Ablation of Calsequestrin-1, Ca2+ unbalance, and susceptibility to heat stroke

Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels. The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation. The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved. Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE

Seasonal variation in preference dictates space use in an invasive generalist

The spatiotemporal distribution of resources is a critical component of realized animal distributions. In agricultural landscapes, space use by generalist consumers is influenced by ephemeral resource availability that may produce behavioral differences across agricultural seasons, resulting in economic and production consequences and increased human-wildlife conflict. Our objective was to assess changes in habitat selection across seasons in an invasive generalist omnivore (feral pigs, Sus scrofa). Hypothesizing that pig space use is primarily driven by forage availability, we predicted strong selection for the most nutritionally beneficial crops and resource types as agricultural seasons progressed. We deployed GPS collars on 13 adult feral pigs in the Mississippi Alluvial Valley to study resource selection in a fragmented agricultural landscape. We estimated resource selection using mixed-effect logistic regression to assess variation in selection across planting, growing, harvest, and fallow seasons

Neoadjuvant eribulin mesylate following anthracycline and taxane in triple negative breast cancer: Results from the HOPE study

Background Eribulin mesylate (E) is indicated for metastatic breast cancer patients previously treated with anthracycline and taxane. We argued that E could also benefit patients eligible for neoadjuvant chemotherapy. Methods Patients with primary triple negative breast cancer 2 cm received doxorubicin 60 mg/m2 and paclitaxel 200 mg/m2 x 4 cycles (AT) followed by E 1.4 mg/m2 x 4 cycles. Primary endpoint was pathological complete response (pCR) rate; secondary and explorative endpoints included clinical/metabolic response rates and safety, and biomarker analysis, respectively. Using a two-stage Simon design, 43 patients were to be included provided that 4 of 13 patients had achieved pCR in the first stage of the study. Results In stage I of the study 13 women were enrolled, median age 43 years, tumor size 2–5 cm in 9/13 (69%), positive nodal status in 8/13 (61%). Main grade 3 adverse event was neutropenia (related to AT and E in 4 and 2 cases, respectively). AT followed by E induced clinical complete + partial responses in 11/13 patients (85%), pCR in 3/13 (23%). Median measurements of maximum standardized uptake value (SUVmax) resulted 13, 3, and 1.9 at baseline, after AT and E, respectively. Complete metabolic response (CMR) occurred after AT and after E in 2 and 3 cases, respectively. Notably, 2 of the 5 (40%) patients with CMR achieved pCR at surgery. Immunostaining of paired pre-/post-treatment tumor specimens showed a reduction of β-catenin, CyclinD1, Zeb-1, and c-myc expression, in the absence of N-cadherin modulation. The study was interrupted at stage I due to the lack of the required patients with pCR. Conclusions Despite the early study closure, preoperative E following AT showed clinical and biological activity in triple negative breast cancer patients. Furthermore, the modulation of β-catenin pathway core proteins, supposedly outside the domain of epithelial–mesenchymal transition, claims for further investigation. Trial registration EU Clinical Trial Register, EudraCT number 2012-004956-12

Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells.Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells

Deletion of small ankyrin 1 (SANK1) isoforms results in structural and functional alterations in aging skeletal muscle fibers

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4\u20136 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12\u201315 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Q\u3b3 charge and L-type Ca2+ current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture

AB0370 UTILITY OF CRP AND ESR IN THE DIAGNOSIS OF GIANT CELL ARTERITIS RELAPSE IN A PHASE 2 TRIAL OF MAVRILIMUMAB

Background:No universally accepted definition of flare currently exists in giant cell arteritis (GCA). Although relapses are defined mostly on clinical grounds (recurrence of GCA-related signs/symptoms), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) help clinicians assess disease activity. In fact, >70% of patients on glucocorticoids (GCs) alone have increased CRP or ESR when the disease is active. In contrast, tocilizumab, given its IL-6-blockade effect in the liver, rapidly reduces CRP and ESR levels, rendering them unreliable for disease activity monitoring. Mavrilimumab – a GM-CSF receptor α inhibitor with demonstrated efficacy in a Phase 2 GCA trial1 – downregulates inflammation upstream of IL-6. We hypothesized that mavrilimumab would not interfere with the utility of CRP and ESR in monitoring disease activity and in identifying GCA relapse.Objectives:To analyze the relationship between CRP/ESR and clinical disease activity in GCA patients treated with mavrilimumab.Methods:New-onset and relapsing GCA patients with active disease were recruited. GC-induced remission (no GCA symptoms and CRP <1 mg/dL or ESR <20 mm/hr) was required by baseline. Patients were randomized 3:2 to mavrilimumab 150 mg or placebo subcutaneously every 2 weeks plus a protocol-defined 26-week prednisone taper. The primary efficacy endpoint was time to relapse by Week 26. Relapse (adjudicated) was defined as recurrent GCA-related signs/symptoms, including new/worsening vasculitis on imaging, concurrent with CRP ≥1 mg/dL and/or ESR ≥30 mm/hr. CRP and ESR were also measured periodically during the trial.This post hoc analysis assessed the association of recurrent GCA-related signs/symptoms with concurrent CRP or ESR elevation post-randomization by treatment arm. We also assessed the proportion of patients with CRP or ESR elevation without GCA-related signs/symptoms up to Week 26.Results:Seventy patients were enrolled (mavrilimumab, N=42; placebo, N=28). The association of CRP or ESR elevation with unequivocal GCA-related signs/symptoms post-randomization was consistent regardless of treatment arm: 8/8 in the mavrilimumab group and 13/13 in the placebo group (Table 1). During relapse, median (range) CRP was 1.8 (1.4 – 8.4) mg/dL (mavrilimumab group) and 1.8 (1.1 – 9.0) mg/dL (placebo group). Corresponding ESR values were 39.5 (30 – 102) mm/hr (mavrilimumab group) and 49 (31 – 101) mm/hr (placebo group). Four mavrilimumab recipients had self-limited, equivocal GCA-related signs/symptoms without concurrent CRP or ESR elevation; all 4 completed the prespecified GC taper by Week 26 without need for rescue GCs, so relapse was not confirmed. At least 1 elevated CRP or ESR value in the absence of GCA-related signs/symptoms was observed in 58.8% of mavrilimumab recipients and 93.3% of placebo recipients by Week 26.Conclusion:The observed association of CRP or ESR elevation with GCA-related signs/symptoms is consistent with the upstream mechanism and supports the utility of the stringent protocol definition of relapse. The frequency and magnitude of CRP and ESR elevations at relapse were similar in both treatment groups, suggesting that CRP and ESR remain useful in assessments of disease activity in mavrilimumab-treated patients. CRP and ESR elevations without GCA-related signs/symptoms occurred more often in placebo recipients.References:[1]Cid, Unizony et al. Arthritis Rheumatol. 2020; 72 (suppl 10)Table 1.CRP and ESR levels in patients with or without GCA relapseAssessment§MavrilimumabPlaceboMavrilimumabPlaceboN=42N=28N=42N=28With RelapseWithout Relapse# of patients8 (19.1)13 (46.4)34 (81.0)15 (53.6) Elevated CRP* or ESR†8 (100.0)13 (100.0)20 (58.8)14 (93.3)  Elevated CRP*7 (87.5)10 (76.9)10 (29.4)11 (73.3)   Median (range) mg/dL1.8 (1.4 - 8.4)1.8 (1.1 - 9.0)2.6 (1.3 – 7.0)2.0 (1.0 – 6.6) Elevated ESR†6 (75.0)9 (69.2)16 (47.1)10 (66.7)  Median (range) mm/hr39.5 (30 - 102)49.0 (31 - 101)41.5 (30 - 110)53.5 (30 - 82)§# (%), except where indicated otherwise.*CRP ≥ 1 mg/dL†ESR ≥ 30 mm/hrDisclosure of Interests:Sebastian Unizony Consultant of: Janssen and Kiniksa, Grant/research support from: Genentech, Maria C. Cid Speakers bureau: Roche and Kiniksa, Paid instructor for: GSK and Vifor, Consultant of: Janssen, GSK, and Abbvie, Grant/research support from: Kiniksa, Elisabeth Brouwer Speakers bureau: Dr. E.Brouwer as an employee of the UMCG received speaker fees and consulting fees from Roche in 2017 2018 which were paid to the UMCG., Consultant of: Dr. E.Brouwer as an employee of the UMCG received speaker fees and consulting fees from Roche in 2017 2018 which were paid to the UMCG., Lorenzo Dagna Speakers bureau: Abbvie, Amgen, Biogen, BMS, Celltrion, Galapagos, Glaxo SmithKline, Novartis, Pfizer, Roche, Sanofi-Genzyme, SOBI, Consultant of: Abbvie, Amgen, Biogen, BMS, Celltrion, Galapagos, Glaxo SmithKline, Novartis, Pfizer, Roche, Sanofi-Genzyme, SOBI; clinical trial for Kiniksa, Grant/research support from: Abbvie, Amgen, BMS, Celltrion, Galapagos, Novartis, Pfizer, Roche, Sanofi-Genzyme, SOBI, Merk Sharp &Dohme, Janssen, Kiniksa, Bhaskar Dasgupta Paid instructor for: Educational grant symposium/workshop for Roche-chugai, Sanofi, and Abbvie, Consultant of: CI UK for the Kiniksa trial, Grant/research support from: Educational grant symposium/workshop for Roche-chugai, Sanofi, and Abbvie, Bernhard Hellmich Consultant of: Honoraria paid to the institution for participation in the clinical trial, Eamonn Molloy: None declared, Carlo Salvarani: None declared, Bruce C. Trapnell Consultant of: Consultant member of DSMB for Kiniksa., Kenneth J Warrington Consultant of: Clinical trial support from Eli Lilly and Kiniksa, Ian Wicks: None declared, Manoj Samant Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, Teresa Zhou Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, Lara Pupim Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, John F. Paolini Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceutical

Efficacy and safety of mavrilimumab in giant cell arteritis: a phase 2, randomised, double-blind, placebo-controlled trial

OBJECTIVES: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is implicated in pathogenesis of giant cell arteritis. We evaluated the efficacy of the GM-CSF receptor antagonist mavrilimumab in maintaining disease remission. METHODS: This phase 2, double-blind, placebo-controlled trial enrolled patients with biopsy-confirmed or imaging-confirmed giant cell arteritis in 50 centres (North America, Europe, Australia). Active disease within 6 weeks of baseline was required for inclusion. Patients in glucocorticoid-induced remission were randomly assigned (3:2 ratio) to mavrilimumab 150 mg or placebo injected subcutaneously every 2 weeks. Both groups received a 26-week prednisone taper. The primary outcome was time to adjudicated flare by week 26. A prespecified secondary efficacy outcome was sustained remission at week 26 by Kaplan-Meier estimation. Safety was also assessed. RESULTS: Of 42 mavrilimumab recipients, flare occurred in 19% (n=8). Of 28 placebo recipients, flare occurred in 46% (n=13). Median time to flare (primary outcome) was 25.1 weeks in the placebo group, but the median was not reached in the mavrilimumab group (HR 0.38; 95% CI 0.15 to 0.92; p=0.026). Sustained remission at week 26 was 83% for mavrilimumab and 50% for placebo recipients (p=0.0038). Adverse events occurred in 78.6% (n=33) of mavrilimumab and 89.3% (n=25) of placebo recipients. No deaths or vision loss occurred in either group. CONCLUSIONS: Mavrilimumab plus 26 weeks of prednisone was superior to placebo plus 26 weeks of prednisone for time to flare by week 26 and sustained remission in patients with giant cell arteritis. Longer treatment is needed to determine response durability and quantify the glucocorticoid-sparing potential of mavrilimumab. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT03827018, Europe (EUdraCT number: 2018-001003-36), and Australia (CT-2018-CTN-01 865-1)

Impact of infectious comorbidity and overall time of hospitalization in total outpatient management of acute myeloid leukemia patients following venetoclax and hypomethylating agents

Venetoclax (VEN) and hypomethylating agent (HMAs) regimens are emerging as the standard of care for unfit for chemotherapy acute myeloid leukemia (AML) patients, but the safety and feasibility of a total outpatient management have not been fully investigated. Fifty-nine AML patients with active disease received VEN and HMAs. Nineteen out of 59 (32.2%) patients received the first cycle as inpatients, whereas 40/59 (67.8%) patients were treated in the outpatient setting. No significant differences were observed with regard to incidence of adverse events (AEs), including tumor lysis syndrome (TLS), and the 30-day and 60-day mortality was comparable. Notably, an infectious prophylaxis inspired to that adopted during intensive chemotherapy resulted in a low infection rate with a reduced bacterial infections incidence in out- versus hospitalized patients (p&nbsp;&lt;.0001). The overall time of hospitalization was significantly shorter in patients who received a total outpatient treatment as compared to those who received the first cycle as inpatients (5.9 vs. 39.7&nbsp;days, p&nbsp;&lt;.0001). Despite the adopted differences in treatment management, the efficacy was similar. These data indicate that a total outpatient management of VEN and HMAs is feasible in AML patients without negatively impacting on treatment efficacy and may yield pharmacoeconomic and quality-of-life benefits

Redistribution of CD8+ T cell subsets in metastatic renal cell carcinoma patients treated with anti-PD-1 therapy

Renal-cell carcinoma (RCC) is responsible for the majority of tumors arising from the kidney parenchyma. Although a progressive improvement in median overall survival has been observed after the introduction of anti-PD-1 therapy, many patients do not benefit from this treatment. Therefore, we have investigated T cell dynamics to find immune modification induced by anti-PD-1 therapy. Here, we show that, after therapy, RCC patients (5 responders and 14 nonresponders) are characterized by a redistribution of different subsets across the memory T cell compartment

Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and na\uefve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM 12 plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies