Mechanism of T cell exhaustion in a chronic environment

T cell exhaustion develops under conditions of antigen-persistence caused by infection with various chronic pathogens, such as human immunodeficiency virus (HIV) and mycobacterium tuberculosis (TB), or by the development of cancer. T cell exhaustion is characterized by stepwise and progressive loss of T cell function, which is probably the main reason for the failed immunological control of chronic pathogens and cancers. Recent observations have detailed some of the intrinsic and extrinsic factors that influence the severity of T cell exhaustion. Duration and magnitude of antigenic activation of T cells might be associated with up-regulation of inhibitory receptors, which is a major intrinsic factor of T cell exhaustion. Extrinsic factors might include the production of suppressive cytokines, T cell priming by either non-professional antigen-presenting cells (APCs) or tolerogenic dendritic cells (DCs), and alteration of regulatory T (Treg) cells. Further investigation of the cellular and molecular processes behind the development of T cell exhaustion can reveal therapeutic targets and strategies for the treatment of chronic infections and cancers. Here, we report the properties and the mechanisms of T cell exhaustion in a chronic environment.     

Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.     

Suppression of the immune response to immunotoxins with anti-CD4 monoclonal antibodies.

Treatment of normal mice with a mAb to CD4 (GK1.5) was explored as a means of inhibiting the antibody response to an immunotoxin. Three days of pretreatment with 200 micrograms of GK1.5 completely abrogated the primary antibody response to a 3-micrograms dose of a mutant diphtheria toxin conjugated to an anti-transferrin receptor antibody. The same dose and schedule of anti-CD4 antibody significantly reduced and delayed, but did not prevent, the anamnestic antitoxin response in animals that had been previously primed to the immunotoxin. Three daily injections of anti-CD4 antibodies followed by weekly doses of immunotoxin resulted in a 3-wk delay in the development of antitoxin antibodies, and the kinetics of the antitoxin response correlated with the kinetics of recovery of CD4+ T cells in the spleen and lymph nodes. The antitoxin response to repeated doses of immunotoxin was completely abrogated when anti-CD4 antibodies were given every 2 wk throughout the course of immunotoxin treatment. Thus, transient depletion of Th cells during treatment can block the immune response to an immunotoxin. There was no evidence of tolerance induction with this regimen.     

Estrogen and n-3 polyunsaturated fatty acid supplementation have a synergistic hypotriglyceridemic effect in ovariectomized rats

The n-3 polyunsaturated fatty acids (PUFAs), EPA and DHA, as well as estrogen have been shown to decrease circulating levels of triglyceride (TG), but their underlying mode of action is unclear. The purpose of this study was to determine the effects of n-3 PUFA consumption and estrogen injection on TG metabolism. Rats (n = 48) were fed a modified AIN-93G diet with 0, 1, or 2 % EPA + DHA relative to the total energy intake during 12 weeks. At 8 weeks, rats were ovariectomized (OVX), and after a 1-week recovery, rats were injected with either 17β-estradiol-3-benzoate (E₂) or corn oil for the last 3 weeks. The n-3 PUFA consumption and E₂ injection independently decreased the hepatic expressions of sterol regulatory element-binding protein 1, acetyl-CoA carboxylase 1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2) (P < 0.05). There were interactions between n-3 PUFA consumption and E₂ injection on hepatic expression of FAS and DGAT2. In addition, n-3 PUFA consumption and E₂ injection up-regulated the expression of AMP-activated protein kinase (AMPK), phosphorylated AMPK, peroxisomal proliferator-activated receptor α, and carnitine palmitoyltransferase 1 in liver and skeletal muscle. E₂ injection increased the expression of estrogen receptor α and β in skeletal muscle and liver, but n-3 PUFA consumption increased the expression of both receptors only in skeletal muscle. The present study suggests that the hypotriglyceridemic effects of n-3 PUFA consumption and E₂ injection could be due to the down-regulation of hepatic TG synthesis and up-regulation of TG oxidation in liver and skeletal muscle in OVX rats.     

Relationship of stigma behaviors and breeding system in three Mazus (Phrymaceae) species with bilobed stigma

Sensitive stigma has been recognized to facilitate outcrossing. We hypothesized that species with different levels of sensitivity might have corresponding differences in components of their breeding system. In this study, three Mazus species with bilobed stigmas were used to test the hypothesis. We explored stigma behaviors of the species in reaction time, recovery time, permanent closing time, and the minimum pollen load causing permanent closure. We investigated floral traits, pollinator type and behavior, pollination intensity, and natural schedule of pollen deposition on stigma. Moreover, we evaluated the mating system of the species by checking seed set after controlled pollination treatments, namely, natural flowers with open pollination, enclosed flowers without pollination, and enclosed flowers with self and outcross hand pollination. Results indicated that stigma of M. pumilus (N. L. Burman) Steenis was not sensitive, whereas stigmas of M. miquelii Makino and M. stachydifolius (Turcz.) Maxim. closed and reopened quickly in response to pollination. Accordingly, hand pollination treatments revealed that seed set of self-spontaneous pollination in M. pumilus was similar to the other treatments. For M. miquelii, outcross pollen resulted in significantly higher seed set than self-pollen.Mazus stachydifolius was self-incompatible. Additionally, the corresponding characteristics in other components of the breeding system for each species were found. Our study indicated that the sensitivity of bilobed stigma might be linked with floral traits and the mating system in a given species. Sensitive stigma should be regarded as an evolutionary mechanism for enhancement of outcrossing.     

Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion)

This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP‐expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two‐cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP‐expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose “prematuration somatic cell fusion” as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.     

Multimodal Discrimination of Alzheimer’s Disease Based on Regional Cortical Atrophy and Hypometabolism

Structural MR image (MRI) and ¹⁸F-Fluorodeoxyglucose-positron emission tomography (FDG-PET) have been widely employed in diagnosis of both Alzheimer’s disease (AD) and mild cognitive impairment (MCI) pathology, which has led to the development of methods to distinguish AD and MCI from normal controls (NC). Synaptic dysfunction leads to a reduction in the rate of metabolism of glucose in the brain and is thought to represent AD progression. FDG-PET has the unique ability to estimate glucose metabolism, providing information on the distribution of hypometabolism. In addition, patients with AD exhibit significant neuronal loss in cerebral regions, and previous AD research has shown that structural MRI can be used to sensitively measure cortical atrophy. In this paper, we introduced a new method to discriminate AD from NC based on complementary information obtained by FDG and MRI. For accurate classification, surface-based features were employed and 12 predefined regions were selected from previous studies based on both MRI and FDG-PET. Partial least square linear discriminant analysis was employed for making diagnoses. We obtained 93.6% classification accuracy, 90.1% sensitivity, and 96.5% specificity in discriminating AD from NC. The classification scheme had an accuracy of 76.5% and sensitivity and specificity of 46.5% and 89.6%, respectively, for discriminating MCI from AD. Our method exhibited a superior classification performance compared with single modal approaches and yielded parallel accuracy to previous multimodal classification studies using MRI and FDG-PET.     

An eukaryotic translation initiation factor,AteIF5A‐2, affects cadmium accumulation and sensitivity in Arabidopsis

Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild‐type Columbia‐0 (Col‐0) with a knockdown mutant of AteIF5A‐2, fbr12‐3 under Cd stress conditions. The results showed that the mutant fbr12‐3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A‐2 makes the mutant more Cd sensitive. Real‐time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12‐3 compared with Col‐0. As a result, an increase in MDA and H₂O₂ content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis.