Graft-versus-host disease (GVHD) remains the major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is caused by donor T cells that mediate host tissue injury through multiple inflammatory mechanisms. Blockade of individual effector molecules has limited efficacy in controlling GVHD. Here, we report that Notch signaling is a potent regulator of T-cell activation, differentiation, and function during acute GVHD. Inhibition of canonical Notch signaling in donor T cells markedly reduced GVHD severity and mortality in mouse models of allogeneic HSCT. Although Notch-deprived T cells proliferated and expanded in response to alloantigens in vivo, their ability to produce interleukin-2 and inflammatory cytokines was defective, and both CD4+ and CD8+ T cells failed to up-regulate selected effector molecules. Notch inhibition decreased the accumulation of alloreactive T cells in the intestine, a key GVHD target organ. However, Notch-deprived alloreactive CD4+ T cells retained significant cytotoxic potential and antileukemic activity, leading to improved overall survival of the recipients. These results identify Notch as a novel essential regulator of pathogenic CD4+ T-cell responses during acute GVHD and suggest that Notch signaling in T cells should be investigated as a therapeutic target after allogeneic HSCT.

Methylation profiling of an 11-gene set in urine sediments provides a sensitive and specific detection of bladder cancer.

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Ethanolamine kinase (EKI) is the first committed step in phosphatidylethanolamine (PtdEtn) biosynthesis via the CDP-ethanolamine pathway. We identify a human cDNA encoding an ethanolamine-specific kinase EKI1 and the structure of the EKI1 gene located on chromosome 12. EKI1 overexpression in COS-7 cells results in a 170-fold increase in ethanolamine kinase-specific activity and accelerates the rate of [ 3 H]ethanolamine incorporation into PtdEtn as a function of the ethanolamine concentration in the culture medium. Acceleration of the CDP-ethanolamine pathway does not result in elevated cellular PtdEtn levels, but rather the excess PtdEtn is degraded to glycerophosphoethanolamine. EKI1 has negligible choline kinase activity in vitro and does not influence phosphatidylcholine biosynthesis. Acceleration of the CDP-ethanolamine pathway also does not change the rate of PtdEtn formation via the decarboxylation of phosphatidylserine. The data demonstrate the existence of separate ethanolamine and choline kinases in mammals and show that ethanolamine kinase can be a rate-controlling step in PtdEtn biosynthesis.Ethanolamine kinase or EKI 1 (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82) catalyzes the first step of PtdEtn biosynthesis via the CDP-Etn pathway. ECT, a cytidylyltransferase, and EPT, an amino alcohol phosphotransferase, catalyze the subsequent two steps, and together these three enzymes constitute the de novo pathway for PtdEtn formation. The decarboxylation of PtdSer is an alternate route for PtdEtn production and is functionally important in cultured cell lines (1, 2), although the CDP-Etn pathway is considered a major route for PtdEtn synthesis in most mammalian tissues (3-6). PtdEtn is an abundant phospholipid in eucaryotic cell membranes, and the ECT reaction is thought to be a major regulator of its synthesis (7). EKI has been proposed as a regulatory step based on theoretical considerations (8). However, experimental investigations result in conflicting conclusions. Experiments performed with rat hepatocytes (4, 9) demonstrate that the supply of ethanolamine limits the rate of PtdEtn production at concentrations below 30 M. Only at higher concentrations of ethanolamine does accumulation of phosphoethanolamine occur, indicating ECT as the rate-limiting enzyme of the pathway. On the contrary, McMaster and Choy (10) report that the EKI step is rate-limiting with increased ethanolamine concentrations using a hamster heart perfusion model. Both studies agree that the rate of PtdEtn synthesis is dependent on the extracellular ethanolamine concentration (9, 10) and that the stimulation of PtdEtn biosynthesis occurs at physiological levels of 20 -50 M (9).There is a long standing discussion as to the number and specificity of the enzymes that catalyze the phosphorylation of ethanolamine and choline. In yeast, there are two enzymes that are able to phosphorylate choline and ethanolamine, and these enzymes are annotated based on their preferred substrate specificities. The CKI enzyme has a specific activit...

Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep IntroductionPathogenic T-cell responses can be detrimental to the host. For example, GVHD is a life-threatening complication after allogeneic BM transplantation (BMT). 1-3 GVHD is caused by donor T cells that attack normal tissues of the recipient. 1-3 Standard immunosuppressive therapy for GVHD lacks efficacy and impairs the antitumor activity. 1,2,4 New approaches are needed to control GVHD.Epigenetic modifications are thought to be important for T-cell immune responses. 5,6 These modifications include histone methylation, DNA methylation, and histone acetylation. 7-10 Histone methylation is the modification of certain amino acids of a histone by adding methyl groups. [8][9][10] Depending on the site and degree of methylation, histone methylation can be activating or repressive. For example, trimethylation of histone H3 at lysine 4 (H3K4me3), H3K36me3, and H3K79me3 are associated with transcriptional activation, whereas H3K9me3, H3K27me3, and H4K20me3 are related to gene repression. 8,9 Recent studies have reported that histone methylation may play important roles in regulating the expression of genes associated with survival, proliferation, and differentiation of Ag-activated T cells. 11,12 Unlike histone methylation, DNA methylation results in global silencing of gene expression, 10 whereas histone acetylation is associated with a relaxing chromatin structure that facilitates transcription. 10 It has been shown that the DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-AzaC) and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) may prevent GVHD in mice through a mechanism of modulating regulatory T cells (Tregs) 13,14 and APCs, 15-17 respectively. However, global modifications of DNA and chromatin structures are found to be associated with toxicities and adverse effects. 10 Thus, novel epigenetic approaches capable of targeting a specific set of genes in alloreactive T cells are desirable for controlling GVHD while minimizing adverse effects.3-Deazaneplanocin A (DZNep) possesses the potent ability to selectively inhibit some histone methylation, such as H3K27me3, H3K4me3, and H4K20me3. 18,19 DZNep is an inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. AdoHcy hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine. 20,21 When this enzyme is inhibited, AdoHcy accumulates in cells, leading to inhibition of the histone methyltransferease (HMT) activity and the subsequent histone methylation inhibition. 20 DZNep acts through a different pathway than DNA methylation inhibitors and HDAC inhibitors. 10,18,19 An Inside Blood analysis of this article appears at the fron...

There are two mammalian genes that encode isoforms of CTP:phosphocholine cytidylyltransferase (CCT), a key rate-controlling step in membrane phospholipid biogenesis. Quantitative determination of the CCT transcripts reveals that CCT␣ is ubiquitously expressed and is found at the highest levels in the testis and lung, with lower levels in the liver and ovary. CCT␤2 is a very minor isoform in most tissues but is significantly expressed in the brain, lung, and gonads. CCT␤3 is the third isoform recently discovered in mice and is expressed in the same tissues as CCT␤2, with its highest level in testes. We investigated the role(s) of CCT␤2 by generating knockout mice. The brains and lungs of mice lacking CCT␤2 expression did not exhibit any overt defects. On the other hand, a large percentage of the CCT␤2 ؊/؊ females were sterile and their ovaries exhibited defective ovarian follicle development. The proportion of female CCT␤2 ؊/؊ mice with defective ovaries increased as the animals aged. The rare litters born from CCT␤2 ؊/؊ ؋ CCT␤2 ؊/0 matings had the normal number of pups. The abnormal ovarian histopathology was characterized by disorganization of the tissue in young adult mice and absence of follicles and ova in older mice, along with interstitial stromal cell hyperplasia which culminated in the emergence of tubulostromal ovarian tumors by 16 months of age. Grossly defective CCT␤2 ؊/؊ ovaries were associated with high follicle-stimulating (FSH) and luteinizing (LH) hormone levels. Male CCT␤2 ؊/0 mice exhibited progressive multifocal testicular degeneration and reduced fertility but had normal FSH and LH levels. Thus, the most notable phenotype of CCT␤2 knockout mice was gonad degeneration and reproductive deficiency. The results indicate that although CCT␤2 is expressed at very low levels compared to the ␣-isoform, loss of CCT␤2 expression causes a breakdown in the gonadal response to hormonal stimulation.Phosphatidylcholine (PtdCho) is a major component of biological membranes in higher eukaryotes and is also secreted by specialized tissues for important extracellular tasks. CTP: phosphocholine cytidylyltransferase (CCT) is a key rate-controlling step in the major biosynthetic pathway leading to PtdCho in most tissues (for reviews, see references 15, 17, and 36). In mammals there are two genes, Pcyt1a (formerly Cptct), located on murine chromosome 16, and Pcyt1b, located on the X chromosome, that encode proteins termed CCT␣ and CCT␤, respectively (35, 38). The two genes exhibit tissuespecific expression, with CCT␣ predominating in most tissues and CCT␤ being most abundant in brain tissue (32). Two transcripts arise from the Pcyt1a gene that encode the identical CCT␣ protein. Alternate splicing of the X-linked Pcyt1b gene directs the synthesis of two mRNAs that encode the CCT␤2 and CCT␤3 isoforms in mice (32,35,38). CCT␤3 is 28 residues shorter at the amino terminus than CCT␤2 due to transcript initiation at an alternate first exon (32). In humans, intron retention gives rise to a CCT␤1 transcript found in the exp...

CoA (coenzyme A) is an essential cofactor that is involved in many metabolic processes. CoA is derived from pantothenate in five biosynthetic reactions. The CoA biosynthetic pathway is regulated by PanKs (pantothenate kinases) and four active isoforms are expressed in mammals. The critical physiological functions of the PanKs are revealed by systematic deletion of the Pank genes in mice.

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