the expression of Pitx2 gene in the left lateral plate mesoderm (LPM) is directly regulated by Xnr1 signaling, which is mediated by Smads and FoxH1. Previous studies suggest that the suppression of Pitx2 gene in the left LPM is a potential cause of cardiac/laterality defects in Oculo-Facio-Cardio-Dental (OFCD) syndrome, which is known to be caused by mutations in BCL6 co-repressor (BCOR) gene. Recently, our work has revealed that the BCL6/BCOR complex blocks Notch-dependent transcriptional activity to protect the expression of Pitx2 in the left LPM from the inhibitory activity of Notch signaling. These studies indicated that uncontrolled Notch activity in the left LPM caused by dysfunction of BCOR may result in cardiac/laterality defects of OFCD syndrome. However, this Notch-dependent inhibitory mechanism of Pitx2 gene transcription still remains unknown. Here we report that transcriptional repressor ESR1, which acts downstream of Notch signaling, inhibits the expression of Pitx2 gene by binding to a left side-specific enhancer (ASE) region in Pitx2 gene and recruiting histone deacetylase 1 (HDAC1) to this region. Once HDAC1 is tethered, histone acetyltransferase p300 is no longer recruited to the Xnr1-dependent transcriptional complex on the ASE region, leading to the suppression of Pitx2 gene in the left LPM. The study presented here uncovers the regulatory mechanism of Pitx2 gene transcription which may contribute to an understanding of pathogenesis of OFCD syndrome.

[Efficacy of WT1 peptide-/MUC-1 peptide-pulsed dendritic cell therapy in 313 patients with a wide range of cancers].

We assessed the efficacy of Wilms' tumor protein-1(WT1)peptide and/or mucin 1(MUC-1)peptide-pulsed dendritic cell(DC)therapy for a wide range of advanced cancers.This retrospective study included 313 patients with advanced cancer who were vaccinated ≥5 times in our clinic between May 2009 and October 2013.T he clinical response was evaluated.This treatment was approved by the ethics panel of our institution.A total of 313 patients were injected an average of 6.0 times with DCs(2.4×10 / 7 cells/injection).Overall, 292 of the 313(93%)patients obtained clinical benefit according to the Response Evaluation Criteria in Solid Tumors(RECIST), version 1.1. The median survival time(MST)from diagnosis and from first being admitted to this institution was 28.4 months and 13.2 months, respectively. Complete response(CR)was achieved in 21 patients(7.2%).Partial response(PR)was achieved in 69 patients(24%).Stable disease(SD)was observed in 107 patients(37%), and progressive disease(PD)was observed in 95 patients(33%).The response rate(RR)and disease control rate(DCR)for each type of cancer were as follows: colorectal cancer(56 patients), 25%and 45%, respectively; lung cancer(39 patients), 31% and 82%, respectively; pancreatic cancer(36 patients), 22% and 64%, respectively; gastric cancer( 34 patients), 32% and 74%; breast cancer(20 patients), 15% and 75%, respectively; ovarian cancer(16 patients), 19% and 50%, respectively; esophageal cancer(15 patients), 20% and 73%, respectively; and others(76 patients), 38% and 67%.These results demonstrated the potential clinical effectiveness of DC-based immunotherapy.

The multiple roles of Notch signaling during left-right patterning.

The establishment of left-right (LR) asymmetry is regulated by intricate signaling mechanisms during embryogenesis and this asymmetry is critical for morphogenesis as well as the positioning of internal organs within the organism. Recent progress including elucidation of ion transporters, leftward nodal flow, and regulation of asymmetric gene expression contributes to our understanding of how the breaking of the symmetry is initiated and how this laterality information is subsequently transmitted to the organ primordium. A number of developmental signaling pathways have been implicated in this complex process. In this review, we will focus on the roles of the Notch signaling pathway during development of LR asymmetry. The Notch signaling pathway is a short-range communication system between neighboring cells. While Notch signaling plays essential roles in regulating the morphogenesis of the node and left-specific expression of Nodal in the lateral plate mesoderm, a hallmark gene in LR patterning, Notch signaling also suppresses the expression of Pitx2 that is a direct downstream target of Nodal during later stages of development. This negative activity of Notch signaling towards left-specific activity was recently shown to be inhibited by the B cell lymphoma 6 (BCL6)/BCL6 co-repressor (BcoR) transcriptional repressor complex in a target-specific manner. The complex regulation of Notch-dependent gene expression for LR asymmetry will be highlighted in this review.

[Prediction of WT1/MUC1 peptide dendritic cell therapy responders by quantification of ex vivo induced mRNA in whole blood].

Cancer immunotherapy has shown much potential, but is not yet beneficial for all patients. Acquired immunity after immunotherapy can be assessed by a variety of methods; however, the methods for the prediction of such responses before treatment are quite limited. To challenge this classic problem, we quantified 17 different leukocyte function-associated mRNAs( IFN-γ,TNFSF1, TNFSF2, TNFSF5, IL-10, TGF ß,CTLA4, PD1, FOXP3, GMCSF, VEGF, IL-8, CCL8, CXCL3, and IL-2) in whole blood after ex vivo stimulation with 7 different agents(PHA, HAG, zymosan, IFN-γ,rIL-2, aTCR, and picibanil) to activate various subsets of leukocytes. The mRNAs were quantified by the Hitachi Chemical Research Center, Inc. Irvine, CA. The clinical outcomes for WT1 peptide-and/or MUC1 peptide-pulsed dendritic cell therapy for advanced cancer (n=26) were CR+PR, 4 cases; SD, 9 cases; and PD, 13 cases. The accuracy of prediction was found to be 100% using the formula developed by multivariate discriminant analysis of the values for ex vivo induced mRNAs. Because the volume of blood needed for this test is less than 1.5 mL, and because cell isolation and culture are not necessary, this method will become a model of personalized medicine diagnostics for cancer immunotherapy.

Hospital Evacuation Implications After the 2016 Kumamoto Earthquake.

During the 2016 Kumamoto earthquake, 10 hospitals took responsibility for complete evacuation, in what has become regarded as one of the largest evacuations of patients in 1 seismic disaster. We aimed to examine the reasons for evacuation and to assess hospital vulnerability as well as preparedness for the earthquake. A multidisciplinary team conducted semi-structured interviews with the hospitals 6 months after the earthquake. The primary reasons for the decision to evacuate hospitals were categorized into 3: 1) Concern for structural safety (4 facilities), 2) Damage to the facility water system (7 facilities), and 3) Cessation of regional water supply (5 facilities).All hospitals decided on immediate evacuation within 30 hours and could not wait for structural engineers to inspect the affected buildings. Damage to sprinklers or water facilities caused severe water shortages and flood, thus requiring weeks to resume inpatient care. The earthquake revealed the vulnerability of rapid building-inspection systems, aging buildings, and water infrastructure.

Abnormal ciliogenesis in decidual stromal cells in recurrent miscarriage.

Primary cilia regulate cellular signaling and are involved in both sensing and transducing extracellular stimuli. A recent study of patients with recurrent miscarriage (RM) identified mutations affecting DYNC2H1, which were involved in ciliary biogenesis. However, there has been no study concerning primary cilia in the decidua. We compared the number and the length of primary cilia in the decidua of 15 patients with unexplained RM with those of 7 pregnant controls who underwent an artificial termination of pregnancy. Immunohistochemistry was performed using antibodies against primary cilia, extravillous trophoblasts (EVTs), macrophages, uterine Natural Killer (uNK) cells, decidual stromal cells, and the activation of TGF-ß and CREB signaling in the decidua of early pregnancy was studied. The density of decidual stromal cells, but not EVTs, macrophages or uNK cells, was found to be significantly higher in the decidua of patients compared to controls. The percentage of ciliated decidual stromal cells was significantly decreased in patients. There was no difference in the primary ciliary length. Regarding TGF-ß signaling, p-Smad2 in these cells was diminished significantly in patients, and most of the TGF-ß-activated decidual stromal cells of both patients and controls had primary cilia. No difference in the activation of CREB was found. Abnormal primary cilia on decidual stromal cells may be one of the explanatory factors for unknown RM. The inactivation of TGF-ß signaling may lead to abnormal ciliogenesis in the decidua.

Abnormal accumulation of OFD1 in endometrial cancer with poor prognosis inhibits ciliogenesis.

The aim of the present study was to examine primary cilia in endometrial tissue during the menstrual cycle and to clarify their morphological changes with different grades of endometrial cancer. Images of fluorescence immunostaining taken by confocal microscopy were used to count the number of primary cilia in normal endometrium and endometrioid carcinoma Grade 1 and Grade 3 specimens. To examine the association between autophagy and ciliogenesis in endometrioid carcinoma, the expression of p62/Sequestosome-1, a selective substrate for autophagy, and oral-facial-digital syndrome 1 protein (OFD1), a protein associated with ciliogenesis, were examined using images of fluorescence immunostaining taken by confocal microscopy. The level of p62 expression was confirmed by western blotting. In proliferative and secretory endometrial stromal cells, the percentage of cells that were ciliated was 7.2 and 32.7% (95% confidence interval=21.61-39.79; P<0.01), and the length of the primary cilia was 1.24 µm and 2.34 µm (0.92-1.26; P<0.01), respectively. In stromal cells of endometrioid carcinoma Grade 1 and Grade 3, the percentage of ciliated cells was 13.5 and 2.9% (7.89-15.05; P<0.001), and the length of the primary cilia was 2.02 and 1.14 µm (0.76-0.99; P<0.001), respectively. In both normal menstrual cycle tissue and endometrial carcinomas, the percentage of primary cilia was lower and their length was shorter in tissues with higher proliferative potential. The expression of OFD1 was significantly higher in Grade 3 compared with Grade 1 as indicated by quantifying the intensity of the fluorescence images (133-12248; P=0.046). To the best of our knowledge, this is the first study concerning the distribution of primary cilia in normal endometrium and endometrial cancer tissues. Overall, fewer ciliated cells in the highly malignant endometrial cancer tissues may be associated not only to the proliferation of cancer cells, but also to the excessive accumulation of OFD1 due to dysfunctional autophagy.

Embryonic lethality of fortilin-null mutant mice by BMP-pathway overactivation.

BACKGROUND: Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. METHODS AND RESULTS: In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin(+/-) mice have the ability to survive and exhibit normal growth, while fortilin(-/-) mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin(+/-) embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin(-/-) embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin(+/-) embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin's message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. CONCLUSION: We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. GENERAL SIGNIFICANCE: Abnormalities in the fortilin gene may be associated with early pregnancy loss.

[WT1 peptide pulsed dendritic cell therapy with activated T lymphocytes therapy for advanced cancers].

We assessed the efficacy of WT1 peptide pulsed dendritic cell (DC) therapy for various advanced cancers. All patients were vaccinated 5 times for 10 weeks with autologous monocytes derived DC and activated T lymphocytes. We treated a total of 26 patients who had HLA-A2402 or/and HLA-A0201. We evaluated 20 of the 26 patients who finished 5-time vaccination (10 men and 10 women, aged 48-81 years, Mean 64 years) and were diagnosed as follows: 3-pancreas cancer, 2-colorectal, 2-breast, 2-esophageal, 2-lung, 2-uterus, 2-ovarian and 5 others. In Clinical response (RECIST), the result was assessed as CR/PR/SD/PD, 0/7/8/5, respectively. Furthermore, the 7 PRs were resulted from 2-colorectal, and one of each was lung, laryngeal, axis, pancreas and smooth muscle sarcoma cancer. The 4 of 7 PR patients were treated with chemotherapy.

The POU homeobox protein Oct-1 regulates radial glia formation downstream of Notch signaling.

Radial glia cells function as guide cells for neuronal migration and a source of neural progenitor cells, and play a crucial role for the development of the central nervous system. To date, several signals have been demonstrated to promote the formation of radial glia cells and Notch signaling is one such signal. However, the mechanism of the signaling hierarchy of radial glia developmental cascade promoted by Notch signaling still remains incomplete. Here we show that Notch signaling promotes Xenopus radial glia formation and that the Notch activation is sufficient for radial glia formation prior to neural tube closure. Moreover, we have identified Oct-1 (POU2f1), a POU transcription factor, as a downstream target of Notch signaling by microarray based screen. We demonstrate that the expression of Oct-1 in the brain is regulated by Notch signaling and that Oct-1 is sufficient and necessary for radial glia formation. Together, Oct-1 is a downstream effector of Notch signaling during radial glia formation.

Novel cross talk of Kruppel-like factor 4 and beta-catenin regulates normal intestinal homeostasis and tumor repression.

Epithelial cells of the intestinal mucosa undergo a continual process of proliferation, differentiation, and apoptosis which is regulated by multiple signaling pathways. The Wnt/beta-catenin pathway plays a critical role in this process. Mutations in the Wnt pathway, however, are associated with colorectal cancers. Krüppel-like factor 4 (KLF4) is an epithelial transcriptional factor that is down-regulated in many colorectal cancers. Here, we show that KLF4 interacts with beta-catenin and represses beta-catenin-mediated gene expression. Moreover, KLF4 inhibits the axis formation of Xenopus embryos and inhibits xenograft tumor growth in athymic nude mice. Our findings suggest that the cross talk of KLF4 and beta-catenin plays a critical role in homeostasis of the normal intestine as well as in tumorigenesis of colorectal cancers.

Calcineurin regulates cyclin D1 stability through dephosphorylation at T286.

The Calcineurin/NFAT (nuclear factor of activated T cells) pathway plays an essential role in the tumorigenic and metastatic properties in breast cancer. The molecular mechanism of the antiproliferative effect of calcineurin inhibition, however, is poorly understood. We found that calcineurin inhibition delayed cell cycle progression at G1/S, and promoted cyclin D1 degradation by inhibiting dephosphorylation at T286. Importantly, overexpression of cyclin D1 partially rescued delayed G1/S progression, thereby revealing cyclin D1 as a key factor downstream of calcineurin inhibition. Cyclin D1 upregulation is observed in human invasive breast cancers, and our findings indicate that dysregulation of T286 phosphorylation could play a role in this phenomenon. We therefore propose that targeting site specific phosphorylation of cyclin D1 could be a potential strategy for clinical intervention of invasive breast cancer.

[Efficacy of activated lymphocytes transfer therapy as a novel maker for serum granulysin level with advanced gastric cancer patients].

INTRODUCTION: Granulysin is a cytotoxic granule protein of cytotoxic T-lymphocytes and natural killer cells. Gastric cancer patients of stage II and stage III were reported to have pre-operative serum granulysin, which is a good prognostic factor. Activated T lymphocytes produce a lot of granulysin in the cell. We hypothesized that activated T lymphocytes transfer therapy may increase in serum granulysin leveles of stage IV and relapsed patients. METHOD: Between April 2002 and December 2007, the patients received a standard therapy with activated lymphocytes transfer therapy. Peripheral blood samples were taken from the patients. Serum granulysin concentrations were measured using a granulysin-specific ELISA kit. RESULT: The average serum granulysin level for Stage IV (n=29) and relapsed patients (n=13) was 3.3 ng/mL. The gastric cancer patients were classified into two groups based on their concentration of serum granulysin level: high group (> or = 3.3 ng/mL); low group (< 3.3 ng/mL). There were no significant differences between these two groups in gender and age. The stage IV high group had a longer survival time than the stage IV low group. After 60 days from the activated T lymphocytes transfer therapy, the average pre-treatment serum granulysin had increased. However, there was no significant difference to note. It is noteworthy that serum granulysin is a novel parameter of patients cancer immunity.