Human Naive T Cells Express Functional CXCL8 and Promote Tumorigenesis.

Naive T cells are thought to be functionally quiescent. In this study, we studied and compared the phenotype, cytokine profile, and potential function of human naive CD4+ T cells in umbilical cord and peripheral blood. We found that naive CD4+ T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8+ naive T cells were preferentially enriched CD31+ T cells and did not express T cell activation markers or typical Th effector cytokines, including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naive T cells retained high levels of CXCL8 expression. Furthermore, we showed that naive T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naive T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses.

Inhibiting Oxidative Phosphorylation In Vivo Restrains Th17 Effector Responses and Ameliorates Murine Colitis.

Integration of signaling and metabolic pathways enables and sustains lymphocyte function. Whereas metabolic changes occurring during T cell activation are well characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. In this study, we defined the bioenergetics of Th17 effector cells generated in vivo. These cells depend on oxidative phosphorylation (OXPHOS) for energy and cytokine production. Mechanistically, the essential role of OXPHOS in Th17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human Th17 cells, including those isolated from Crohn disease patients, and it is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in Th17-driven diseases.

Programmed death-1 controls T cell survival by regulating oxidative metabolism.

The coinhibitory receptor programmed death-1 (PD-1) maintains immune homeostasis by negatively regulating T cell function and survival. Blockade of PD-1 increases the severity of graft-versus-host disease (GVHD), but the interplay between PD-1 inhibition and T cell metabolism is not well studied. We found that both murine and human alloreactive T cells concomitantly upregulated PD-1 expression and increased levels of reactive oxygen species (ROS) following allogeneic bone marrow transplantation. This PD-1(Hi)ROS(Hi) phenotype was specific to alloreactive T cells and was not observed in syngeneic T cells during homeostatic proliferation. Blockade of PD-1 signaling decreased both mitochondrial H2O2 and total cellular ROS levels, and PD-1-driven increases in ROS were dependent upon the oxidation of fatty acids, because treatment with etomoxir nullified changes in ROS levels following PD-1 blockade. Downstream of PD-1, elevated ROS levels impaired T cell survival in a process reversed by antioxidants. Furthermore, PD-1-driven changes in ROS were fundamental to establishing a cell's susceptibility to subsequent metabolic inhibition, because blockade of PD-1 decreased the efficacy of later F1F0-ATP synthase modulation. These data indicate that PD-1 facilitates apoptosis in alloreactive T cells by increasing ROS in a process dependent upon the oxidation of fat. In addition, blockade of PD-1 undermines the potential for subsequent metabolic inhibition, an important consideration given the increasing use of anti-PD-1 therapies in the clinic.

An automated phenotype-based microscopy screen to identify pro-longevity interventions acting through mitochondria in C. elegans.

Mitochondria are multifunctional organelles that play a central role in cellular homeostasis. Severe mitochondrial dysfunction leads to life-threatening diseases in humans and accelerates the aging process. Surprisingly, moderate reduction of mitochondrial function in different species has anti-aging effects. High-throughput screenings in the nematode Caenorhabditis elegans lead to the identification of several pro-longevity genetic and pharmacological interventions. Large-scale screens, however, are manual, subjective, time consuming and costly. These limitations could be reduced by the identification of automatically quantifiable biomarkers of healthy aging. In this study we exploit the distinct and reproducible phenotypes described in C. elegans upon different levels of mitochondrial alteration to develop an automated high-content strategy to identify new potential pro-longevity interventions. Utilizing the microscopy platform Cellomics ArrayScan Reader, we optimize a workflow to automatically and reliably quantify the discrete phenotypic readouts associated with different degrees of silencing of mitochondrial respiratory chain regulatory proteins, and validate the approach with mitochondrial-targeting drugs known to extend lifespan in C. elegans. Finally, we report that a new mitochondrial ATPase modulator matches our screening phenotypic criteria and extends nematode's lifespan thus providing the proof of principle that our strategy could be exploited to identify novel mitochondrial-targeted drugs with pro-longevity activity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

Resveratrol inhibits ovarian tumor growth in an in vivo mouse model.

BACKGROUND: Resveratrol inhibits the growth of ovarian carcinoma cells in vitro through the inhibition of glucose metabolism and the induction of both autophagy and apoptosis. In the current study, we investigated the metabolic and therapeutic effects of resveratrol in vivo. METHODS: A fluorescent xenograft mouse model of ovarian cancer was used. Mice were treated with cisplatin, resveratrol, or vehicle alone. Tumor burden was assessed using whole-body imaging. The effect of resveratrol on glucose uptake in vivo was determined using micro-positron emission tomography scanning. To determine whether resveratrol could inhibit tumor regrowth, tumor-bearing mice were treated with cisplatin followed by either daily resveratrol or vehicle. Autophagic response in resected tumors taken from mice treated with resveratrol was examined by transmission electron microscopy. Glycolysis and mitochondrial respiration in ovarian tumor cells after treatment with resveratrol was assessed. RESULTS: Mice treated with resveratrol and cisplatin were found to have a significantly reduced tumor burden compared with control animals (P<.001). Resveratrol-treated mice demonstrated a marked decrease in tumor uptake of glucose compared with controls. After treatment with cisplatin, "maintenance" resveratrol resulted in the suppression of tumor regrowth compared with mice receiving vehicle alone (P<.01). Tumors resected from mice treated with resveratrol exhibited autophagosomes consistent with the induction of autophagy. Treatment with resveratrol inhibited glycolytic response in ovarian tumor cells with high baseline glycolytic rates. CONCLUSIONS: Treatment with resveratrol inhibits glucose uptake and has a significant antineoplastic effect in a preclinical mouse model of ovarian cancer. Resveratrol treatment suppresses tumor regrowth after therapy with cisplatin, suggesting that this agent has the potential to prolong disease-free survival. Cancer 2016;122:722-729. © 2015 American Cancer Society.

Vestibular Mast Cell Density in Vulvodynia: A Case-Controlled Study.

OBJECTIVES: To identify whether mast cell densities in vulvar biopsies from the vestibule are associated with vulvodynia. METHODS: We enrolled 100 women aged 19 to 59 years with confirmed vulvodynia cases, 100 racially matched controls, and 100 black control women. All had vulvar biopsies performed at the 7 o'clock position of the vestibule, which were then immunostained to detect c-KIT protein. The numbers of c-KIT positive mast cells per ×400 magnification field were manually counted, and t tests and logistic regression were used to assess the association with case-control status. RESULTS: Of the biopsies, 235 were adequate samples for c-KIT testing for mast cells. The mast cell density was substantially lower in black control women (13.9 ± 10.9) in comparison to white control women (22.5 ± 13.2 p < 0.001): hence the analysis was confined to white cases and racially matched control women. Compared with racially matched controls, cases were younger, more likely to be married, and reported a higher household income. The average number of mast cells per ×400 magnification field overall was 19.1 ± 13.2 (range, 0-62). There was no difference in the mast cell count between racially matched cases (22.4 ± 13.9 per ×400 field) and controls (22.5 ± 13.2) in either the univariate or multivariable analyses. Within the group of cases, there was no difference in mast cell density based on the presence or absence of a variety of urogenital symptoms. CONCLUSIONS: No difference in mast cell density in biopsies of the vestibule was found between white cases and racially matched controls. Black control women have a lower mast cell density compared with white control women.

Distinct metabolic programs in activated T cells: opportunities for selective immunomodulation.

For several decades, it has been known that T-cell activation in vitro leads to increased glycolytic metabolism that fuels proliferation and effector function. Recently, this simple model has been complicated by the observation that different T-cell subsets differentially regulate fundamental metabolic pathways under the control of distinct molecular regulators. Although the majority of these data have been generated in vitro, several recent studies have documented the metabolism of T cells activated in vivo. Here, we review the recent data surrounding the differential regulation of metabolism by distinct T-cell subsets in vitro and in vivo and discuss how differential metabolic regulation might facilitate T-cell function vis-à-vis proliferation, survival, and energy production. We further discuss the important therapeutic implications of differential metabolism across T-cell subsets and review recent successes in exploiting lymphocyte metabolism to treat immune-mediated diseases.

National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: V. The 2014 Ancillary Therapy and Supportive Care Working Group Report.

The 2006 National Institutes of Health (NIH) Consensus paper presented recommendations by the Ancillary Therapy and Supportive Care Working Group to support clinical research trials in chronic graft-versus-host disease (GVHD). Topics covered in that inaugural effort included the prevention and management of infections and common complications of chronic GVHD, as well as recommendations for patient education and appropriate follow-up. Given the new literature that has emerged during the past 8 years, we made further organ-specific refinements to these guidelines. Minimum frequencies are suggested for monitoring key parameters relevant to chronic GVHD during systemic immunosuppressive therapy and, thereafter, referral to existing late effects consensus guidelines is advised. Using the framework of the prior consensus, the 2014 NIH recommendations are organized by organ or other relevant systems and graded according to the strength and quality of supporting evidence.

Effector T cells require fatty acid metabolism during murine graft-versus-host disease.

Activated T cells require increased energy to proliferate and mediate effector functions, but the metabolic changes that occur in T cells following stimulation in vivo are poorly understood, particularly in the context of inflammation. We have previously shown that T cells activated during graft-versus-host disease (GVHD) primarily rely on oxidative phosphorylation to synthesize adenosine 5'-triphosphate. Here, we demonstrate that alloreactive effector T cells (Teff) use fatty acids (FAs) as a fuel source to support their in vivo activation. Alloreactive T cells increased FA transport, elevated levels of FA oxidation enzymes, up-regulated transcriptional coactivators to drive oxidative metabolism, and increased their rates of FA oxidation. Importantly, increases in FA transport and up-regulation of FA oxidation machinery occurred specifically in T cells during GVHD and were not seen in Teff following acute activation. Pharmacological blockade of FA oxidation decreased the survival of alloreactive T cells but did not influence the survival of T cells during normal immune reconstitution. These studies suggest that pathways controlling FA metabolism might serve as therapeutic targets to treat GVHD and other T-cell-mediated immune diseases.

Anaplerotic metabolism of alloreactive T cells provides a metabolic approach to treat graft-versus-host disease.

T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic (13)C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders.

First-in-human safety, tolerability, and pharmacokinetic results of DFV890, an oral low-molecular-weight NLRP3 inhibitor.

This first-in-human study evaluated the safety, tolerability, single- and multiple-dose pharmacokinetic profiles with dietary influence, and pharmacodynamics (PD) of DFV890, an oral NLRP3 inhibitor, in healthy participants. In total, 122 participants were enrolled into a three-part trial including single and 2-week multiple ascending oral doses (SAD and MAD, respectively) of DFV890, and were randomized (3:1) to DFV890 or placebo (SAD [3-600 mg] and MAD [fasted: 10-200 mg, once-daily or fed: 25 and 50 mg, twice-daily]). DFV890 was generally well-tolerated. Neither deaths nor serious adverse events were reported. A less than dose-proportional increase in exposure was observed with the initially used crystalline suspension (3-300 mg); however, an adjusted suspension formulation using spray-dried dispersion (SDD; 100-600 mg) confirmed dose-proportional increase in exposure. Relative bioavailability between crystalline suspension and tablets, and food effect were evaluated at 100 mg. Under fasting conditions, Cmax of the tablet yielded 78% compared with the crystalline suspension, and both formulations showed comparable AUC. The fed condition led to a 2.05- and 1.49-fold increase in Cmax and AUC0-last compared with the fasting condition. The median IC50 and IC90 for ex-vivo lipopolysaccharide-stimulated interleukin IL-1ß release inhibition (PD) were 61 (90% CI: 50, 70) and 1340 ng/mL (90% CI: 1190, 1490). Crystalline tablets of 100 mg once-daily or 25 mg twice-daily were sufficient to maintain ~90% of the IL-1ß release inhibition over 24 h at steady state. Data support dose and formulation selection for further development in diseases, in which an overactivated NLRP3 represents the underlying pathophysiology.

NLRP3 Inhibition Leads to Impaired Mucosal Fibroblast Function in Patients with Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD) are characterized by mucosal inflammation and sequential fibrosis formation, but the exact role of the hyperactive NLRP3 inflammasome in these processes is unclear. Thus, we studied the expression and function of the NLRP3 inflammasome in the context of inflammation and fibrosis in IBD. METHODS: We analysed intestinal NLRP3 expression in mucosal immune cells and fibroblasts from IBD patients and NLRP3-associated gene expression via single-cell RNA sequencing and microarray analyses. Furthermore, cytokine secretion of NLRP3 inhibitor treated blood and mucosal cells, as well as proliferation, collagen production, and cell death of NLRP3 inhibitor treated intestinal fibroblasts from IBD patients were studied. RESULTS: We found increased NLRP3 expression in the inflamed mucosa of IBD patients and NLRP3 inhibition led to reduced IL-1ß and IL-18 production in blood cells and diminished the bioactive form of mucosal IL-1ß. Single cell analysis identified overlapping expression patterns of NLRP3 and IL-1ß in classically activated intestinal macrophages and we also detected NLRP3 expression in CD163+ macrophages. In addition, NLRP3 expression was also found in intestinal fibroblasts from IBD patients. Inhibition of NLRP3 led to reduced proliferation of intestinal fibroblasts, which was associated with a marked decrease in production of collagen type I and type VI in IBD patients. Moreover, NLRP3 inhibition in intestinal fibroblasts induced autophagy, a cellular process involved in collagen degradation. CONCLUSIONS: In the presented study, we demonstrate that inhibiting NLRP3 might pave the way for novel therapeutic approaches in IBD, especially to prevent the severe complication of intestinal fibrosis formation.

Niclosamide Enema for Active Distal Ulcerative Colitis: A Phase 1, Open-Label Study.

BACKGROUND: Oral and rectal formulations of 5-aminosalicylic acid are the first-line therapy for mild-to-moderate, distal ulcerative colitis (UC), but such a treatment is not effective in one-third of patients. Niclosamide is a small molecule, developed and approved as an orally administered drug to treat helminthic infections, with an excellent safety profile. Preclinical work showed that niclosamide is an anti-inflammatory agent, thereby providing the rationale to explore its safety and efficacy in patients with UC. This phase 1, open-label trial was aimed at assessing the safety of niclosamide formulated as an enema in patients with mild-to-moderate, distal UC, who relapsed on maintenance therapy with oral and/or rectal 5-aminosalicylic acid. METHODS: Seventeen patients with active UC received niclosamide enema (150 mg/60 mL) twice a day for 6 weeks. The primary endpoint was the safety of niclosamide treatment. Secondary endpoints included clinical remission and improvements in endoscopic Mayo/histologic scores. Endoscopic remission percentages exclude participants meeting criteria at baseline for endoscopic remission. RESULTS: Niclosamide was well tolerated by all 17 patients that were enrolled and treated. No serious adverse event was registered. Fifteen mild adverse events were registered in 6 patients and considered to be unrelated to the treatment. Clinical remission was achieved in 10 (59%) of 17 patients. Improvements of endoscopic Mayo score and histologic Geboes score were seen in 7 (58%) of 12 and 7 (41.2%) of 17 patients, respectively. CONCLUSIONS: Niclosamide enema treatment is safe and well tolerated. Niclosamide improves clinical symptoms and endoscopic/histologic signs of UC; however, appropriately designed placebo-controlled clinical trials are required to confirm efficacy.

OXPHOS promotes apoptotic resistance and cellular persistence in TH17 cells in the periphery and tumor microenvironment.

T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (TH17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. TH17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that TH17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates TH17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in TH17s. We found that TH17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS TH17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS TH17s. By contrast, glycolytic TH17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected TH17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.

CLU blocks HDACI-mediated killing of neuroblastoma.

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation.

Glucose deprivation activates AMPK and induces cell death through modulation of Akt in ovarian cancer cells.

OBJECTIVES: Upregulation of glycolysis has been demonstrated in multiple tumor types. Glucose deprivation results in diminished intracellular ATP; this is counteracted by AMPK activation during energy deficiency to restore ATP levels. We sought to determine whether glucose deprivation could induce cytotoxicity in ovarian cancer cells through activation of AMPK, and whether AMPK activators could mimic glucose deprivation induced cytotoxicity. METHODS: Sensitivity to 2DG induced cytotoxicity and glucose deprivation was determined in a panel of ovarian cancer cells. Cellular growth rate, rate of glucose uptake, and response to glucose deprivation were determined. Expression of Glut-1, HIF1-α, AMPK and Akt was determined by immunoblotting. RESULTS: Incubation of ovarian cancer cells with glucose-free media, 2-DG and AMPK activators resulted in cell death. The glycolytic phenotype of ovarian cancer cells was present in both normoxic and hypoxic conditions, and did not correlate with HIF1-α expression levels. Sensitivity to glucose deprivation was independent of growth rate, rate of glucose uptake, and appeared to be dependent upon constitutive activation of Akt. Glucose deprivation resulted in activation of AMPK and inhibition of Akt phosphorylation. Treatment with AMPK activators resulted in AMPK activation, Akt inhibition, and induced cell death in ovarian cancer cells. CONCLUSIONS: Ovarian cancer cells are glycolytic as compared to normal, untransformed cells, and are sensitive to glucose deprivation. Because ovarian cancer cells are dependent upon glucose for growth and survival, treatment with AMPK activators that mimic glucose deprivation may result in broad clinical benefits to ovarian cancer patients.

Ku70 acetylation in neuroblastoma pathogenesis and therapy.

Neuroblastoma is a cancer that occurs in children. It develops from stem cells that normally give rise to parts of the peripheral nervous system and adrenal glands. Although most children with localized neuroblastoma are cured, children with wide-spread disease have a small chance of survival even after surgery, chemotherapy, radiation and bone marrow transplantation. Ten to fifteen percent of patients die from treatment complications, and long-term survival is less than 30%. Although contemporary molecular tumor marker discoveries have improved prognostication, few have led to new therapeutic approaches. To solve this problem, we are working to understand which molecules in the stem cells from which this cancer arises malfunction to cause neuroblastoma and apply this information to develop new models to treat this disease. Our efforts have focused on the functional regulation of a protein called Ku70, which coordinately regulates DNA repair and cell death. We propose that the incorrect balance between these two activities underlies this cancer's development, and that re-balancing with drug therapy offers a way to treat this disease.

Apoptotic signaling activated by modulation of the F0F1-ATPase: implications for selective killing of autoimmune lymphocytes.

7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a proapoptotic 1,4-benzodiazepine that potently suppresses disease in the murine model of lupus by selectively killing pathogenic lymphocytes. In MRL/MpJ-Fas(lpr) (MRL-lpr) mice, Bz-423 overcomes deficient expression of the Fas death receptor and hyperactivation of antiapoptotic phosphatidylinositol 3-kinase (PI3K)-Akt signaling to specifically kill pathogenic CD4(+) T cells. Bz-423 binds to the oligomycin-sensitivity-conferring protein component of the mitochondrial F(0)F(1)-ATPase, which modulates the enzyme leading to formation of superoxide by the mitochondrial respiratory chain. Scavenging this reactive oxygen species blocks all subsequent components of the apoptotic cascade. To gain insight into how apoptotic signaling activated by Bz-423-induced superoxide contributes to the selective depletion of MRL-lpr CD4(+) T cells, we characterized the death mechanism in a CD4(+) T cell leukemia line (Jurkat). Although Bz-423-induced superoxide indirectly inactivates Akt, this response is not required for T cell death. Apoptosis instead results from parallel increases in levels of the proapoptotic Bcl-2 proteins Noxa and Bak leading to specific activation of Bak, mitochondrial outer membrane permeabilization, and a commitment to apoptosis. By directly up-regulating proteins that trigger loss of mitochondrial outer membrane integrity, Bz-423 bypasses defective Fas function and antiapoptotic PI3K-Akt signaling in MRL-lpr CD4(+) T cells. Moreover, because disease-associated abnormalities should sensitize autoreactive CD4(+) T cells to transcriptional up-regulation of Noxa by redox signals and to Bak-dependent apoptosis, the apoptotic mechanism elucidated in Jurkat cells provides important clues into the cell-type- and disease-selective effects of Bz-423 in MRL-lpr mice.

Efficient nonparametric belief propagation for pose estimation and manipulation of articulated objects.

Robots working in human environments often encounter a wide range of articulated objects, such as tools, cabinets, and other jointed objects. Such articulated objects can take an infinite number of possible poses, as a point in a potentially high-dimensional continuous space. A robot must perceive this continuous pose to manipulate the object to a desired pose. This problem of perception and manipulation of articulated objects remains a challenge due to its high dimensionality and multimodal uncertainty. Here, we describe a factored approach to estimate the poses of articulated objects using an efficient approach to nonparametric belief propagation. We consider inputs as geometrical models with articulation constraints and observed RGBD (red, green, blue, and depth) sensor data. The described framework produces object-part pose beliefs iteratively. The problem is formulated as a pairwise Markov random field (MRF), where each hidden node (continuous pose variable) is an observed object-part's pose and the edges denote the articulation constraints between the parts. We describe articulated pose estimation by a "pull" message passing algorithm for nonparametric belief propagation (PMPNBP) and evaluate its convergence properties over scenes with articulated objects. Robot experiments are provided to demonstrate the necessity of maintaining beliefs to perform goal-driven manipulation tasks.

Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax.

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.