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Chloroquine is an antimalarial agent commonly used as an autophagy inhibitor by preventing autolysosomal fusion. Autophagy is an important cellular process through which defective cytoplasmic structures are recycled and degraded that has been implicated in aging neuromuscular effects. We recently showed that acute chloroquine treatment blunts the maximal forces generated by the diaphragm muscle, likely by impairing neuromuscular transmission generally consistent with aging effects in the diaphragm muscle. In addition, old mice show increased accumulation of autophagy proteins in phrenic motor neurons, consistent with impaired autophagy flux. In the present study, we hypothesized that chloroquine treatment results in accumulation of bulk endosomes pre-synaptically. Each hemidiaphragm with attached phrenic nerve was excised from 24-month-old male and female C57BL/6 mice to evaluate frequency dependent (0 vs 80 Hz stimulation) uptake of the styryl dye FM4-64, given that 80-Hz stimulation frequency induces bulk endocytosis after synaptic vesicle retrieval. Diaphragm-phrenic nerve preparations were incubated in 5 μM FM4-64 for 1 h prior to 6-min nerve stimulation (0.5 ms supramaximal pulses in 80 Hz, 200 ms trains delivered at 40% duty cycle) followed by a 5-min post-stimulation incubation. FM4-64 uptake at diaphragm neuromuscular junctions (NMJs) was measured using confocal microscopy. Preparations were randomly assigned to co-incubation with chloroquine (50 μM) or vehicle (Rees-Simpson solution). There was minimal FM4-64 uptake in the absence of phrenic nerve stimulation, consistent with the activity-dependent uptake of the styryl dye. With 80 Hz stimulation, high levels of FM4-64 uptake were evident in both the chloroquine and vehicle-treated groups without significant differences across groups. FM4-64 fluorescence was evident throughout the presynpatic terminal with few areas of dense accumulation, consistent with retention of endosomes. There was no evidence of greater endosome accumulation with chloroquine treatment. Of note, a slight increase in FM4-64 uptake was evident in the chloroquine treated groups compared to vehicle in the non-stimulated NMJs. These results are consistent with FM4-64 uptake reflecting bulk endocytosis that mainly occurs during high frequency stimulation. The impairment in autophagy flux following chloroquine treatment is expected to result in endosome accumulation but it is possible that the stimulation protocol was insuffcient to elucidate this possibility. Collectively, the findings of this study are consistent with impaired autophagy at diaphragm presynaptic terminals which may underlie neuromuscular deficits observed in old age. Supported by NIH grant R01 AG057052 and the Mayo Clinic. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In rats, there is a strong association between the emergence of mature diaphragm muscle (DIAm) myosin heavy chain isoform expression and the expansion of phrenic motor neuron (PhMN) size (from ~21-28 days postnatal (P)), alongside the manifestation of mature morphological and functional properties. In the Spastic ( Spa) mutant mouse, mutated glycine receptors lead to a spasticity phenotype emerging between P14-28, concomitant with DIAm and PhMN development. In adult Spa mice, DIAm weakness, fiber atrophy and loss of larger PhMNs is evident. We aim to characterize whether these DIAm and PhMN deficits occur contemporaneously to spasticity. Using wildtype (WT) and Spa mice at P14, P21, P28, and P90 (maturity) we evaluated DIAm and PhMN physiologic properties. Ex vivo DIAm strips were used for functional assessments of maximum specific force (Po) and fatigue. DIAm strips were also fresh-frozen, and 8 μm cross-sections were cut and stained with H&E to evaluate cross-sectional area (CSA) of individual DIAm fibers. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 μm) and prepared for confocal imaging and PhMN assessment. In WT mice, DIAm specific force increased at each age (P14, P21, P28 and P90), whilst in Spa mice, DIAm specific force was reduced from P21 compared to WT. Similarly, DIAm fiber CSA was reduced in Spa compared to WT mice at P21, P28 and P90. The number of PhMNs was similar between genotypes at P21, with a reduction in the number of PhMNs in Spa mice compared to WT evident at P28 continuing to P90. In Spa mice, PhMN size was smaller compared to WT at P21, unchanged at P28 and reduced at P90. The changes in Spa DIAm and PhMNs during the first postnatal month are consistent with the emergence of spasticity from P14 to P28. As most changes occur at ~P21, we are optimistic that interventions prior to weaning may promote the normal growth of DIAm and survival of PhMNs and may stave off later life DIAm behavioral deficits. HL166204 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract Background Perioperative morbidity and mortality remain a point of concern for the Associating Liver Partition and Portal Vein Ligation for Staged hepatectomy (ALPPS) procedure. Post-hepatectomy liver failure (PHLF) after the second step poses the most significant risk factor for futile outcome. Recovery of liver function after the first step of ALPPS is critically important to allow for sufficient liver function after this surgery. The APRI+ALBI score has been proposed as an easily assessable score to monitor liver function and its dynamic recovery. Aims We explored if the APRI+ALBI score was able to assess liver function recovery after the first step of ALPPS and allow for optimal timing of the 2nd step. Methods Based on the ALPPS registry, patients from 2012 to 2020 with available APRI+ALBI score were included. Postoperative outcomes (PHLF A – C, PHLF B + C, 90-day mortality and severe morbidity) were defined as per standard definitions. The APRI/ALBI score was monitored perioperatively. Results Overall, 464 patients from 16 international participating centers in the ALPPS registry were included. Clinically relevant PHLF (B + C) was observed in 7.5 % and of these 63 % of patients ultimately died due to liver failure. The APRI+ALBI score immediately increased after each surgical intervention and continuously decrease thereafter. Failure of the APRI+ALBI score to decrease until the 2nd step of ALPPS predicted PHLF B+C (p = 0.001; AUC 0.78). Particularly patients with high APRI+ALBI scores and short interstage intervals were at risk to develop PHLF B+C. Conclusion The APRI+ALBI score objectifies liver function recovery after the 1st step of ALPPS and failure to sufficiently decreased is associated with a higher risk for PHLF B+C. This might allow for optimized planning of the 2nd step of ALPPS to allow for sufficient liver function recovery to minimize the risk for PHLF B+C and associated futile outcome.

Purpose: To investigate the molecular characteristics of and potential for precision medicine in KRAS wildtype pancreatic ductal adenocarcinoma (PDAC). Patients and Methods: We investigated 27 patients with KRASWT PDAC at our institution. Clinical data were obtained via chart review. Tumor specimens for each subject were interrogated for somatic single nucleotide variants, insertion and deletions, and copy number variants by DNA sequencing. Gene fusions were detected from RNA-seq. A patient-derived organoid (PDO) was developed from a patient with a MET translocation and expanded ex vivo to predict therapeutic sensitivity prior to enrollment in a phase 2 clinical trial. Results: Transcriptomic analysis showed our cohort may be stratified by the relative gene expression of the KRAS signaling cascade. The PDO derived from our patient harboring a TFG-MET rearrangement was found to have in vitro sensitivity to the multi-tyrosine kinase inhibitor crizotinib. The patient was enrolled in the phase 2 SPARTA clinical trial and received monotherapy with vebrelitinib, a c-MET inhibitor, and achieved a partial and durable response. Conclusions: KRASWT PDAC is molecularly distinct from KRASMUT and enriched with potentially actionable genetic variants. In our study, transcriptomic profiling revealed that the KRAS signaling cascade may play a key role in KRASWT PDAC. Our report of a KRASWT PDAC patient with TFG-MET rearrangement who responded to a cMET inhibitor further supports the pursuit of precision oncology in this sub-population. Identification of targetable mutations, perhaps through approaches like RNA-seq, can help enable precision-driven approaches to select optimal treatment based on tumor characteristics.

Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of neurological autoimmune diseases is promising, but CAR T cell kinetics and immune alterations after treatment are poorly understood. Here, we performed single-cell multi-omics sequencing of paired cerebrospinal fluid (CSF) and blood samples from patients with neuromyelitis optica spectrum disorder (NMOSD) treated with anti–B cell maturation antigen (BCMA) CAR T cells. Proliferating cytotoxic-like CD8 + CAR T cell clones were identified as the main effectors in autoimmunity. Anti-BCMA CAR T cells with enhanced features of chemotaxis efficiently crossed the blood-CSF barrier, eliminated plasmablasts and plasma cells in the CSF, and suppressed neuroinflammation. The CD44-expressing early memory phenotype in infusion products was potentially associated with CAR T cell persistence in autoimmunity. Moreover, CAR T cells from patients with NMOSD displayed distinctive features of suppressed cytotoxicity compared with those from hematological malignancies. Thus, we provide mechanistic insights into CAR T cell function in patients with neurological autoimmune disease.

Objective Previous studies indicate that eosinophils are recruited into the allograft following orthotopic liver transplantation and protect from ischaemia reperfusion (IR) injury. In the current studies, we aim to explore whether their protective function could outlast during liver repair. Design Eosinophil-deficient mice and adoptive transfer of bone marrow-derived eosinophils (bmEos) were employed to investigate the effects of eosinophils on tissue repair and regeneration after hepatic IR injury. Aside from exogenous cytokine or neutralising antibody treatments, mechanistic studies made use of a panel of mouse models of eosinophil-specific IL-4/IL-13-deletion, cell-specific IL-4rα-deletion in liver macrophages and hepatocytes and macrophage-specific deletion of heparin-binding epidermal growth factor-like growth factor (hb-egf). Result We observed that eosinophils persisted over a week following hepatic IR injury. Their peak accumulation coincided with that of hepatocyte proliferation. Functional studies showed that eosinophil deficiency was associated with a dramatic delay in liver repair, which was normalised by the adoptive transfer of bmEos. Mechanistic studies demonstrated that eosinophil-derived IL-4, but not IL-13, was critically involved in the reparative function of these cells. The data further revealed a selective role of macrophage-dependent IL-4 signalling in liver regeneration. Eosinophil-derived IL-4 stimulated macrophages to produce HB-EGF. Moreover, macrophage-specific hb-egf deletion impaired hepatocyte regeneration after IR injury. Conclusion Together, these studies uncovered an indispensable role of eosinophils in liver repair after acute injury and identified a novel crosstalk between eosinophils and macrophages through the IL-4/HB-EGF axis.

Abstract Background: Based on preclinical data showing addition of CDK4/6 inhibitors to gemcitabine is synergistic, ribociclib was evaluated in combination with gemcitabine to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT). Methods: In this single arm multicohort phase I trial, we evaluated the safety and efficacy of Ribociclib plus Gemcitabine in patients with advanced solid tumors. Patients received Gemcitabine intravenously on days 1 and 8 followed by Ribociclib days 8–14, with treatment repeated every 3 weeks. Results: The study enrolled 43 patients between October 2017 and September 2019. The escalation phase (19 patients) determined the MTD and recommended phase II dose (RP2D) to be ribociclib 800mg daily and gemcitabine 1000mg/m2 for the expansion phase (24 patients). One patient experienced Grade 4 thrombocytopenia. Eleven patients experienced Grade 3 adverse events (AE), the most common being neutropenia, thrombocytopenia, and anemia. No partial or complete responses were observed. 15/22 (68%) of efficacy evaluable patients who received the MTD achieved best response of stable disease. Conclusions: The addition of Ribociclib to Gemcitabine was tolerated well and yielded stability of tumors in both cohorts. Ribociclib and gemcitabine could have synergistic activity in certain tumor types, and our data provides support for the combination. Clinical Trial Registration: NCT03237390

Introduction: The predictive value of left atrial (LA) volume and reservoir function (ResF) post-atrial fibrillation (AF) ablation has been scarcely studied. Hypothesis: Abnormal LA volume and ResF post-ablation are associated with adverse cardiovascular events. Methods: We analyzed LA maximum (LAVImax) and minimum (LAVImin) volume index, and 2 measures of ResF, emptying fraction (LAEF) and expansion index (LAEI), prospectively measured at 3-month post-procedure in patients in sinus rhythm who underwent AF ablation between 2007-2016. Composite endpoint (CE) at follow up was all-cause mortality, cardiac hospitalization (heart failure or acute ischemic event) and stroke. Results: 792 patients were studied ( age 60±10 years, 25% women ; Table 1) . Over 7.5 years [IQR 3.0-9.7], 96 (12%) patients experienced CE, which was more prevalent in patients with enlarged LA and reduced ResF. Impaired LA volumes and ResF were each independently associated with CE ( Fig.1 ), even after adjustment for clinical, demographic and echo characteristics (Model A, Fig.2 ) (LAVImax ≥35 ml/m 2 : HR 2.15 [95% CI, 1.35-3.43], p=0.001; LAVImin≥20.5 ml/m 2 : HR 2.88 [1.77-4.69], p<0.0001; LAEF<39%: HR 2.00 [1.31-3.05], p=0.001; LAEI<65%: HR 1.95 [1.27-3.01], p=0.003). ResF measures (LAEF and LAEI) were incremental to LAVImax (all p<0.05), but not LAVImin (all p>0.05; Fig.2 ). In patients with normal LA (LAVImax <35 ml/m 2 ; n=408), increased LAVImin (≥20.5 ml/m 2 ) remained associated with CE (HR 3.11 [1.45-6.67], p=0.004) and cardiac hospitalization (HR 5.81 [1.87-18.01], p=0.002). These findings were independent of AF recurrence. Conclusions: In these patients, enlarged LA and impaired ResF at 3-month post-ablation were strongly associated with adverse cardiovascular outcomes, regardless of AF recurrence. Measures of LA ResF were incremental to LAVImax. Notably, LAVImin showed the strongest associations and even identified a high-risk subgroup among patients with non-dilated LA.