No abstract

ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2(-*)), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2(-*), or an antagonist of the BK(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2(-*) generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.

To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD ‘up’, two-RBD ‘down’ state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFNγ+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca(2+)-sensitive K(+) (mtK(Ca)) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK(Ca) channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K(+) influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (DeltaPsi(m)). To test this hypothesis, we measured effects of NS-1619, a putative mtK(Ca) channel opener, and valinomycin, a K(+) ionophore, on mitochondrial respiration, DeltaPsi(m), and ROS generation in guinea pig heart mitochondria. NS-1619 (30 microM) increased state 2 and 4 respiration by 5.2 +/- 0.9 and 7.3 +/- 0.9 nmol O(2).min(-1).mg protein(-1), respectively, with the NADH-linked substrate pyruvate and by 7.5 +/- 1.4 and 11.6 +/- 2.9 nmol O(2).min(-1).mg protein(-1), respectively, with the FADH(2)-linked substrate succinate (+ rotenone); these effects were abolished by the mtK(Ca) channel blocker paxilline. DeltaPsi(m) was not decreased by 10-30 microM NS-1619 with either substrate, but H(2)O(2) release was increased by 44.8% (65.9 +/- 2.7% by 30 muM NS-1619 vs. 21.1 +/- 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H(2)O(2) release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained DeltaPsi(m), despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K(+) influx during states 2 and 4 increases mitochondrial respiration while maintaining DeltaPsi(m); this allows singlet electron uptake by O(2) and ROS generation.

IL-17-producing CD4 1 T cells (Th17) have been classified as a new T helper cell subset. Using an IL-17 fate mapping mouse strain, which genetically fixes the memory of IL-17 expression, we demonstrate that IL-17A/F-expressing T helper cells generated either in vitro or in vivo are not a stable T-cell subset. Upon adoptive transfer of IL-17F-reporterpositive Th17 cells to RAG-deficient or WT animals, encephalitogenic Th17 cells partially lose IL-17 expression and upregulate IFN-c. Additionally, we show that Th1 cells can convert in vivo to IL-17A/IFN-c-coexpressing cells in the mesenteric lymph nodes (mLN). Our data classify IL-17A and IL-17F as cytokines produced transiently in response to the local microenvironment, thus showing that IL-17 expression does not define an end-stage T helper cell subset. IntroductionSince the finding that IL-23 and not IL-12 is necessary for active induction of EAE [1,2], the previously common dogma for the pathogenesis of the disease has changed. Th17 cells, which were soon thereafter shown to depend on IL-23 [3,4], are now regarded as major initiators of pathogenesis in a number of disease models and human conditions. Th17 achieve their pathogenic phenotype by secreting cytokines which in turn induces the surrounding tissue to secrete chemokines and other cytokines important for the immigration of potentially pathogenic leukocytes such as granulocytes and lymphocytes [5].In a previous landmark EAE study, Th17 cells that were expanded in the presence of IL-23 were shown to be extremely efficient in inducing passive EAE [4]. Low amounts of transferred cells (150 000) were able to induce EAE in SJL/J animals. This finding together with the full resistance of IL-23-deficient animals in response to active EAE induction [2] cemented the idea of Th17 cells as a major pathogenic cell population in EAE. This was further supported by the discovery that Th17 can be very efficiently generated in vitro when naïve CD4 1 T cells are activated in the presence of and that IL-6 is necessary for EAE induction [9][10][11][12]. Furthermore, transgenic expression of TGF-b in T cells enhanced EAE severity [6]. Another milestone for à These authors contributed equally to this work. 3336Frontline this hypothesis was the finding that RORgt deficiency led to a major lack of Th17 cells and to a near complete resistance against active EAE, even in the presence of extensive CNS infiltration [13]. Other transfer studies in the SJL/J mouse using IL-23 expanded encephalitogenic cells found an enhanced infiltration of granulocytes concomitant with EAE development compared to transfer of IL-12 expanded T cells [5,14], further supporting a specific role for Th17 cells in autoimmunity. Given the previous lack of suitable Th17 reporter strains, these studies relied on transfer of in vitro generated Th17 cells of a heterogenous nature, rather than a pure Th17 population.Recently, the encephalitogenicity of Th17 cells was challenged by O'Connor et al., who showed that transferring myelin oligodendrocyte glycoprotei...

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1β), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.

BackgroundMultiple sclerosis (MS) is often accompanied by optic nerve inflammation. And some patients experience permanent vision loss. We examined if the grade of optic nerve infiltration and demyelination affects the severity of clinical signs in an experimental autoimmune encephalomyelitis (EAE) model. The loss of retinal ganglion cells (RGC) and alterations in glia activity were also investigated.MethodsC57BL/6 mice were immunized with peptide MOG35-55 in complete Freund’s adjuvant (CFA) and controls received PBS in CFA. Then 23 days post immunization eyes were prepared for flatmounts and stained with Nissl to evaluated neuronal density. Clinical EAE symptoms as well as cell infiltration and demyelination in the optic nerve were examined. Retinal sections were stained with hematoxylin and eosin and silver stain. Immunohistochemistry was used to label RGCs (Brn-3a), apoptotic cells (caspase 3), macroglia (glial fibrillary acidic protein (GFAP)), microglia (Iba1), macrophages (F 4/80) and interleukin-6 (IL-6) secretion.ResultsEAE symptoms started at day 8 and peaked at day 15. Cell infiltrations (P = 0.0047) and demyelination (P = 0.0018) of EAE nerves correlated with the clinical score (r > 0.8). EAE led to a significant loss of RGCs (P< 0.0001). Significantly more caspase 3+ cells were noted in these animals (P = 0.0222). They showed an increased expression of GFAP (P< 0.0002) and a higher number of microglial cells (P< 0.0001). Also more macrophages and IL-6 secretion were observed in EAE mice.ConclusionsMOG immunization leads to optic neuritis and RGC loss. EAE severity is related to the severity of optic nerve inflammation and demyelination. EAE not only affects activation of apoptotic signals, but also causes a glial response in the retina.

Mitochondrial oxygen tension (mitoPO(2)) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO(2) in vivo exists. Here we report in vivo measurement of mitoPO(2) and the recovery of mitoPO(2) histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO(2) in rat liver in vivo. The results demonstrate mitoPO(2) values of approximately 30-40 mmHg. mitoPO(2) was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO(2) distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications.