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Hepatic dysfunction is a common clinical complication in malaria, although its pathogenesis remains largely unknown. Using a variety of in vivo and ex vivo approaches, we have shown for the first time that malarial infection induces hepatic apoptosis through augmentation of oxidative stress. Apoptosis in hepatocyte has been confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin-nick-end labeling assay (TUNEL) and caspase-3 activation. Gene expression analysis using RT-PCR indicates the significant down-regulation of Bcl-2 and up-regulation of Bax expression in liver of malaria infected mice suggesting the involvement of mitochondrial pathway of apoptosis. The levels of Fas expression and caspase-8 activity in infected liver were same as that of uninfected control mice indicating death receptor (Fas) pathway did not contribute to liver apoptosis during malarial infection. Moreover, evidence has been presented by confocal microscopy to show the translocation of Bax from cytosol to mitochondria in apoptotic hepatocyte, resulting in opening of permeability transition pores, which in turn decreases mitochondrial membrane potential and induces cytochrome c release into cytosol. Malarial infection induces the generation of hydroxyl radical (*OH) in liver, which may be responsible for the induction of oxidative stress and apoptosis as administration of *OH specific antioxidant as well as spin trap, alpha-phenyl-tert-butyl-nitrone in malaria-infected mice significantly inhibits the development of oxidative stress as well as induction of apoptosis. Thus, results suggest the implication of oxidative stress induced-mitochondrial pathway of apoptosis in the pathophysiology of hepatic dysfunction in malaria.

We showed earlier that malarial infection significantly induces liver apoptosis mediated by oxidative stress mechanisms. Thus, a nontoxic antioxidant-antiapoptotic molecule may be beneficial for hepatoprotection. Melatonin remarkably prevents hepatocyte apoptosis in mice induced during malaria as indicated by caspase 3 and TUNEL assays as well as transmission electron microscopy (TEM) of the liver tissue. The mitochondrial apoptotic pathway, which plays a critical role in liver cell death during malarial infection, was almost completely suppressed by melatonin as it corrects both the overexpression of Bax and down-regulation of bcl-2 as revealed by semiquantitative RT-PCR. Fluorometric studies using JC-1 documented that melatonin also restores mitochondrial transmembrane potential (DeltaPsim) in malaria-infected mice liver. The antiapoptotic effect of melatonin is associated with its antioxidant role because melatonin protects liver from oxidative stress induced during malaria by scavenging the hydroxyl radicals, preventing the depletion of reduced glutathione, inhibiting lipid peroxidation and protein carbonyl formation. The effective antioxidant dose of melatonin to protect liver from oxidative stress during malaria is 20 times lower than that of known antioxidants, vitamin C and vitamin E. Apoptosis of hepatocytes during malarial infection is well correlated with dysfunction of the liver while melatonin offers hepatoprotective effects as indicated by different liver function tests. Thus, melatonin may well be effective in combating oxidative stress-induced apoptosis and liver damage during malaria infection.

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Autoimmune regulator (Aire) has a unique expression pattern in thymic medullary epithelial cells (mTECs), in which it plays a critical role in the activation of tissue-specific antigens. The expression of Aire in mTECs is activated by receptor activator of nuclear factor κB (RANK) signaling; however, the molecular mechanism behind this activation is unknown. Here, we characterize a conserved noncoding sequence 1 (CNS1) containing two NF-κB binding sites upstream of the Aire coding region. We show that CNS1-deficient mice lack thymic expression of Aire and share several features of Aire-knockout mice, including downregulation of Aire-dependent genes, impaired terminal differentiation of the mTEC population, and reduced production of thymic Treg cells. In addition, we show that CNS1 is indispensable for RANK-induced Aire expression and that CNS1 is activated by NF-κB pathway complexes containing RelA. Together, our results indicate that CNS1 is a critical link between RANK signaling, NF-κB activation, and thymic expression of Aire.Keywords: Autoimmune regulator · Enhancer · NF-κB · Receptor activator of nuclear factor κB · Thymic medulla See accompanying Commentary by Mitsuru MatsumotoAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionCentral tolerance is achieved in the thymus by selection of a T-cell repertoire that is nonreactive to self. In the thymus, developing T cells, termed thymocytes, interact with cortical thymic epithelial cells and thymic medullary epithelial cells (cTECs and mTECs) and dendritic cells that present antigens during the positive and negative selection processes [1,2]. Due to their unique ability to express a wide range of peripheral tissue-specific antigens (TSAs), mTECs are believed to play a central role in the negative selection of self-reactive thymocytes and thereby securing tolerance to self-proteins [3,4]. The promiscuous expression of peripheral antigens in mTECs is largely controlled by autoimmune regulator Correspondence: Dr. Pärt Peterson e-mail: part.peterson@ut.ee (Aire) that constitutively promotes the transcription of hundreds of tissue-specific genes [5][6][7][8]. The importance of Aire in negative selection is exemplified by the mutations in the human Aire gene, which cause a monogenic disorder called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy [9][10][11]. Similar to human autoimmune polyendocrinopathy-candidiasisectodermal dystrophy patients, Aire-deficient mice exhibit a multiorgan autoimmune phenotype [5], although some clear differences exist including a lack of endocrine organ involvement, relatively mild disease, and the lack of high-titer neutralizing auto-Abs to type 1 interferons and interleukin 22 [12,13].The Aire protein has a unique and highly specific expression profile. Aire is rapidly upregulated for a few days [14,15] [17,24,[26][27][28]. RANK ligation activates both the classical and alternative pathways of NF-κB signaling [29]. The classical pathway involves t...

Choline kinase is the first enzyme in the Kennedy pathway (CDP-choline pathway) for the biosynthesis of the most essential phospholipid, phosphatidylcholine, in Plasmodium falciparum. In addition, choline kinase also plays a pivotal role in trapping essential polar head group choline inside the malaria parasite. Recently, Plasmodium falciparum choline kinase (PfCK) has been cloned, overexpressed, and purified. However, the function of this enzyme in parasite growth and survival has not been evaluated owing to the lack of a suitable inhibitor. Purified recombinant PfCK enabled us to identify an inhibitor of PfCK, hexadecyltrimethylammonium bromide (HDTAB), which has a very close structural resemblance to hexadecylphosphocholine (miltefosin), the well-known antiproliferative and antileishmanial drug. HDTAB inhibited PfCK in a dose-dependent manner and offered very potent antimalarial activity in vitro against Plasmodium falciparum. Moreover, HDTAB exhibited profound antimalarial activity in vivo against the rodent malaria parasite Plasmodium yoelii (N-67 strain). Interestingly, parasites at the trophozoite and schizont stages were found to be particularly sensitive to HDTAB. The stage-specific antimalarial effect of HDTAB correlated well with the expression pattern of PfCK in P. falciparum, which was observed by reverse transcription-PCR and immunofluorescence microscopy. Furthermore, the antimalarial activity of HDTAB paralleled the decrease in phosphatidylcholine content, which was found to correlate with the decreased phosphocholine generation. These results suggest that inhibition of choline kinase by HDTAB leads to decreased phosphocholine, which in turn causes a decrease in phosphatidylcholine biosynthesis, resulting in death of the parasite.

We have investigated the mechanism of antiapoptotic and cell renewal effects of lansoprazole, a proton pump inhibitor, to protect and heal gastric mucosal injury in vivo induced by indomethacin, a non-steroidal anti-inflammatory drug (NSAID). Lansoprazole prevents indomethacin-induced gastric damage by blocking activation of mitochondrial and Fas pathways of apoptosis. Lansoprazole prevents indomethacin-induced up-regulation of proapoptotic Bax and Bak and down-regulation of antiapoptotic Bcl-2 and Bcl xL to maintain the normal proapoptotic/antiapoptotic ratio and thereby arrests indomethacin-induced mitochondrial translocation of Bax and collapse of mitochondrial membrane potential followed by cytochrome c release and caspase-9 activation. Lansoprazole also inhibits indomethacin-induced Fas-mediated mucosal cell death by down-regulating Fas or FasL expression and inhibiting caspase-8 activation. Lansoprazole favors mucosal cell renewal simultaneously by stimulating gene expression of prosurvival proliferating cell nuclear antigen, survivin, epidermal growth factor, and basic fibroblast growth factor. The up-regulation of Flt-1 further indicates that lansoprazole activates vascular epidermal growth factor-mediated controlled angiogenesis to repair gastric mucosa. Lansoprazole also stimulates the healing of already formed ulcers induced by indomethacin. Time course study of healing indicates that it switches off the mitochondrial death pathway completely but not the Fas pathway. However, lansoprazole heals mucosal lesions almost completely after overcoming the persisting Fas pathway, probably by favoring the prosurvival genes expression. This study thus provides the detailed mechanism of antiapoptotic and prosurvival effects of lansoprazole for offering gastroprotection against indomethacin-induced gastropathy.Non-steroidal anti-inflammatory drugs (NSAIDs), 2 commonly used for the treatment of arthritis and other musculoskeletal disorders are considered to be one of the most important causative factors for gastric damage (1-3). Although various mechanisms have been suggested for NSAID-induced gastric ulcer (4 -7), recent studies suggest that increased apoptotic cell death and simultaneous block of mucosal cell renewal play major roles in the development of mucosal lesion (8 -10). Healthy gastric mucosa is always under equilibrium between cell death and cell renewal (11, 12) and mucosal injury is developed when this balance is disturbed due to an increase in apoptosis and/or inhibition of cell proliferation (11,12). NSAIDs are shown to induce apoptosis in gastric mucosal cells through reactive oxygen species generation, cytochrome c release, activation of caspase-3, inhibition of survivin expression, and induction of Ca 2ϩ signaling (8,10,13,14). Caspase-3 activation is generally mediated through two main pathways, viz. the mitochondrial (internal) and death receptor (external) pathways (15, 16). In the mitochondrial pathway, up-regulation or activation of pro-apoptotic proteins and/or down-regulation or inactivat...