The cis-Regulatory Atlas of the Mouse Immune System.

A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.

A microRNA expression and regulatory element activity atlas of the mouse immune system.

To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system.

Parsing the Interferon Transcriptional Network and Its Disease Associations.

Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical "interferon signature genes" that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry.

The transcriptional regulator Aire binds to and activates super-enhancers.

Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. We found that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized to and activated long chromatin stretches that were overloaded with transcriptional regulators, known as super-enhancers. We also identified topoisomerase 1 as a cardinal Aire partner that colocalized on super-enhancers and was required for the interaction of Aire with all of its other associates. We propose a model that entails looping of super-enhancers to efficiently deliver Aire-containing complexes to local and distal transcriptional start sites.

Aire Controls Heterogeneity of Medullary Thymic Epithelial Cells for the Expression of Self-Antigens.

The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire's primary targets while distinguishing them from the secondary targets.

Dissecting the Immune System through Gene Regulation.

The immune system plays a dual role in human health, functioning both as a protector against pathogens and, at times, as a contributor to disease. This feature emphasizes the importance to uncover the underlying causes of its malfunctions, necessitating an in-depth analysis in both pathological and physiological conditions to better understand the immune system and immune disorders. Recent advances in scientific technology have enabled extensive investigations into gene regulation, a crucial mechanism governing cellular functionality. Studying gene regulatory mechanisms within the immune system is a promising avenue for enhancing our understanding of immune cells and the immune system as a whole. The gene regulatory mechanisms, revealed through various methodologies, and their implications in the field of immunology are discussed in this chapter.

Quantification of Casein in Baked Food Products by Selective Analysis of Phosphorylated Peptides Using Fluorous Derivatization with Liquid Chromatography-Tandem Mass Spectrometry Method.

Casein is one of the allergen proteins present in milk. Therefore, a quantification method for the selective analysis of casein using fluorous derivatization with LC-tandem mass spectrometry (LC-MS/MS) was developed. After two allergen proteins (αS1-casein and ß-casein) extracted from baked sugar cookies were tryptic digested, the obtained phosphorylated peptides were selectively derivatized by ß-elimination with Ba(NO3)2 under basic condition and Michael addition with perfluoroalkylthiol (1H,1H,2H,2H-perfluorooctanethiol, PFOT). In this study, YKVPQLEIVPN(pSer)AQQR (104-119 fragment from αS1-casein) and FQ(pSer)EEQQQTEDELQDK (33-48 fragment from ß-casein) obtained by tryptic digestion were selected as target peptides. The phosphorylated serine residue in each peptide was converted to a perfluoroalkyl group by derivatization. The obtained fluorous-derivatized peptides were analyzed by LC-MS/MS, to which a fluorous LC column was connected. Therefore, it was possible to analyze casein without being affected by the matrix components in the baked food sample. When the present method was applied to cookies with arbitrary amounts of αS1-casein and ß-casein, the obtained quantification values were in good agreement with the arbitrary amounts spiked. The quantification limits of αS1- and ß-casein in cookie analysis were 246 and 152 ng/g, respectively. Hence, this method can be used to analyze trace amounts of allergen proteins present in the baked food.

Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis.

Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested ß-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer)EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via ß-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to ß-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics.

The discovery of novel 3-aryl-indazole derivatives as peripherally restricted pan-Trk inhibitors for the treatment of pain.

The design, synthesis, and biological evaluation of novel 3-aryl-indazole derivatives as peripherally selective pan-Trk inhibitors are described. Three strategies were used to obtain a potent compound exhibiting low central nervous system (CNS) penetration and high plasma exposure: 1) a structure-based drug design (SBDD) approach was used to improve potency; 2) a substrate for an efflux transporter for lowering brain penetration was explored; and 3) the most basic pKa (pKa-MB) value was used as an indicator to identify compounds with good membrane permeability. This enabled the identification of the peripherally targeted 17c with the potency, kinase-selectivity, and plasma exposure required to demonstrate in vivo efficacy in a Complete Freund's adjuvant (CFA)-induced thermal hypersensitivity model.

Network pharmacology of JAK inhibitors.

Small-molecule inhibitors of the Janus kinase family (JAKis) are clinically efficacious in multiple autoimmune diseases, albeit with increased risk of certain infections. Their precise mechanism of action is unclear, with JAKs being signaling hubs for several cytokines. We assessed the in vivo impact of pan- and isoform-specific JAKi in mice by immunologic and genomic profiling. Effects were broad across the immunogenomic network, with overlap between inhibitors. Natural killer (NK) cell and macrophage homeostasis were most immediately perturbed, with network-level analysis revealing a rewiring of coregulated modules of NK cell transcripts. The repression of IFN signature genes after repeated JAKi treatment continued even after drug clearance, with persistent changes in chromatin accessibility and phospho-STAT responsiveness to IFN. Thus, clinical use and future development of JAKi might need to balance effects on immunological networks, rather than expect that JAKis affect a particular cytokine response and be cued to long-lasting epigenomic modifications rather than by short-term pharmacokinetics.

Assessment of Anticancer Drug Effects on Pancreatic Cancer Cells under Glucose-Depleted Conditions Using Intracellular and Extracellular Amino Acid Metabolomics.

Previously, we developed a method to evaluate states of cells treated with anticancer drugs via the comprehensive analysis of amino acids, termed amino acid metabolomics. In the present study, we evaluated the effects of the anticancer drugs, gemcitabine hydrochloride and pyrvinium pamoate, on the proliferation of a pancreatic cancer cell line (PANC-1) under hypoglycemic conditions using amino acid metabolomics. Intracellular and extracellular amino acid profiles of PANC-1 were determined by hydrophilic interaction chromatography-tandem mass spectrometry with simple pretreatment. Changes to the drugs' anticancer effects resulting from glucose starvation conditions were presented in score plots obtained from principal component analyses. In particular, the analysis of intracellular amino acids was found to be the superior approach because the results allowed a clearer assessment of the cell state. Further, orthogonal partial least squares discriminant analysis was performed to search for amino acid candidates that discriminate with anticancer drug-treated PANC-1 cells. We identified several amino acids that might be able to distinguish the drug-treated group from the control group. These results might provide a better understanding of the mechanisms underlying cell responses such as drug resistance or austerity. The present study is the first to evaluate the efficacy of anticancer drugs under glucose starvation based on the analysis of the variation of extracellular and intracellular amino acid profiles in vitro.

Brd4 bridges the transcriptional regulators, Aire and P-TEFb, to promote elongation of peripheral-tissue antigen transcripts in thymic stromal cells.

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.

Risk factors for Propionibacterium acnes infection after neurosurgery: A case-control study.

Propionibacterium acnes is increasingly known as a causative organism for post-neurosurgical infection; however, no clinical studies have examined the risk factors associated with P. acnes infections. Clinical data obtained from 14 cases of P. acnes infection and 28 controls infected with other pathogens were analyzed. Craniotomy, malignancy, and prolonged duration of operation were significantly associated with the onset of P. acnes infection. No fatal cases were reported.

Latent TGF-ß binding protein-2 is essential for the development of ciliary zonule microfibrils.

Latent TGF-ß-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

A novel JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl(2,6-dichlorophenyl)acetate, suppresses helper T cell differentiation in vitro and collagen-induced arthritis in vivo.

Th17 cells, which have been implicated in autoimmune diseases including rheumatoid arthritis (RA), require the JAK-STAT3 pathway for their differentiation and functions. Recently, JAK inhibitors have been developed as a therapeutic drug for RA. However, the current JAK inhibitors are not optimized to STAT3 compared with other STATs. In this study, we found a new lead compound of a small molecule JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl (2,6-dichlorophenyl)acetate, which inhibits STAT3 as efficiently as other STATs. This compound, named JI069, was selected by STAT3 reporter assay in combination with an in silico docking model. JI069 inhibited gp130 signaling by inducing dissociation between gp130 and JAK1. In HEK293T cells and primary T cells, JI069 suppressed STAT3 activation as efficiently as other STATs, including STAT1, STAT5, and STAT6. JI069 effectively suppressed Th1, Th2, and Th17 differentiation while strongly promoted iTreg differentiation. JI069 suppressed symptoms of the collagen-induced arthritis (CIA) model in mice, and inhibited the cytokine production from T cells as well as the STAT3 phosphorylation of synovial cells. These data suggest that JI069 is a new type of JAK inhibitor which has potential for the treatment of immunological disorders.

Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells.

Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

[Stent-Assisted Coil Embolization of a Dissecting Aneurysm of the Posterior Cerebral Artery: A Case Report].

Dissecting aneurysms of the posterior cerebral artery (PCA) are rare, especially those at the P1 segment. Here, we describe the case of a 57-year-old woman with a subarachnoid hemorrhage (SAH). Computed tomography angiography (CTA) and digital subtraction angiography (DSA) revealed a small (3 mm) dissecting aneurysm with the typical pearl-and-string sign at the right P1 segment. Fourteen days after onset, the patient developed aphasia. DSA revealed vasospasm of the right middle cerebral artery, and we performed endovascular treatment by the intra-arterial injection of 1-(5-isoquinolinesulfonyl) homopiperazine. After this treatment, the patient's symptoms recovered immediately. Vertebral angiography revealed enlargement of the dissecting aneurysm (up to 7 mm diameter). We started a loading dose of 300 mg aspirin and 400 mg clopidogrel after observing growth of the aneurysm. Fifteen days after onset, we performed a stent-assisted coil embolization, and obtained nearly complete obliteration of the aneurysm with preserved patency of the parent artery. Six-month follow-up DSA demonstrated complete occlusion of the aneurysm with good patency of the stented PCA; the patient was at modified Rankin Scale 1. In the treatment of ruptured dissecting aneurysms, parent vessel occlusion (PVO) with aneurysm is common. However, PVO may cause both cerebral infarction of the distal area and perforator occlusion of the occluded vessel. Stent-assisted coil embolization can preserve parent vessel flow and obliterate the aneurysm. Stents offer a therapeutic alternative for PCA dissecting aneurysms, especially when PVO cannot be tolerated.

[Methods of preventing phlebitis induced by infusion of fosaprepitant].

At our hospital, we use aprepitant for nausea and vomiting when administering highly emetic anticancer agents, according to "Guidelines for the Appropriate Use of Antiemetic Agents" given by the Japan Society of Clinical Oncology. We initiated the intravenous administration of fosaprepitant for better compliance compared with aprepitant; however, we observed phlebitis after the infusion of fosaprepitant. Therefore, we investigated measures to reduce phlebitis associated with the infusion of fosaprepitant. For the first premedication, fosaprepitant (150 mg) was dissolved in 100 mL of saline and administered for 30 minutes; 1 of 2 patients showed grade 4 phlebitis. For the modified premedication, fosaprepitant, dexamethasone, and 5- HT(3) antagonist were dissolved in 100 mL of saline and administered for 30 minutes. The modified premedication was administered to a total of 27 patients; 5 patients developed mild phlebitis (grade 1), but infusion could be continued by treating their phlebitis with a hot pack. We used a combination of dexamethasone and 5-HT(3) antagonist with fosaprepitant as a modified premedication in order to avoid drug-induced vascular damage, which resulted in the pH decreasing to 6.20-7.55 (close to neutral) and a shorter infusion time.

Liquid chromatography/tandem mass spectrometry with fluorous derivatization method for selective analysis of sialyl oligosaccharides.

RATIONALE: A separation-oriented derivatization method using a specific fluorous affinity between perfluoroalkyl-containing compounds was applied to selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of sialyl oligosaccharides. The perfluoroalkyl-labeled sialyl oligosaccharides could be selectively retained on an LC column with the perfluoroalkyl-modified stationary phase and effectively distinguished from non-derivatized species. METHODS: Sialyl oligosaccharides (3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, and disialyllacto-N-tetraose) were derivatized with 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecylamine via amidation in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (condensation reagent). The obtained derivatives were directly injected onto the fluorous LC column without any pretreatments and then detected by positive electrospray ionization MS/MS. RESULTS: The method enabled accurate determination of the sialyl oligosaccharides in biological samples such as human urine and human milk, because there was no interference with matrix-induced effects during LC/MS/MS analysis. The limits of detection of the examined sialyl oligosaccharides, defined as signal-to-noise (S/N) = 3, were in the range 0.033-0.13 nM. Accuracy in the range 95.6-108% was achieved, and the precision (relative standard deviation) was within 9.4%. CONCLUSIONS: This method enabled highly selective and sensitive analysis of sialyl oligosaccharides, enabling accurate measurement of even their trace amounts in biological matrices. The proposed method may prove to be a powerful tool for the analysis of various sialyl oligosaccharides.

[A case of intracranial primary leptomeningeal lymphoma].

Primary leptomeningeal lymphoma(PLML)is a neoplastic meningitis of lymphomatous origin without parenchymal central nervous system(CNS)disease or a systemic tumor. We report a case of PLML that presented with epileptic seizure, and review relevant literature. A 27-year-old man was brought to the emergency department with an epileptic seizure. Two months later, he was again brought to the emergency department with an epileptic seizure. MRI showed enhanced lesions on the surface of the right cerebellar hemisphere, right parietal sulci, and interhemispheric surface of the frontal lobes. We performed an open biopsy and diagnosed the patient with diffuse large B-cell lymphoma of the leptomeninges on the basis of histological findings. The patient was initially treated with chemotherapy including high-dose methotrexate(MTX). Because remission was not achieved by chemotherapy, the patient was treated with whole-brain radiation therapy. After onset, the patient survived for 2 years without recurrence. PLML is a particularly rare type of primary CNS lymphoma. The outcome of PLML, compared with general primary CNS lymphoma, is reported to be very poor because chemotherapy including MTX is ineffective.