Gotowa bibliografia na temat „Calcineurin B2”

Endurance training leads to many adaptational changes in several tissues. In skeletal muscle, fatty acid usage is enhanced and mitochondrial content is increased. The exact molecular mechanisms regulating these functional and structural changes remain to be elucidated. Contractile activity-induced metabolic perturbation has repeatedly been shown to be important for the induction of mitochondrial biogenesis. Recent reports suggest that the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α)/mitochondrial transcription factor A (Tfam) pathway is involved in exercise-induced mitochondrial biogenesis. In the present study, nine healthy men performed two 45-min bouts of one-legged knee extension exercise: one bout with restricted blood flow, and the other with nonrestricted blood flow to the working muscle. Muscle biopsies were obtained from the vastus lateralis muscle before exercise and at 0, 30, 120, and 360 min after the exercise bout. Biopsies were analyzed for whole muscle, as well as fiber-type specific mRNA expression of myocyte-enriched calcineurin interacting protein (MCIP)-1, PGC-1α, and downstream mitochondrial transcription factors. A novel finding was that, in human skeletal muscle, PGC-1α mRNA increased more after exercise with restricted blood flow than in the nonrestricted condition. No changes were observed for the mRNA of NRF-1, Tfam, mitochondrial transcription factor B1, and mitochondrial transcription factor B2. Muscle fiber type I and type II did not differ in the basal PGC-1α mRNA levels or in the expression increase after ischemic training. Another novel finding was that there was no difference between the restricted and nonrestricted exercise conditions in the increase of MCIP-1 mRNA, a marker for calcineurin activation. This suggests that calcineurin may be activated by exercise in humans and does not exclude that calcineurin could play a role in PGC-1 transcription activation in human skeletal muscle.

Calcineurin inhibitors like FK506 (tacrolimus) are routinely used for immunosuppression following transplantation. Its use is limited by many side effects, including renal tubular acidosis (RTA), mainly of the distal type. In this study, rats were treated with FK506 and at baseline (after 9 days) systemic acid-base status was similar to that in control animals. However, FK506-treated rats given NH4Cl in the drinking water for 2 days developed a more severe metabolic acidosis than control animals. Urine pH was more alkaline, but net acid excretion was normal. After 7 days of acid load, all differences related to acid-base homeostasis were equalized in both groups. Protein abundance of type IIa Na-Pi cotransporter, type 3 Na+/H+ exchanger, and electrogenic Na+-bicarbonate cotransporter, and both a4 and B2 subunits of the vacuolar H+-ATPase were reduced under baseline conditions, while induction of metabolic acidosis enhanced protein abundance of these transporters in FK506-treated animals. In parallel, protein expression of AE1 was reduced at baseline and increased together with pendrin during NH4Cl loading in FK506 rats. Protein abundance of the Na+-bicarbonate cotransporter NBCn1 was reduced under baseline conditions but remained downregulated during metabolic acidosis. Morphological analysis revealed an increase in the relative number of non-type A intercalated cells in the connecting tubule and cortical collecting duct at the expense of principal cells. Additionally, subcellular distribution of the a4 subunit of the vacuolar H+-ATPase was affected by FK506 with less luminal localization in the connecting tubule and outer medullary collecting duct. These data suggest that FK506 impacts on several major acid-base transport proteins in the kidney, and its use is associated with transient metabolic acidosis and altered expression of key renal acid-base transport proteins.

Patients with myelomonocytic and monocytic AML are often resistant to current standards of care. LILRB4, an immunosuppressive myeloid checkpoint, is expressed in myelomonocytic and monocytic AML and CMML. Preclinical evaluation of a novel anti-LILRB4 IgG1 monoclonal antibody, IO-202, demonstrated 3 mechanisms of action: 1) activation of effector T-cells; 2) prevention of leukemic blast infiltration; and 3) killing of LILRB4-high blasts via ADCC and ADCP. IO-202 exhibits high affinity binding to human LILRB4, blocking APOE binding and activation of LILRB4. Inhibiting this pathway activates T-cell cytotoxicity against leukemic cells, reversing T-cell suppression mediated by AML cells in vitro. In a syngeneic mouse AML model, IO-202 increases CD8+ T cells, resulting in tumor growth inhibition. In mouse xenograft models, IO-202 inhibits AML tumor growth, prolongs survival, and blocks tissue infiltration of leukemia cells. IO-202 depletes leukemic cells expressing high levels of LILRB4 in vitro through ADCC and ADCP. In cynomolgus monkeys IO-202 exhibited the expected PK profile. In a GLP 4-week repeated-dose IV toxicity study with a 10-week recovery period in cynomolgus monkeys, IO-202 was well-tolerated at all doses tested, up to 180 mg/kg/dose. IO-202-related microscopic findings were noted in the brain, spleen and lung; the microscopic finding of mild multifocal gliosis at ≥60 mg/kg/dose was considered adverse. At the end of the recovery period, all findings showed signs of complete or partial reversibility. TheNOAEL was 20 mg/kg, providing a sufficient safety margin for the clinical starting dose of 0.03 mg/kg. The nonclinical assessment provides a comprehensive understanding of IO-202 and is adequate to support its evaluation in the proposed Phase 1 study. Study Design and Methods Major inclusion criteria for the phase 1 study (NCT04372433) include: 1) Age > 18; 2) Relapsed AML with myelomonocytic or monoblastic/monocytic differentiation or CMML and has failed treatment with available active therapies; 3) adequate renal/hepatic function; and 4) ECOG 0-2. Major exclusion criteria include: 1) HSCT within 60 days, on calcineurin inhibitors, or chronic GVHD; 2) chemotherapy, radiotherapy, or investigational agents within 7 days; 3) significant cardiac disease; 4) active infection; 5) uncontrolled CNS leukemia; and 6) hyperleukocytosis (> 25 x 109/L, although hydroxyurea is permitted). The primary objective is assessment of safety and tolerability at increasing doses of IO-202 to estimate the MTD or MAD, and select the RP2D. Secondary objectives include characterizing PK, immunogenicity, and preliminary anti-leukemia activity. Biomarker evaluation will include changes in blasts and immune cells, cytokine profiling, LILRB4 expression level and receptor occupancy, and MRD assessment. Dose escalation will employ an accelerated titration scheme to minimize exposure to low-dose levels, with conversion to a 3+3 design with the occurrence of > Grade 2 AEs. The RP2D will be determined by the totality of data including the MAD or MTD, PK, PD, and AEs during a 28-day DLT evaluation period. After RP2D determination, an expansion cohort will be enrolled to assess the preliminary efficacy of IO-202 in R/R myelomonocytic and monocytic AML. In addition to descriptive statistics for AEs, PK, PD, and response rates, a Bayesian estimation of DLTs will be performed to further assess safety. If there is a >80% probability of DLTs being > 20%, the study will be paused to further evaluate the safety findings. A planned protocol amendment will evaluate combination therapies with IO-202, including IO-202 + azacitidine. Disclosures DiNardo: Jazz: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Novartis: Consultancy; Calithera: Research Funding; MedImmune: Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; ImmuneOnc: Honoraria; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria, Research Funding; Syros: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Pollyea:Karyopharm: Consultancy; Novartis: Consultancy; Genentech: Consultancy; Agios: Consultancy; Celgene/BMS: Consultancy; Pfizer: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Consultancy; Abbvie: Consultancy, Research Funding; Janssen: Consultancy; 47: Consultancy, Research Funding; Amgen: Consultancy; Glycomimetics: Other; Syros: Consultancy; Syndax: Consultancy. Konopleva:Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Forty-Seven: Consultancy, Research Funding; Calithera: Research Funding; Ablynx: Research Funding; Cellectis: Research Funding; Genentech: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Sanofi: Research Funding; Kisoji: Consultancy; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; AstraZeneca: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Eli Lilly: Research Funding; Agios: Research Funding. Hong:Roche: Current equity holder in publicly-traded company; Immune-Onc Therapeutics, Inc.: Current Employment. Huang:Immune Onc Therapeutics, Inc: Current Employment; Roche: Current equity holder in publicly-traded company; Exelixis: Current equity holder in publicly-traded company. Song:Immune Onc Therapeutics: Current Employment; Roche: Current equity holder in publicly-traded company. Wieland:Immune-Onc Therapeutics: Consultancy. Woodard:Immune-Onc Therapeutics, Inc: Current Employment; Bellicum, Inc: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Liao:Immune Onc Therapeutics: Current Employment. Zhang:Immune-Onc Therapeutics: Research Funding. Patel:Celgene: Consultancy, Speakers Bureau; DAVA Pharmaceuticals: Honoraria; France Foundation: Honoraria; Agios: Consultancy. Aribi:Seattle Genetics: Consultancy.

Myofibrillogenesis is a complex process involving assembly of many structural proteins in an orchestrated spatio-temporal manner to form a highly ordered contractile sarcomeric unit. Mutations in the proteins involved in muscle contraction and function lead to myopathic conditions in human. Hence, understanding the etiology of these diseases and genes involved may help in accurate diagnosis, prognosis and exploration of possible therapeutics. Molecular players and signaling pathways of myogenesis are highly conserved across phyla, enabling us to exploit indirect flight muscles (IFM) of Drosophila melanogaster as a model to study muscle development and function. IFM is the only fibrillar muscle which has considerable functional similarity to vertebrate cardiac muscles. It also enables the analyses of all stages of muscle development from its earliest stages of fusion of the imaginal myoblasts to fully differentiated muscle with its assembled contractile apparatus. Perturbance of developmental process in IFM leads to flightless flies with dysfunctional muscle. High throughput mutant screens, designed to isolate flightless flies have led to the identification of large number of genetic loci which are involved in muscle patterning and myofibrillogenesis, thus giving useful insights into the structural and functional aspects of fibre formation. One such classical mutant, flightless H (fliH), isolated during mutagenesis screen leads to IFM degeneration after fibres are formed normally. This interesting phenomenon is designated as muscle hypercontraction and is comparable to hypertrophic cardiomyopathies in humans. The muscle hypercontraction phenotype in this mutant was found to be temperature dependent and development of the process initiated at later stages of pupation. Cellular events associated with the IFM hypercontraction were followed up through development using this mutant. Further, interaction of fliH allele with other genetic backgrounds gave valuable insights on mechanisms of causation of muscle hypercontraction. Genetics played a pivotal role in identifying the mutant locus. The mutation was genetically mapped to the regulatory region of the wupA gene which was confirmed by sequencing data. The wupA gene codes for Troponin I (TnI), an inhibitory component of the troponin-tropomyosin complex of thin filaments. The mutation leads to reduced level of TnI transcript and hence reduced amount of protein, as a consequence, troponin complex formation is impeded leading to uninhibited acto-myosin interactions, thus causing muscle fibre breakdown. Our study reveals that fliH is a unique allele which confers temperature sensitive muscle phenotype. This is the first mutation found in the regulatory region of any structural gene which is temperature dependant and leads to muscle hypercontraction. This study also emphasizes that stoichiometry of structural proteins is important for proper functioning of muscle. Apart from mutations in sarcomeric genes, perturbations in calcium signaling also affect muscle functioning and lead to development of cardiac hypertrophy and failure. Hence, the role of calcineurin β-subunit (canB2), a calcium dependant protein phosphatase, in muscle was analyzed. Studies involving overexpression of canB2 in IFM showed that it leads to muscle hypercontraction. In addition, characterization of one of the new allele generated for the present study confirmed presence of muscle tearing and sarcomeric structure abnormality. canB2 alleles genetically interact with other hypercontracting alleles and enhance the hypercontraction phenotype. Overall, present study will help us to understand how genetic predisposition can enhance or suppress muscle hypercontraction. In a reverse genetics approach, role of muscle LIM protein, Beadex (Bx) in IFM was analyzed, as point mutations and loss of function alleles of LIM genes are associated with cardiomyopathies in humans. Immuno-histochemistry showed that Bx is expressed in myoblasts associated with wing imaginal disc which gives rise to IFM. Expression is also seen in developing IFM and in the neurons innervating the IFM. However, unlike the other known LIM proteins in Drosophila, Bx was not adhered to muscle fibre and showed predominant cytosolic localization. Targeted knockout and over-expression in muscles showed fibre rupturing and Z-disc deformities. Our results suggest that Bx may be involved in mechano-sensory stress signaling pathway like the other LIM proteins in humans and proper maintenance of the sarcomeric structure. Thus, present study elucidates the role of three loci namely: wupA, canB2 and Bx in proper muscle development and function. All the three loci code for proteins having orthologues in higher vertebrates and have been implicated in the pathogenesis of cardiomyopathies and/or skeletal myopathies in humans. Overall, such studies involving analyses of genes implicated in muscle development and function will help in exploring disease pathways which may help in derivation of new therapeutic strategies.