Case study observational research: inflammatory cytokines in the bronchial epithelial lining fluid of COVID-19 patients with acute hypoxemic respiratory failure.

BACKGROUND: In this study, the concentrations of inflammatory cytokines were measured in the bronchial epithelial lining fluid (ELF) and plasma in patients with acute hypoxemic respiratory failure (AHRF) secondary to severe coronavirus disease 2019 (COVID-19). METHODS: We comprehensively analyzed the concentrations of 25 cytokines in the ELF and plasma of 27 COVID-19 AHRF patients. ELF was collected using the bronchial microsampling method through an endotracheal tube just after patients were intubated for mechanical ventilation. RESULTS: Compared with those in healthy volunteers, the concentrations of interleukin (IL)-6 (median 27.6 pmol/L), IL-8 (1045.1 pmol/L), IL-17A (0.8 pmol/L), IL-25 (1.5 pmol/L), and IL-31 (42.3 pmol/L) were significantly greater in the ELF of COVID-19 patients than in that of volunteers. The concentrations of MCP-1 and MIP-1ß were significantly greater in the plasma of COVID-19 patients than in that of volunteers. The ELF/plasma ratio of IL-8 was the highest among the 25 cytokines, with a median of 737, and the ELF/plasma ratio of IL-6 (median: 218), IL-1ß (202), IL-31 (169), MCP-1 (81), MIP-1ß (55), and TNF-α (47) were lower. CONCLUSIONS: The ELF concentrations of IL-6, IL-8, IL-17A, IL-25, and IL-31 were significantly increased in COVID-19 patients. Although high levels of MIP-1 and MIP-1ß were also detected in the blood samples collected simultaneously with the ELF samples, the results indicated that lung inflammation was highly compartmentalized. Our study demonstrated that a comprehensive analysis of cytokines in the ELF is a feasible approach for understanding lung inflammation and systemic interactions in patients with severe pneumonia.

Reciprocal expression of the immune response genes CXCR3 and IFI44L as module hubs are associated with patient survivals in primary central nervous system lymphoma.

PURPOSE: Here, we investigated expression modules reflecting the reciprocal expression of the cancer microenvironment and immune response-related genes associated with poor prognosis in primary central nervous system lymphoma (PCNSL). METHODS: Weighted gene coexpression network analysis revealed representative modules, including neurogenesis, immune response, anti-virus, microenvironment, gene expression and translation, extracellular matrix, morphogenesis, and cell adhesion in the transcriptome data of 31 PCNSL samples. RESULTS : Gene expression networks were also reflected by protein-protein interaction networks. In particular, some of the hub genes were highly expressed in patients with PCNSL with prognoses as follows: AQP4, SLC1A3, GFAP, CXCL9, CXCL10, GBP2, IFI6, OAS2, IFIT3, DCN, LRP1, and LUM with good prognosis; and STAT1, IFITM3, GZMB, ISG15, LY6E, TGFB1, PLAUR, MMP4, FTH1, PLAU, CSF3R, FGR, POSTN, CCR7, TAS1R3, small ribosomal subunit genes, and collagen type 1/3/4/6 genes with poor prognosis. Furthermore, prognosis prediction formulae were constructed using the Cox proportional-hazards regression model, which demonstrated that the IP-10 receptor gene CXCR3 and type I interferon-induced protein gene IFI44L could predict patient survival in PCNSL. CONCLUSION: These results indicate that the differential expression and balance of immune response and microenvironment genes may be required for PCNSL tumor growth or prognosis prediction, which would help understanding the mechanism of tumorigenesis and potential therapeutic targets in PCNSL.

Differential expression of N-linked oligosaccharides in methotrexate-resistant primary central nervous system lymphoma cells.

BACKGROUND: Oligosaccharides of glycoprotein, particularly negatively-charged sialylated N-glycans, on the surface of lymphomas play important roles in cell-cell interactions and bind immunoglobulin-like lectins, causing inflammatory responses and bioregulation. However, their characterizations have largely been unknown in central nervous system (CNS) lymphoma. METHODS: Here, we investigated expression patterns of N-linked oligosaccharides of glycoproteins in cells derived from CNS lymphomas and clinical specimens. RESULTS: We first generated methotrexate (MTX)-resistant cells derived from HKBML and TK as CNS lymphoma, and RAJI as non-CNS lymphoma and determined N-linked oligosaccharide structures in these cells and other non-CNS lymphoma-derived cells including A4/FUK, OYB, and HBL1. Major components of the total oligosaccharides were high-mannose type N-glycans, whose level increased in MTX-resistant HKBML and TK but decreased in MTX-resistant RAJI. We also detected sialylated biantennary galactosylated N-glycans with α1,6-fucosylation, A2G2F, and A2G2FB from HKBML, TK, and RAJI. Sialylated A4G4F was specifically isolated from RAJI. However, the ratios of these sialylated N-glycans slightly decreased against MTX-resistant compared to non-resistant cells. Interestingly, almost all complex-type oligosaccharides were α2,6-sialylated. DISCUSSION: This is the first study for the expression profile of N-oligosaccharides on MTX-resistant primary CNS lymphoma-derived cells HKBML and TK, and tumor tissues resected from patients with CNS lymphoma, CONCLUSION: These results propose a possibility that the differential expression of high-mannose types and sialylated A2G2F, A2G2FB, and A4G4F on the surface of CNS lymphomas may provide a hint for targets for diagnoses and treatments of the oligosaccharide type-specific lymphomas.

Cell-type-specific sensitivity of bortezomib in the methotrexate-resistant primary central nervous system lymphoma cells.

BACKGROUND: Methotrexate (MTX) is used in first-line treatment of primary central nervous system lymphoma (PCNSL), but most cases result in relapse-acquired resistance to MTX. However, only few studies have reported on internal changes and chemotherapies in PCNSL. METHODS: In this study, we generated two MTX-resistant PCNSL cell lines, designated MTX-HKBML and MTX-TK, in addition to a MTX-resistant Burkitt lymphoma cell line, designated MTX-RAJI. We examined gene expression changes and drug sensitivity to a proteasome inhibitor, bortezomib, in these cells. RESULTS: Cytotoxic tests revealed that the 50% inhibitory concentration for MTX in MTX-HKBML is markedly higher than that in the other two cell lines. Expression of the genes in MTX and folate metabolisms, including gamma-glutamyl hydrolase and dihydrofolate reductase, are upregulated in both MTX-HKBML and MTX-TK, whereas the gene expression of folylpolyglutamate synthetase, thymidylate synthase, and methylenetetrahydrofolate dehydrogenase 1 were upregulated and downregulated in MTX-HKBML and MTX-TK, respectively, on the other hand, bortezomib sensitivity was observed in MTX-TK, as compared with control TK, but not in MTX-HKBML. CONCLUSION: These results indicate the cell-type-specific changes downstream of metabolic pathways for MTX and folate, bortezomib sensitivity, and purine and pyrimidine syntheses, in each PCNSL cell line. The MTX-resistant lymphoma cell lines established may be useful for in vitro relapse models for MTX and development of salvage chemotherapy and drug discovery.

Trilateral retinoblastoma: A systematic review of 211 cases.

We conducted a systematic review of 72 studies to characterize trilateral retinoblastomas. Kaplan-Meier analysis was used to estimate survival, and statistical significance was assessed by using a log-rank test. We analyzed 211 cases of trilateral retinoblastomas. The average age of onset of retinoblastoma was 0.79 ± 1.38 years, and the average latency period between the onset of retinoblastomas and trilateral retinoblastomas was 1.49 ± 1.76 years. The brain tumors were found before the retinoblastoma diagnosis in 6 cases (3.1%), concurrently in 61 cases (32.1%), and after the retinoblastoma diagnosis in 123 cases (64.7%). Pineal tumors were found in 155 cases (73.4%) and sellar tumors in 46 cases (21.8%). The overall median survival was 10.3 months (95% CI, 8.5-13) and the 5-year survival rate was 15.7%. Central nervous system symptoms were variable and associated with shorter survival in univariate and multivariate analyses. The survival time in patients who received high-dose chemotherapy with stem cell transplant was significantly longer (p = 0.0067) than that of with or without conventional chemotherapy. Twelve long-term survivors were reported, and of these, six patients were treated with high-dose chemotherapy with stem cell transplant and six patients were treated with conventional chemotherapy. It is important that survivors continue to undergo regular medical surveillance in order to detect trilateral retinoblastoma at a potentially curative stage. Trilateral retinoblastoma patients with an irradiation history had shorter survival than those without irradiation history for retinoblastoma. High-dose chemotherapy should be considered as a potential treatment option for trilateral retinoblastomas.

Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors.

Hepatocellular carcinoma (HCC) accounts for a large proportion of liver cancer cases and has an extremely poor prognosis. Therefore, novel innovative therapies for HCC are strongly desired. As gene therapy tools for HCC, 2 hepatic transcription factors (TF), HNF4A and HNF1A, have been used to suppress proliferation and to extinguish cancer-specific characteristics of target cells. However, our present data demonstrated that single transduction of HNF4A or HNF1A had only a limited effect on suppression of HCC cell proliferation. Thus, in this study, we examined whether combinations of TF could show more effective antitumor activity, and found that combinatorial transduction of 3 hepatic TF, HNF4A, HNF1A and FOXA3, suppressed HCC cell proliferation more stably than single transduction of these TF. The combinatorial transduction also suppressed cancer-specific phenotypes, such as anchorage-independent growth in culture and tumorigenicity after transplantation into mice. HCC cell lines transduced with the 3 TF did not recover their proliferative property after withdrawal of anticancer drugs, indicating that combinatorial expression of the 3 TF suppressed the growth of all cell subtypes within the HCC cell lines, including cancer stem-like cells. Transcriptome analyses revealed that the expression levels of a specific gene set involved in cell proliferation were only decreased in HCC cells overexpressing all 3 TF. Moreover, combined transduction of the 3 TF could facilitate hepatic differentiation of HCC cell lines. Our strategy for inducing stable inhibition and functional differentiation of tumor cells using a defined set of TF will become an effective therapeutic strategy for various types of cancers.

Suppression of lethal-7b and miR-125a/b Maturation by Lin28b Enables Maintenance of Stem Cell Properties in Hepatoblasts.

UNLABELLED: In liver development, hepatoblasts that act as hepatic stem/progenitor cells proliferate and differentiate into both hepatocytes and cholangiocytes to form liver tissues. Although numerous factors contribute to this event, little is known about the roles of microRNAs in hepatoblast proliferation and differentiation. In this study, we focused on the lineage-28 (Lin28) family proteins, which are required for microRNA regulation in pluripotent stem cells and cancer cells, and investigated their roles as regulatory factors for the properties of hepatoblasts. CONCLUSION: Lin28b was specifically expressed in hepatoblasts, and its suppression induced growth arrest and cholangiocyte differentiation of hepatoblasts; mechanistically, Lin28b positively regulates the expression of Lin28b itself and cell cycle-related proteins in hepatoblasts by suppressing the maturation of target microRNAs, lethal-7b and miR-125a/b, enabling maintenance of the stem cell properties of hepatoblasts, such as their capabilities for proliferation and bi-lineage differentiation, during liver development. (Hepatology 2016;64:245-260).

Dynamic three-dimensional morphogenesis of intrahepatic bile ducts in mouse liver development.

UNLABELLED: During liver development, biliary epithelial cells differentiated from bipotential hepatic progenitor cells (hepatoblasts) form a cell layer, called the ductal plate surrounding portal veins (PVs), and develop into intrahepatic bile ducts (IBDs) following developmental programs. Because IBDs make duct structures in the liver, it is necessary to perform sequential and three-dimensional (3D) analyses from the early stages of liver development to address the process of morphogenesis in detail. However, to date, the development of IBDs has mainly been investigated using tissue sections in two-dimensional planes, and examinations of the 3D morphogenesis and quantitative analyses based on morphometrics have not been performed. Therefore, in this study, we simulated the solid structures of IBDs from mouse embryos to adults in silico, analyzed the subjects for the length and number of developing duct structures, number of predicted connections, and discrete distance from the PV, and examined the developmental process of the IBD in detail in a quantitative manner. CONCLUSIONS: Through quantitative analyses with spatiotemporal observations using a 3D structural reconstruction model and morphometrics, we succeeded in constructing a 3D dynamic model of bile duct formation. Because the 3D reconstruction technique used in this study is available for analyzing solid structures in tissues that are difficult to approach, it shows promise for wide use in the fields of biology and medicine.

Regulation of organogenesis and stem cell properties by T-box transcription factors.

T-box transcription factors containing the common DNA-binding domain T-box contribute to the organization of multiple tissues in vertebrates and invertebrates. In mammals, 17 T-box genes are divided into five subfamilies depending on their amino acid homology. The proper distribution and expression of individual T-box transcription factors in different tissues enable regulation of the proliferation and differentiation of tissue-specific stem cells and progenitor cells in a suitable time schedule for tissue organization. Consequently, uncontrollable expressions of T-box genes induce abnormal tissue organization, and eventually cause various diseases with malformation and malfunction of tissues and organs. Furthermore, some T-box transcription factors are essential for maintaining embryonic stem cell pluripotency, improving the quality of induced pluripotent stem cells, and inducing cell-lineage conversion of differentiated cells. These lines of evidence indicate fundamental roles of T-box transcription factors in tissue organization and stem cell properties, and suggest that these transcription factors will be useful for developing therapeutic approaches in regenerative medicine.

Potential marker subset of blood-circulating cytokines on hematopoietic progenitor-to-Th1 pathway in COVID-19.

In this study, we analyzed a relatively large subset of proteins, including 109 kinds of blood-circulating cytokines, and precisely described a cytokine storm in the expression level and the range of fluctuations during hospitalization for COVID-19. Of the proteins analyzed in COVID-19, approximately 70% were detected with Bonferroni-corrected significant differences in comparison with disease severity, clinical outcome, long-term hospitalization, and disease progression and recovery. Specifically, IP-10, sTNF-R1, sTNF-R2, sCD30, sCD163, HGF, SCYB16, IL-16, MIG, SDF-1, and fractalkine were found to be major components of the COVID-19 cytokine storm. Moreover, the 11 cytokines (i.e., SDF-1, SCYB16, sCD30, IL-11, IL-18, IL-8, IFN-γ, TNF-α, sTNF-R2, M-CSF, and I-309) were associated with the infection, mortality, disease progression and recovery, and long-term hospitalization. Increased expression of these cytokines could be explained in sequential pathways from hematopoietic progenitor cell differentiation to Th1-derived hyperinflammation in COVID-19, which might also develop a novel strategy for COVID-19 therapy with recombinant interleukins and anti-chemokine drugs.

Immune-related zinc finger gene ZFAT is an essential transcriptional regulator for hematopoietic differentiation in blood islands.

TAL1 plays pivotal roles in vascular and hematopoietic developments through the complex with LMO2 and GATA1. Hemangioblasts, which have a differentiation potential for both endothelial and hematopoietic lineages, arise in the primitive streak and migrate into the yolk sac to form blood islands, where primitive hematopoiesis occurs. ZFAT (a zinc-finger gene in autoimmune thyroid disease susceptibility region/an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook) was originally identified as an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook, and is highly conserved among species. ZFAT is thought to be a critical transcription factor involved in immune-regulation and apoptosis; however, developmental roles for ZFAT remain unknown. Here we show that Zfat-deficient (Zfat(-/-)) mice are embryonic-lethal, with impaired differentiation of hematopoietic progenitor cells in blood islands, where ZFAT is exactly expressed. Expression levels of Tal1, Lmo2, and Gata1 in Zfat(-/-) yolk sacs are much reduced compared with those of wild-type mice, and ChIP-PCR analysis revealed that ZFAT binds promoter regions for these genes in vivo. Furthermore, profound reduction in TAL1, LMO2, and GATA1 protein expressions are observed in Zfat(-/-) blood islands. Taken together, these results suggest that ZFAT is indispensable for mouse embryonic development and functions as a critical transcription factor for primitive hematopoiesis through direct-regulation of Tal1, Lmo2, and Gata1. Elucidation of ZFAT functions in hematopoiesis might lead to a better understanding of transcriptional networks in differentiation and cellular programs of hematopoietic lineage and provide useful information for applied medicine in stem cell therapy.

Identification of independent risk loci for Graves' disease within the MHC in the Japanese population.

To identify genetic variants that confer the risk of Graves' disease (GD) in the Japanese population, we conducted a two-stage genome-wide association study (GWAS) using 1119 Japanese individuals with GD and 2718 unrelated controls, and a subsequent replication study using independent 432 GD cases and 1157 controls. We identified 34 single nucleotide polymorphisms (SNPs) to be significantly associated with GD in the GWAS phase. Twenty-two out of 34 SNPs remained positive in the replication study. All 22 SNPs were located within the major histocompatibility complex (MHC) locus on chromosome 6p21. No strong long-range linkage disequilibrium (LD) was observed among the 22 SNPs, indicating independent involvement of multiple loci within the MHC with the risk of GD. Multivariate stepwise logistic regression analysis selected rs3893464, rs4313034, rs3132613, rs4248154, rs2273017, rs9394159 and rs4713693, as markers for independent risk loci for GD. The analysis of LD between these seven SNPs and tagging SNPs for GD-associated human leukocyte antigen (HLA) alleles in the Japanese population (HLA-DPB1(*)0501 and HLA-A(*)0206) demonstrated that all of and five of seven SNPs were not in strong LD with HLA-DPB1(*)0501 and HLA-A(*)0206, respectively. Although causal variants remain to be identified, our results demonstrate the existence of multiple GD susceptibility loci within the MHC region.

ZFAT is a critical molecule for cell survival in mouse embryonic fibroblasts.

ZFAT was originally identified as an immune-related transcriptional regulator containing 18 C2H2-type zinc-finger domains and one AT-hook. ZFAT is highly conserved among species and functions as an anti-apoptotic molecule in the lymphoblastic leukemia cell line, MOLT-4. We recently demonstrated that ZFAT is an essential molecule for hematopoietic differentiation in blood islands through the direct regulation of particular transcriptional factors, including Tal1, for endothelial cell assembly, and for the branch point formation of capillary-like structures. However, the molecular mechanisms underlying the anti-apoptotic function of ZFAT remain unknown. Here, we report that ZFAT knockdown by small interfering RNA induced apoptosis in mouse embryonic fibroblasts (MEFs). This response had been similarly observed for MOLT-4 cells. To explore the molecular mechanisms for ZFAT in anti-apoptotic function in both MEFs and MOLT-4 cells, microarray expression analysis and quantitative RT-PCR were done. Of interest was that Bcl-2 and Il6st were identified as commonly down-regulated genes by the depletion of ZFAT for both MEFs and MOLT-4 cells. These results suggest that ZFAT is a critical molecule for cell survival in MEFs and MOLT-4 cells at least in part through the regulation of the apoptosis involved in the BCL-2- and IL6st-mediated pathways. Further elucidation of the molecular functions for ZFAT might shed light on the cellular programs in the mesoderm-derived cells.

Survival prediction based on the gene expression associated with cancer morphology and microenvironment in primary central nervous system lymphoma.

Dysregulation of cell morphology and cell-cell interaction results in cancer cell growth, migration, invasion, and metastasis. Besides, a balance between the extracellular matrix (ECM) and matrix metalloprotease (MMP) is required for cancer cell morphology and angiogenesis. Here, we determined gene signatures associated with the morphology and microenvironment of primary central nervous system lymphoma (PCNSL) to enable prognosis prediction. Next-generation sequencing (NGS) on 31 PCNSL samples revealed gene signatures as follows: ACTA2, ACTR10, CAPG, CORO1C, KRT17, and PALLD in cytoskeleton, CDH5, CLSTN1, ITGA10, ITGAX, ITGB7, ITGA8, FAT4, ITGAE, CDH10, ITGAM, ITGB6, and CDH18 in adhesion, COL8A2, FBN1, LAMB3, and LAMA2 in ECM, ADAM22, ADAM28, MMP11, and MMP24 in MMP. Prognosis prediction formulas with the gene expression values and the Cox regression model clearly divided survival curves of the subgroups in each status. Furthermore, collagen genes contributed to gene network formation in glasso, suggesting that the ECM balance controls the PCNSL microenvironment. Finally, the comprehensive balance of morphology and microenvironment enabled prognosis prediction by a combinatorial expression of 8 representative genes, including KRT17, CDH10, CDH18, COL8A2, ADAM22, ADAM28, MMP11, and MMP24. Besides, these genes could also diagnose PCNSL cell types with MTX resistances in vitro. These results would not only facilitate the understanding of biology of PCNSL but also consider targeting pathways for anti-cancer treatment in personalized precision medicine in PCNSL.

ZFAT is essential for endothelial cell assembly and the branch point formation of capillary-like structures in an angiogenesis model.

ZFAT, originally identified as a susceptibility gene for autoimmune thyroid disease, encodes a transcriptional regulator with one AT-hook and 18 C(2)H(2)-type zinc-finger domains. It is highly conserved among species. Here, we demonstrate that ZFAT is clearly expressed in human umbilical vein endothelial cells (HUVECs). Furthermore, we show that endothelial cell assembly and the branch point formation of capillary-like structures in HUVECs is impaired by the reduction of ZFAT expression through the use of ZFAT-miRNAs, whereas differences in cell proliferation or apoptotic features were not observed after the reduction in ZFAT expression. These results suggest that ZFAT may have critical roles in the capillary-like network formation that is involved in vascular remodeling. Elucidating the ZFAT-mediated transcriptional network will lead to a better understanding of the molecular mechanisms of angiogenesis.

Metabolome Analysis Reveals Excessive Glycolysis via PI3K/AKT/mTOR and RAS/MAPK Signaling in Methotrexate-Resistant Primary CNS Lymphoma-Derived Cells.

PURPOSE: Metabolome analysis is an emerging method that provides insight into intracellular and physiologic responses. Methotrexate (MTX) is an antifolate that suppresses DNA syntheses by inhibiting dihydrofolate reductase. High-dose methotrexate treatment with deferred radiotherapy is a standard protocol in primary central nervous system lymphoma (PCNSL) treatments. However, most cases come to relapse-acquired resistance, in which the role of metabolic pathways is largely unknown. EXPERIMENTAL DESIGN: Metabolome analysis in methotrexate-resistant PCNSL-derived cells (designated as TK-MTX and HKBML-MTX) was performed to detect alternative metabolites and pathways. RESULTS: The metabolomic analyses using capillary electrophoresis-time-of-flight mass spectrometry detected 188 and 169 peaks in TK- and HKBML-derived cells, respectively, including suppression of central carbon metabolism, lipid metabolism, nucleic acid metabolism, urea cycle, branched chain and aromatic amino acids, and coenzyme metabolism. Particularly, whole suppressive metabolic pathways were demonstrated in TK-MTX, whereas HKBML-MTX indicated partially enhanced pathways of the urea cycle, amino acid metabolism, and coenzyme metabolism. Reciprocally detected metabolites for glycolysis, including induced glucose and reduced glycogen, and induced lactate and reduced pyruvate, in addition to increased lactate dehydrogenase activity, which is involved in Warburg effect. Thereby, ATP was increased in both methotrexate-resistant PCNSL-derived cells. Furthermore, we specifically found that PI3K/AKT/mTOR and RAS/MAPK signaling pathways were activated in TK-MTX but not in HKBML-MTX by growth rate with inhibitors and gene expression analysis, suggestive of cell type-specific methotrexate-resistant metabolic pathways. CONCLUSIONS: These results can help us understand targeted therapies with selective anticancer drugs in recurrent CNS lymphoma-acquired resistance against methotrexate.

GSEA-assisted gene signatures valid for combinations of prognostic markers in PCNSL.

Primary central nervous system lymphoma (PCNSL) is a brain malignant non-Hodgkin's B-cell lymphoma. The standard treatments are high-dose methotrexate (MTX)-based chemotherapies and deferred whole brain radiotherapy. However, MTX resistance-dependent global expression and signaling pathway changes and their relationship with prognoses have not yet been elucidated. Here, we conducted a global expression analysis with next-generation sequencing and gene set enrichment analysis (GSEA) in MTX-resistant PCNSL cell lines (HKBML-MTX and TK-MTX) and PCNSL tissues. In rank scores, genes listed in HKBML-MTX and TK-MTX were enriched in PCNSL with poor prognoses. In fold changes, a part of differentially-expressed genes in PCNSL tissues were also detected in HKBML-MTX and TK-MTX cells; FOXD2-AS1 and MMP19 were commonly expressed in both HKBML-MTX and TK-MTX, FABP5 and CD70 were HKBML-MTX-specifically expressed, and CLCN2, HOXB9, INE1, and LRP5L were TK-MTX-specifically expressed, which may provide a combination of prognostic markers on MTX-sensitivities in PCNSL. Additionally, PCNSL subgroups, divided with hierarchical clustering and Kaplan-Meier methods, included twenty commonly expressed genes in both HKBML-MTX and TK-MTX, ten HKBML-MTX-specifically expressed genes, and two TK-MTX-specifically expressed genes. These results suggest that the GSEA-assisted gene signatures can provide a combination for prognostic markers in recurrent PCNSL with MTX resistances.

miR-101, miR-548b, miR-554, and miR-1202 are reliable prognosis predictors of the miRNAs associated with cancer immunity in primary central nervous system lymphoma.

MicroRNAs (miRNAs) inhibit protein function by silencing the translation of target mRNAs. However, in primary central nervous system lymphoma (PCNSL), the expression and functions of miRNAs are inadequately known. Here, we examined the expression of 847 miRNAs in 40 PCNSL patients with a microarray and investigated for the miRNA predictors associated with cancer immunity-related genes such as T helper cell type 1/2 (Th-1/Th-2) and regulatory T cell (T-reg) status, and stimulatory and inhibitory checkpoint genes, for prognosis prediction in PCNSL. The aim of this study is to find promising prognosis markers based on the miRNA expression in PCNSL. We detected 334 miRNAs related to 66 cancer immunity-related genes in the microarray profiling. Variable importance measured by the random survival forest analysis and Cox proportional hazards regression model elucidated that 11 miRNAs successfully constitute the survival formulae dividing the Kaplan-Meier curve of the respective PCNSL subgroups. On the other hand, univariate analysis shortlisted 23 miRNAs for overall survival times, with four miRNAs clearly dividing the survival curves-miR-101/548b/554/1202. These miRNAs regulated Th-1/Th-2 status, T-reg cell status, and immune checkpoints. The miRNAs were also associated with gene ontology terms as Ras/MAP-kinase, ubiquitin ligase, PRC2 and acetylation, CDK, and phosphorylation, and several diseases including acquired immunodeficiency syndrome, glioma, and those related to blood and hippocampus with statistical significance. In conclusion, the results demonstrated that the four miRNAs comprising miR-101/548b/554/1202 associated with cancer immunity can be a useful prognostic marker in PCNSL and would help us understand target pathways for PCNSL treatments.

The increased expression of periostin during early stages of prostate cancer and advanced stages of cancer stroma.

BACKGROUND: Three-dimensional culture (3DC) is a relevant in vitro model used to study prostate development and carcinogenesis. Recent studies have indicated that 3DC-associated genes would be more sensitive as prognostic markers for cancer; however, no 3DC-associated genes in prostate cancer (CaP) have thus far been elucidated. METHODS: Candidate 3DC-associated genes in non-malignant prostatic epithelial cells, RWPE-1 and TA2, were selected, based on a comparison of microarray gene expression data between cells grown in two-dimensional culture (2DC) and in 3DC. To extract CaP-associated genes among the 3DC-associated genes, gene expression levels from the microdissected tissue samples were compared between 20 well-differentiated or 20 poorly differentiated CaP, as well as matched normal prostate epithelium. The expression levels of CaP-associated genes in 3DC were validated by quantitative RT-PCR using TA2 cells in 2DC and 3DC. Protein expression of periostin (POSTN), which is one of the 3DC CaP-associated genes, was further evaluated in the clinical samples by immunohistochemistry. RESULTS: Several genes including POSTN were identified as CaP-associated genes using a 3DC system. Immunohistochemical analyses revealed that POSTN expression was increased in the early stages of CaP (Gleason score 6-7), but not in the advanced stages of CaP. Furthermore, the positive ratio observed for the expression of POSTN in tumor-associated stroma was significantly correlated with the degree of malignancy. CONCLUSIONS: POSTN, one of the 3DC-associated genes, may serve as a potential biomarker for predicting the pathological grade and prognosis of CaP. Prostate 69: 1398-1403, 2009. (c) 2009 Wiley-Liss, Inc.

ZFAT expression in B and T lymphocytes and identification of ZFAT-regulated genes.

The human ZFAT gene encodes a 1243-amino-acid protein containing one AT hook and 18 C2H2 zinc finger domains, which are highly conserved among ZFAT orthologues from fish to mammalian species. Consistent with the presence of multiple predicted nuclear localization signals, endogenous ZFAT protein was found to be localized to the nucleus. In the mouse tissues examined by Western blotting, ZFAT was found to be expressed in thymus, spleen, and lymph nodes, but not in other tissues, including bone marrow. Furthermore, ZFAT protein was found to be up-regulated during the transition from CD4(-)CD8(-) to CD4(+)CD8(+) thymocytes and to be expressed only in B and T lymphocytes in peripheral lymphoid tissues. Expression array analyses demonstrated that genes that are down-regulated upon ZFAT overexpression in mouse Ba/F3 cells are significantly enriched for those functionally related to immune responses. These results suggest that ZFAT functions as a critical transcriptional regulator in B and T lymphocytes.