CD4<Superscript>+</Superscript> T cell-mediated immunity against prion proteins

The prion protein (PrP(C)) is essential for susceptibility to transmissible spongiform encephalopathies. A specific conformer of this protein (PrP(Sc)) is, according to the 'protein only' hypothesis, the principal or only component of the infectious agent, designated prion. Transmission of prions between species is often inefficient, resulting in low attack rates and/or prolonged incubation times and is ascribed to a 'species barrier' caused by differences in the amino acid sequence of PrP between recipient and donor. In this report, we demonstrate that these differences in amino acid sequence result in presentation of distinct peptides on major histocompatibility complex class II molecules. These peptides result in activation of specific CD4+ T cells which leads to the induction of an effective immune response against foreign PrP as demonstrated by antibody production. Therefore, CD4+ T cells represent a crucial component of the immune system to distinguish between foreign and self PrP.     

Memory CD4<Superscript>+</Superscript> T Cells: fate determination,positive feedback and plasticity

Naïve CD4⁺ T cells undergo massive cell proliferation upon encountering their cognate ligand. This proliferation depends upon appropriate cues from the antigen-presenting cells that have processed the antigen and present the peptide to the T cells, and requires the establishment of a cytokine environment that can support such proliferation. Expansion of antigen-specific CD4⁺ T cells needs to be coupled with differentiation into one of several effector/regulatory phenotypes if the priming event is to result in cells that can initially act to control the particular pathogen that elicited the response, and later to serve as memory cells to insure an appropriate response upon reintroduction of the pathogen. Here, we discuss the initiation of T helper lineage commitment, the positive feedback regulation by the cytokine environment to enhance and stabilize the differentiation into distinct T helper subsets, and the biological significance of CD4⁺ T cell plasticity and long-term CD4⁺ T cell memory.     

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na⁺, K⁺-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.     

Involvement of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and its inhibitors in HuR-mediated cytokine mRNA stabilization in lung epithelial cells

Increasing evidence demonstrates that Na⁺, K⁺-ATPase plays an important role in pulmonary inflammation, but the mechanism remains largely unknown. In this study, we used cardiotonic steroids as Na⁺, K⁺-ATPase inhibitors to explore the possible involvement of Na⁺, K⁺-ATPase in pulmonary epithelial inflammation. The results demonstrated that mice after ouabain inhalation developed cyclooxygenase-2-dependent acute lung inflammation. The in vitro experiments further confirmed that Na⁺, K⁺-ATPase inhibitors significantly stimulated cyclooxygenase-2 expression in lung epithelial cells of human or murine origin, the process of which was participated by multiple cis-elements and trans-acting factors. Most importantly, we first described here that Na⁺, K⁺-ATPase inhibitors could evoke a significant Hu antigen R nuclear export in lung epithelial cells, which stabilized cyclooxygenase-2 mRNA by binding with a proximal AU-rich element within its 3′-untranslated region. In conclusion, HuR-mediated mRNA stabilization opens new avenues in understanding the importance of Na⁺, K⁺-ATPase, as well as its inhibitors in inflammation.     

C-peptide stimulates Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na⁺, K⁺-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na⁺, K⁺-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37°C for 10 min stimulated ⁸⁶Rb⁺ uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive ⁸⁶Rb⁺ uptake, C-peptide increased subunit phosphorylation and basolateral membrane (BLM) abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits. The increase in BLM abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits was accompanied by depletion of ₁ and ₁ subunits from the endosomal compartments. C-peptide action on Na⁺, K⁺-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na⁺, K⁺-ATPase activation, phosphorylation of ₁-subunit and translocation of ₁ and ₁ subunits to the BLM were abolished by a MEK1/2 inhibitor (20 M PD98059). C-peptide stimulation of ⁸⁶Rb⁺ uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 M GF109203X). C-peptide stimulated phosphorylation of human Na⁺, K⁺-ATPase subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the subunit of Na⁺, K⁺-ATPase.Received 15 June 2004; received after revision 14 September 2004; accepted 14 September 2004     

The membrane domain of vacuolar H<Superscript>+</Superscript>ATPase: a crucial player in neurotransmitter exocytotic release

V-ATPases are multimeric enzymes made of two sectors, a V1 catalytic domain and a V0 membrane domain. They accumulate protons in various intracellular organelles. Acidification of synaptic vesicles by V-ATPase energizes the accumulation of neurotransmitters in these storage organelles and is therefore required for efficient synaptic transmission. In addition to this well-accepted role, functional studies have unraveled additional hidden roles of V0 in neurotransmitter exocytosis that are independent of the transport of protons. V0 interacts with SNAREs and calmodulin, and perturbing these interactions affects neurotransmitter release. Here, we discuss these data in relation with previous results obtained in reconstituted membranes and on yeast vacuole fusion. We propose that V0 could be a sensor of intra-vesicular pH that controls the exocytotic machinery, probably regulating SNARE complex assembly during the synaptic vesicle priming step, and that, during the membrane fusion step, V0 might favor lipid mixing and fusion pore stability.     

Regulation of the H<Superscript>+</Superscript>-ATP synthase by IF1: a role in mitohormesis

The mitochondrial H⁺-ATP synthase is a primary hub of cellular homeostasis by providing the energy required to sustain cellular activity and regulating the production of signaling molecules that reprogram nuclear activity needed for adaption to changing cues. Herein, we summarize findings regarding the regulation of the activity of the H⁺-ATP synthase by its physiological inhibitor, the ATPase inhibitory factor 1 (IF1) and their functional role in cellular homeostasis. First, we outline the structure and the main molecular mechanisms that regulate the activity of the enzyme. Next, we describe the molecular biology of IF1 and summarize the regulation of IF1 expression and activity as an inhibitor of the H⁺-ATP synthase emphasizing the role of IF1 as a main driver of energy rewiring and cellular signaling in cancer. Findings in transgenic mice in vivo indicate that the overexpression of IF1 is sufficient to reprogram energy metabolism to an enhanced glycolysis and activate reactive oxygen species (ROS)-dependent signaling pathways that promote cell survival. These findings are placed in the context of mitohormesis, a program in which a mild mitochondrial stress triggers adaptive cytoprotective mechanisms that improve lifespan. In this regard, we emphasize the role played by the H⁺-ATP synthase in modulating signaling pathways that activate the mitohormetic response, namely ATP, ROS and target of rapamycin (TOR). Overall, we aim to highlight the relevant role of the H⁺-ATP synthase and of IF1 in cellular physiology and the need of additional studies to decipher their contributions to aging and age-related diseases.     

Structural variations in wheat HKT1;5 underpin differences in Na<Superscript>+</Superscript> transport capacity

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na⁺) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na⁺-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K _m) for the Na⁺ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D⁴⁷¹/a gap and D⁴⁷⁴/G⁴⁷³ that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na⁺ exclusion in wheat that leads to an improved salinity tolerance in the field.     

Using a recombinant bispecific antibody to block Na<Superscript>+</Superscript>-channel toxins protects against experimental scorpion envenoming

In recent years, several molecular engineering methods of designing bispecific antibodies in various formats have been developed. Tandem-scFvs comprising two scFvs fused together via a peptide are 55-kDa molecules, and are one of the most promising and most straightforward approaches to bispecific antibody production. We report an attempt to design more effective antivenoms to the Androctonus australis scorpion using murine scFvs as building blocks to create a unique bispecific molecule that neutralizes the potent neurotoxins AahI and AahII. The tandem-scFv was produced in recombinant bacteria, purified by immobilized metal ion affinity chromatography, and analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, mass spectrometry, and direct and competitive radioimmunoassay. In vivo, it neutralized the binding of the AahI and AahII toxins to their receptor, and protected mice against experimental envenomation. The findings reported here highlight the potential of recombinant antibody fragments for protecting against scorpion venom toxicity. Received 8 September 2006; received after revision 10 November 2006; accepted 27 November 2006     

ATP-induced Ca<Superscript>2+</Superscript>-signalling mechanisms in the regulation of mesenchymal stem cell migration

The ability of cells to migrate to the destined tissues or lesions is crucial for physiological processes from tissue morphogenesis, homeostasis and immune responses, and also for stem cell-based regenerative medicines. Cytosolic Ca²⁺ is a primary second messenger in the control and regulation of a wide range of cell functions including cell migration. Extracellular ATP, together with the cognate receptors on the cell surface, ligand-gated ion channel P2X receptors and a subset of G-protein-coupled P2Y receptors, represents common autocrine and/or paracrine Ca²⁺ signalling mechanisms. The P2X receptor ion channels mediate extracellular Ca²⁺ influx, whereas stimulation of the P2Y receptors triggers intracellular Ca²⁺ release from the endoplasmic reticulum (ER), and activation of both type of receptors thus can elevate the cytosolic Ca²⁺ concentration ([Ca²⁺]_c), albeit with different kinetics and capacity. Reduction in the ER Ca²⁺ level following the P2Y receptor activation can further induce store-operated Ca²⁺ entry as a distinct Ca²⁺ influx pathway that contributes in ATP-induced increase in the [Ca²⁺]_c. Mesenchymal stem cells (MSC) are a group of multipotent stem cells that grow from adult tissues and hold promising applications in tissue engineering and cell-based therapies treating a great and diverse number of diseases. There is increasing evidence to show constitutive or evoked ATP release from stem cells themselves or mature cells in the close vicinity. In this review, we discuss the mechanisms for ATP release and clearance, the receptors and ion channels participating in ATP-induced Ca²⁺ signalling and the roles of such signalling mechanisms in mediating ATP-induced regulation of MSC migration.     

Major contribution of codominant CD8 and CD4 T cell epitopes to the human cytomegalovirus-specific T cell repertoire

Human cytomegalovirus (HCMV) infection or reactivation is a cause of morbidity and mortality in immunocompromised individuals. In immunocompetent individuals, in contrast, HCMV is successfully controlled by specific CD8 and CD4 T cells. Knowledge of CD8 and CD4 T cell epitopes from HCMV and their immunodominant features is crucial for the generation of epitope-specific T cells for adoptive immunotherapy and for the development of a peptide-based HCMV vaccine. Therefore, we investigated the natural frequencies of a large number of CD8 and CD4 T cell epitopes, including 10 novel ones. We determined several epitopes as immunodominant. Surprisingly, no clear hierarchies were found for CD8 T cell epitopes, indicating codominance. These results will be valuable for adoptive transfer strategies and support initiatives towards development of a peptide-based HCMV vaccine.Received 12 August 2004; received after revision 24 September 2004; accepted 29 October 2004 These authors contributed equally to this work.     

Identification of Treg-like cells in <Emphasis Type="Italic">Tetraodon</Emphasis>: insight into the origin of regulatory T subsets during early vertebrate evolution

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg cells) are critical for the maintenance of peripheral tolerance, and the suppression of autoimmune diseases and even tumors. Although Treg cells are well characterized in humans, little is known regarding their existence or occurrence in ancient vertebrates. In the present study, we report on the molecular and functional characterization of a Treg-like subset with the phenotype CD4-2⁺CD25-like⁺Foxp3-like⁺ from a pufferfish (Tetraodon nigroviridis) model. Functional studies showed that depletion of this subset produced an enhanced mixed lymphocyte reaction (MLR) and nonspecific cytotoxic cell (NCC) activity in vitro, as well as inflammation of the intestine in vivo. The data presented here will not only enrich the knowledge of fish immunology but will also be beneficial for a better cross-species understanding of the evolutionary history of the Treg family and Treg-mediated regulatory networks in cellular immunity.     

Ca<Superscript>2+</Superscript> signals,cell membrane disintegration,and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca²⁺. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT₂₉, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca²⁺, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca²⁺, which was paralleled by the activation of outwardly rectifying Cl^? currents, which were typical for TMEM16F/ANO6. Ca²⁺ oscillations were due to Ca²⁺ release from endoplasmic reticulum, and were independent of extracellular Ca²⁺. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl^? currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.     

Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca<Superscript>2+</Superscript> fluxes in oxidative cell death

Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca²⁺ fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca²⁺ influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca²⁺ fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and proteins.     

Polyisoprenyl glycolipids as targets of CD1-mediated T cell responses

T cells are well known to recognize peptide antigens presented by major histocompatibility (MHC) class I or class II molecules. More recently, the CD1 family of antigen-presenting molecules has been shown to present both mammalian and microbial glycolipid antigens for specific recognition by T cells. Human CD1c proteins mediate T cell recognition of polyisoprenyl glycolipids, evolutionarily conserved phosphoglycolipids, which function in glycan synthesis pathways. This family of antigenic molecules is particularly attractive for the study of the molecular features that control T cell recognition of self and foreign glycolipids because natural polyisoprenols from mammals, fungi, protozoa, mycobacteria and eubacteria differ in structure. Moreover, these naturally occurring structural differences can influence their recognition by CD1c-restricted T cells. This review of the structural diversity and evolutionary relationships of polyisoprenoid glycolipids emphasizes those features of polyisoprenyl glycolipid biosynthesis that are relevant to their functions as targets of CD1-mediated T cell responses. Received 16 March 2001; received after revision 19 April 2001; accepted 23 April 2001     

<Emphasis Type="Italic">The Art of War:</Emphasis> Innate and adaptive immune responses

Research over the last several years has greatly advanced our understanding of the mechanisms by which the immune system functions. There exist two main branches of immunity, termed innate and adaptive immunity. Innate immunity uses the genetic memory of germline-encoded receptors to recognize the molecular patterns of common pathogens. Adaptive immunity, akin to somatic memory, is a complex system by which the body learns to recognize a pathogens unique antigens and builds an antigen specific response to destroy it. The effective development of the overall immune response depends on careful interplay and regulation between innate and adaptive immunity. Here we review our current understanding of how these integrated systems distinguish targets against which a response is appropriate and neutralize potentially pathogenic challenges.Received 8 May 2003; accepted 2 June 2003     

GPR39: a Zn<Superscript>2+</Superscript>-activated G protein-coupled receptor that regulates pancreatic,gastrointestinal and neuronal functions

GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn²⁺ has been shown to be a potent stimulator of GPR39 activity via the Gα_q, Gα_12/13 and Gα_s pathways. The potency and specificity of Zn²⁺ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn²⁺ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.     

Changes in calpastatin localization and expression during calpain activation: a new mechanism for the regulation of intracellular Ca<Superscript>2+</Superscript>-dependent proteolysis

The amount of calpastatin directly available in cytosol is under the control of [Ca²⁺] and [cyclic AMP]. Prolonged calpain activation also promotes degradation of calpastatin. The fluctuation of calpastatin concentration in cell soluble fraction is accompanied by an initial decrease in calpastatin gene expression, followed by a fivefold increase in its expression when the inhibitor protein is degraded. This process can be conceptualized as a mechanism to regulate calpastatin availability in the cell. This conclusion is supported by the fact that calpain, the other component of this proteolytic system, undergoes changes in its levels of expression in a much more limited manner. Furthermore, this process can be observed both in cells exposed to different natural stimuli, or in other cell lines. Modification of calpastatin gene expression might represent a new tool for the in vivo control of the regulatory machinery required for the modulation of Ca²⁺-dependent proteolysis.Received 18 July 2003; received after revision 3 September 2003; accepted 23 September 2003