[Background] It is recently reported that microRNAs (miRNAs) are stably present in serum and potentially useful in the diagnosis and evaluation of treatment of cancer. [Materials and Methods] Serum samples of breast cancer before treatment (n=1280) between 2008 and 2014 were obtained from National Cancer Center Hospital and controls (n=3348) were obtained from collaborative institutes. Additionally, the serum sample of patients who received neoadjuvant chemotherapy (NAC) and surgery between last chemotherapy administration and surgery were collected. A comprehensive quantitative expression analysis of miRNA was performed using the by DNA chip “3D-Gene® (Toray Industries Inc.)” Clinicopathological data was retrieved from medical records. Pathological complete response (pCR) was defined as the absence of residual invasive and in situ cancer of the resected breast specimen and all sampled regional lymph nodes. [Results] Serum samples before treatment of breast cancer patients (n=74), non-cancer controls and patients with other cancers were used (n=2007) in a training set. The rest except for samples after NAC were used in a test set. The formula with the combination of five miRNAs (miR-1246, miR-1307-3p, miR-4634, miR-6861-5p, and miR-6875-5p) was found to be able to detect breast cancer (BCmiR set). BCmiR set had a sensitivity of 97.3%, specificity of 82.9% and accuracy of 89.7% in the test cohort. BCmiR set could detect breast cancer in the non-invasive stage (sensitivity of 98.0% for Tis). In the breast cancer patients, 91 patients received NAC and surgery. Median age of NAC patients was 49 years (range 28-77). Forty-two patients were hormone receptor-positive (HR+) and HER2-negative (HER2-), 24 were HR+ and HER2-positive (HER2+), 11 were hormone receptor-negative (HR-) and HER2+ and 14 were HR- and HER2-. pCR was observed in 19 (20.9%) of NAC patients. pCR in each subtypes were 3 (7.7%) in HR+ and HER2-, 6 (33.3%) in HR+ and HER2+, 4 (57.1%) in HR- and HER2+ and 3 (27.3%) in HR- and HER2-. Serum after NAC were obtained from 19 pCR patients and 71 non-pCR patients. When we applied BCmiR set to the serum samples after NAC, the average diagnostic index (cut-off value=0) in pCR patients was significantly lower than that in non-pCR patients (pCR, -0.30±0.84; non-pCR, 0.31±1.15; p=0.03). In fact, 57.9% of pCR patients were classified into non-breast cancer. However, 40.8% of non-pCR patients were misclassified into non-breast cancer. [Conclusion] The combination of five miRNAs (BCmiR set) measured from serum can be used to detect breast cancer. BCmiR set has potential to predict pCR in patients who received NAC. The further analysis to predict pCR is underway and further results will be presented in the symposium. Citation Format: Shimomura A, Shiino S, Kawauchi J, Takizawa S, Sakamoto H, Shimizu C, Takeshita F, Niida S, Kinoshita T, Tamura K, Ochiya T. Detecting early breast cancer by the combination of five serum microRNAs and its possibility of prediction of pathological complete response in neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-04.

Results: Of 69 plasma cfDNA samples collected from patients (colorectal cancer, 28; NSCLC, 18, breast cancer, 12; melanoma, 11) at disease progression, methylation scores were consistent with the presence of cancer in 84% (58/69); while in 79% (46/58) of plasma cfDNA samples, methylation scores accurately classified the underlying cancer type. High methylation scores in plasma cfDNA samples collected at disease progression compared to low methylation scores were associated with shorter survival irrespective of the tumor type (3.9 vs 10.4 months, P< 0.001, confirmed on multivariable analysis). In addition, high methylation scores in plasma cfDNA samples collected at progression vs low methylation scores were associated with shorter time to treatment failure (TTF) on systemic therapy (1.6 vs. 2.8 months, P1⁄4 0.007). There was a positive correlation between RECIST response (%) to systemic therapy and methylation scores (r 0.32, P1⁄4 0.03). Methylations score before therapy (median 87.28) were higher than methylation scores on therapy (median 9.49, P1⁄4 0.001).

Background: Recent studies have reported that serum microRNAs (miRNAs) are potentially useful biomarkers for diagnosis and treatment of cancer. However, its utility for detecting esophageal squamous cell carcinoma (ESCC) has not been investigated yet. The aims of this study are to identify circulating serum miRNAs for ESCC detection. Methods: We comprehensively analyzed serum miRNA expression profiles of 595 patients with ESCC, and 5051 non-cancer individuals using microarray (3D-Gene, Toray). Serum of non-cancer individuals was obtained from Biobank of NCGG and Health check up clinic (Yokohama Minoru clinic). Serum of ESCC patients was collected before starting any treatment such as radiotherapy, surgery and chemotherapy. Analyzed samples were randomly divided into discovery and validation sets. Serum miRNA levels were compared between ESCC and non-ESCC patients. Fisher's linear discriminant analysis was performed to construct the discriminant model for ESCC detection. Measured values of each miRNAs were extrapolated into the discriminant formulas. We performed ROC analysis to evaluate the diagnostic ability of these formulas in each validation cohort. Results: In discovery set, 300 patients of ESCC was compared to 300 individuals of non-cancer control. We picked up 3 miRNAs, named miR-A, miR-B and miR-C, for ESCC diagnosis. Their AUC for detection of ESCC was 0.967, 0.873 and 0.650, respectively in ROC analysis. We constructed the discriminant model, called EC index, using these 3 miRNAs in discovery set. In validation set which contain 295 pts of ESCC and 4751 person of non-cancer control, EC index showed the AUC, sensitivity and specificity of the discriminant formula was 0.99, 0.98 and 0.95, respectively. Conclusions: We identified novel serum miRNAs for ESCC detection. Our discriminant using these miRNAs can diagnose ESCC. Clinical trial identification: NCCH2016-249 Legal entity responsible for the study: National Cancer Center Funding: Japan Agency for Medical Research and Development Disclosure: K. Kato: Research fund: ONO, MSD, Shionogi. J. Kawauchi, S. Takizawa: Employee of Tray company. T. Ochiya: Grant/Research funding from: Kyowa Medex, Kewpie Corporation, Takeda, Rohto Pharmaceutical Co., Ltd., Japan Atherosclerosis Research Foundation, Inter Stem, BioMimetics Sympathies. All other authors have declared no conflicts of interest.

T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c+ cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c+ cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c+ cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.

The innate immune system plays an important role systemically and locally in infectious and inflammatory diseases. Vaccines, vaccine adjuvants and anti-inflammatory drugs were developed by understanding mechanisms of the innate immune system and causative factors of infection and inflammatory diseases. Pattern-recognition receptors, such as Toll-like receptors, retinoic acid-inducible gene I (RIG-I)-like helicases and nucleotide-binding oligomerization domain(NOD)-like receptors, and their downstream signals have great potential as targets of therapeutics because they are involved in numerous diseases. Furthermore, proteolytic systems such as autophagy and immunoproteasomes play important roles in the innate immune system, making them potential therapeutic targets also. By taking advantage of the immune system, humankind has made a great effort to develop new therapeutic and preventive medicines. Accordingly, we have reported several studies on the development of vaccines and adjuvants based on novel mechanistic strategies. Additionally, we have elucidated the mechanism underlying an interaction between innate immunity and the endocrine system. This review introduces the possible use of innate immune molecules for the development of immunomodulatory drugs and the involvement of the immune system in endocrine metabolic diseases to discuss future applications of innate immune molecules to therapeutics of various inflammatory diseases.

receptor or its genetic deletion profoundly reduced innate NK cell activation and DC maturation with poly IC. For example the DC failed to acquire any immunstimulatory activity. In addition, while gag-specific CD4+ T cell responses were totally dependent upon type I interferon, they were independent on IFN-g and IL-12. After poly IC stimulation, IFNb was produced by DEC-205+ DCs but also monocytes and stromal cells. Type I IFN induction was highly dependent on MDA5 helicase in these cell types, but DEC+DCs also used TLR3. Surprisingly, bone marrow chimera experiments indicated that nonhematopoietic cells were the main source of type I IFN, but both bone marrow derived and nonhematopoietic cells contributed significantly to the adaptive CD4+ Th1 response, These experiments provide a means to establish the consequences of pattern recognition and cytokine production on DCs in vivo, and demonstrate a critical need for type I interferon to link innate with adaptive immunity during the strong adjuvant role of poly IC and a clinically feasible protein vaccine.

Correction to: Cancer Gene Therapy advance online publication 26 June 2009; doi:10.1038/cgt.2009.43 In this article, Figure 3 was missing the labels for the x-axis and should have appeared as shown below.

Mycobacterium leprae is an intracellular pathogen that survives within the phagosome of host macrophages. Several host factors are involved in producing tolerance, while others are responsible for killing the mycobacterium. Tryptophan aspartate-containing coat protein (TACO; also known as CORO1A or coronin-1) inhibits the phagosome maturation that allows intracellular parasitization. In addition, the Toll-like receptor (TLR) activates the innate immune response. Both CORO1A and TLR-2 co-localize on the phagosomal membrane in the dermal lesions of patients with lepromatous leprosy. Therefore, we hypothesized that CORO1A and TLR-2 might interact functionally. This hypothesis was tested by investigating the effect of CORO1A in TLR-2-mediated signalling and, inversely, the effect of TLR-2-mediated signalling on CORO1A expression. We found that CORO1A suppresses TLR-mediated signal activation in human macrophages, and that TLR2-mediated activation of the innate immune response resulted in suppression of CORO1A expression. However, M. leprae infection inhibited the TLR-2-mediated CORO1A suppression and nuclear factor-kappaB activation. These results suggest that the balance between TLR-2-mediated signalling and CORO1A expression will be key in determining the fate of M. leprae following infection.

MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, noncoding small RNAs that act as post-transcriptional gene regulators. Experimental evidence has shown that miRNAs can play roles as oncogenes or tumor suppressor genes, suggesting their contribution to cancer development and progression. Expression profiles of human miRNAs demonstrated that many miRNAs are deregulated in cancers and are differentially expressed in normal tissues and cancers. Therefore, miRNA profiling is used to create signatures for a variety of cancers, indicating that the profile will help further establish molecular diagnosis, prognosis and therapy using miRNAs. This paper introduces the aberrant expression of miRNAs in human cancer, and discusses the potential of these miRNAs as biomarkers and targets/molecules for molecular therapy.

It appears that the adult pancreas has limited regenerative ability following beta cell destruction by streptozotocin (STZ). However, it is not clear if this limitation is due to an inability to respond to, rather than an absence of, regenerative stimuli. In this study we aimed to uncouple the regenerative signal from the regenerative response by using an exogenous stem cell source to detect regenerative stimuli produced by the STZ-injured pancreas at physiological blood glucose levels.

The innate immune system recognizes nucleic acids during infection or tissue damage; however, the mechanisms of intracellular recognition of DNA have not been fully elucidated. Here we show that intracellular administration of double-stranded B-form DNA (B-DNA) triggered antiviral responses including production of type I interferons and chemokines independently of Toll-like receptors or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding interferon-beta through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-kappaB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of interferon-beta and NF-kappaB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders.

Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. This study examines the safety and immunogenicity of a replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) in BALB/c mice and rhesus monkeys. This novel Ad5/35 vector showed minimal hepatotoxicity after intramuscular administration with the novel Ad5/35 vector. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use.

Under conventional conditions, NC/Nga mice spontaneously develop an atopic dermatitis (AD)-like skin lesion accompanied by immunoglobulin E (IgE) hyperproduction and the expression of T helper 2 (Th2) cytokines. CpG DNA activates a strong interferon-gamma (IFN-gamma)-dominated T helper 1 (Th1) response, while inhibiting Th2-dependent allergies. In this study, we examined whether CpG oligodeoxynucleotide (ODN) could prevent the development of the skin lesions in NC/Nga mice. Sixteen of 26 NC/Nga mice did not exhibit dermatitis after CpG ODN was administered intraperitoneally every 2 wk for a total of five times. CpG ODN administration induced IFN-gamma production, which inhibited the production of Th2 cytokines (interleukin (IL)-4, IL-5, and IL-13) in both spleen and lymph node cells and culminated in a decrease in the serum IgE level. These data suggest that the CpG ODN has a therapeutic effect against AD; however, some mice (10 of 26) treated with CpG ODN exhibited an exacerbation of dermatitis accompanied by the hyperproduction of IFN-gamma, although Th2 cytokines were suppressed. These results suggest that the suppression of Th2 cytokines may not completely prevent dermatitis and that IFN-gamma may play a role in developing dermatitis in some NC/Nga mice.

Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-alpha in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-alpha production was abolished in Irak-1-deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-kappaB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1-deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-alpha induction in pDCs.

Synthetic immunostimulatory nucleic acids such as CpG DNA are being harnessed therapeutically as vaccine adjuvants, anticancer or antiallergic agents. Efforts to identify nucleic acid-based agents capable of more specifically modulating the immune system are being developed. The current study identifies a novel class of single-stranded oligoribonucleotides (ORN) containing unmethylated CpG motifs and a poly(G) run at the 3' end (CpG ORN) that directly stimulate human CD14+CD11c+ monocytes but not dendritic cells or B cells. CpG ORN activate NF-kappaB and p38 MAPK, resulting in IL-6 and IL-12 production and costimulatory molecule up-regulation but not IFNalpha. Methylation of cytosine at the 5' portion in core CpG motif abrogates such activation. TLR3, 7, 8, or 9 alone did not confer response to CpG ORN, in contrast to previously reported respective nucleic acid ligands. These data suggest that CpG ORN represent a novel class of synthetic immunostimulatory nucleic acids with distinct target cells, receptors, and functions from that of previously known immunomodulatory nucleic acids.