Citations

Abstract BackgroundNiemann–Pick disease type C (NPC) is a rare, progressive, neurodegenerative disease associated with neurovisceral manifestations resulting from lysosomal dysfunction and aberrant lipid accumulation. A multicentre, prospective observational study (ClinicalTrials.gov ID:NCT02435030) of individuals with genetically confirmed NPC1 or NPC2 receiving routine clinical care was conducted, to prospectively characterize and measure NPC disease progression and to investigate potential NPC-related biomarkers versus healthy individuals. Progression was measured using the abbreviated 5-domain NPC Clinical Severity Scale (NPCCSS), 17-domain NPCCSS and NPC clinical database (NPC-cdb) score. Cholesterol esterification and heat shock protein 70 (HSP70) levels were assessed from peripheral blood mononuclear cells (PBMCs), cholestane-3β,5α-,6β-triol (cholestane-triol) from serum, and unesterified cholesterol from both PBMCs and skin biopsy samples. The inter- and intra-rater reliability of the 5-domain NPCCSS was assessed by 13 expert clinicians’ rating of four participants via video recordings, repeated after ≥3 weeks. Intraclass correlation coefficients (ICCs) were calculated.ResultsOf the 36 individuals with NPC (2–18 years) enrolled, 31 (86.1%) completed the 6–14-month observation period; 30/36 (83.3%) were receiving miglustat as part of routine clinical care. A mean (±SD) increase in 5-domain NPCCSS scores of 1.4 (±2.9) was observed, corresponding to an annualized progression rate of 1.5. On the 17-domain NPCCSS, a mean (±SD) progression of 2.7 (±4.0) was reported. Compared with healthy individuals, the NPC population had significantly lower levels of cholesterol esterification (p<0.0001), HSP70 (p<0.0001) and skin unesterified cholesterol (p=0.0006). Cholestane-triol levels were significantly higher in individuals with NPC versus healthy individuals (p=0.008) and correlated with the 5-domain NPCCSS (Spearman’s correlation coefficient=0.265, p=0.0411). The 5-domain NPCCSS showed high ICC agreement in inter-rater reliability (ICC=0.995) and intra-rater reliability (ICC=0.937).ConclusionsProgression rates observed were consistent with other reports on disease progression in NPC. The 5-domain NPCCSS reliability study supports its use as an abbreviated alternative to the 17-domain NPCCSS that focuses on the most relevant domains of the disease. The data support the use of cholestane-triol as a disease monitoring biomarker and the novel methods of measuring unesterified cholesterol could be applicable to support NPC diagnosis. Levels of HSP70 in individuals with NPC were significantly decreased compared with healthy individuals.Trial Registration: CT-ORZY-NPC-001: ClincalTrials.gov NCT02435030, Registered 6 May 2015, https://clinicaltrials.gov/ct2/show/NCT02435030; EudraCT 2014-005194-37, Registered 28 April 2015, https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-005194-37/DE. OR-REL-NPC-01: Unregistered.

Abstract Background: Deregulation of ceramide and sphingomyelinlevels have been suggested tocontribute tothe pathogenesis of Alzheimer’s disease (AD).Ceramide transfer proteins (CERTs) are ceramide carriers, crucial for ceramide and sphingomyelin balance in cells.Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain.Methods: The plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTLwith amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescencein HEK cells.The recombinant CERTL protein wasemployed to study interaction of CERTLwith amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes inAβ toxicity in neuroblastoma cells. CERTLwas overexpressed in neurons by adeno associatedvirus (AAV) in a familial mouse model of familial AD (5xFAD). Ten weeks after transduction animal were challenged with behavior tests for memory, anxiety and locomotion. At week twelve brains were investigated for sphingolipid levels by mass spectrometry, plaques and neuroinflammation by immunohistochemistry, gene expression and/or immunoassay.Results:Here, we report that CERTL, binds to APP, modifies Aβ aggregation and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male transgenic mice, modelling familial AD (5xFAD). CERTLin vivo over-expression hasa mild effect on animal locomotion and decreases Aβ formation and modulates microglia by decreasing their pro-inflammatory phenotype.Conclusion: Our results demonstratea crucial role of CERTL in regulatingceramidelevels in the brain, in amyloid plaque formation and neuroinflammation,thereby opening research avenuesfor therapeutic targets of AD and other neurodegenerative diseases.

Purpose Unprofessional behavior, which can include failure to engage, dishonest and/or disrespectful behavior, and poor self-awareness, can be demonstrated by medical trainees and practicing physicians. In the authors’ experience, these types of behaviors are associated with exposure to adverse childhood experiences (ACEs). Given this overlap, the authors studied the percentage of ACEs among trainees and physicians referred for fitness-for-duty evaluations and patterns between the types of ACEs experienced and the reason for referral. Method A final sample of 123 cases of U.S. trainees and physicians who had been referred to a Midwestern center for assessment and/or remediation of professionalism issues from 2013 to 2018 was created. Included professionalism lapses fell within 3 categories: boundary violation, disruptive behavior, or potential substance use disorder concerns. All participants completed a psychosocial developmental interview, which includes questions about ACE exposure. Overall rate of reported ACEs and types of ACEs reported were explored. Results Eighty-six (70%) participants reported at least 1 ACE, while 27 (22%) reported 4 or more. Compared with national data, these results show significantly higher occurrence rates of 1 or more ACEs and a lower occurrence rate of 0 ACEs. ACEs that predicted reasons for referral were physical or sexual abuse, feeling unwanted or unloved, witnessing abuse of their mother or stepmother, or caretaker substance use. Conclusions In this sample, ACE exposure was associated with professionalism issues. Remediating individuals with professionalism issues and exposure to ACEs can be complicated by heightened responses to stressful stimuli, difficulties with collaboration and trust, and decreased self-efficacy. Adoption of a trauma-informed medical education approach may help those that have been impacted by trauma rebuild a sense of control and empowerment. The findings of this study may be useful predictors in identifying those at risk of problematic behavior and recidivism before a sentinel event.

Measurement of sperm oxidative-antioxidant indicators is widely used in the assessment and detection of biochemical causes of male infertility. The main purpose of this study was to identify biomarkers that assist in diagnostics and monitoring of male reproductive potential. We performed the assessment of oxidative-antioxidant malondialdehyde (MDA), glutathione (GSH), and total redox antioxidant potential (TRAP) indicators in seminal plasma, seminogram, clinical condition, and lifestyle of people with reproductive problems. The combined assessment of GSH and TRAP as potential biomarkers of male infertility in semen plasma was characterized by the highest total sensitivity and specificity. Furthermore, we provide evidence that male reproductive potential is significantly correlated with basic sperm parameters, sperm cell membrane integrity, their morphology, lifestyle, eating habits, occupation, and mental health. Our results provide evidence on the importance of oxidative stress and defense against free radicals in diagnosing and monitoring men with infertility that are consistent with previously conducted research. We provide an alternative approach on the possibility of interpreting the combination of the biomarkers that can bring benefits to a multi-threaded approach to the diagnosis and treatment of male infertility.

Abstract Background Prosthetic joint infection (PJI) is a potentially limb-threatening complication of total knee arthroplasty. Phage therapy is a promising strategy to manage such infections including those involving antibiotic-resistant microbes, and to target microbial biofilms. Experience with phage therapy for infections associated with retained hardware is limited. A 62-year-old diabetic man with a history of right total knee arthroplasty 11 years prior who had suffered multiple episodes of prosthetic knee infection despite numerous surgeries and prolonged courses of antibiotics, with progressive clinical worsening and development of severe allergies to antibiotics, had been offered limb amputation for persistent right prosthetic knee infection due to Klebsiella pneumoniae complex. Intravenous phage therapy was initiated as a limb-salvaging intervention. Methods The patient received 40 intravenous doses of a single phage (KpJH46Φ2) targeting his bacterial isolate, alongside continued minocycline (which he had been receiving when he developed increasing pain, swelling, and erythema prior to initiation of phage therapy). Serial cytokine and biomarker measurements were performed before, during, and after treatment. The in vitro anti-biofilm activity of KpJH46Φ2, minocycline and the combination thereof was evaluated against a preformed biofilm of the patient’s isolate and determined by safranin staining. Results Phage therapy resulted in resolution of local symptoms and signs of infection and recovery of function. The patient did not experience treatment-related adverse effects and remained asymptomatic 34 weeks after completing treatment while still receiving minocycline. A trend in biofilm biomass reduction was noted 22 hours after exposure to KpJH46Φ2 (P = .063). The addition of phage was associated with a satisfactory outcome in this case of intractable biofilm-associated prosthetic knee infection. Pending further studies to assess its efficacy and safety, phage therapy holds promise for treatment of device-associated infections.

Abstract Introduction Voluntary sleep curtailment decreases insulin sensitivity. However, molecular mechanisms underlying impaired insulin signaling are not completely understood. To gain molecular insights, we examined the transcription of known cellular insulin-signaling regulators in adipose tissue obtained from subjects undergoing experimental sleep restriction. Methods Nineteen healthy subjects (males: 11; age: 19 - 36 years; BMI: 24.5 ± 3.6 kg/m2) underwent a normal (9 hours/night) and a restricted sleep (4 hours/night) sequence in a random order. Each inpatient stay included a screen visit followed by 4 days of acclimation and 9 days of experimental phase consisting of a controlled sleep opportunity. Eucaloric diet was provided to ensure weight maintenance. Abdominal fat sample was obtained at the screen and end of the experimental phase. mRNA was quantified by RT-PCR. Fasting morning blood draws were obtained at the end of acclimation and experimental phases. Mixed models were used for analysis. Results mRNA expression did not differ in normal and restricted sleep condition for SOCS3 [changes in normal Vs. restricted sleep, 0.02 (0.11, -0.01) Vs. 0.01 (0.12, -0.07), p=0.33], PTEN [-0.22 (0.18, -0.39) Vs. -0.10 (0.21, -0.20), p=0.22], PTB1B [-0.003 (0.04, -0.07) Vs. -0.01 (0.06, -0.06), p=0.74] and Cav-1 [-2.45 (0.78, -8.39) Vs. -6.31 (1.68, -8.29), p=0.34]. Further, transcription of insulin-receptor also did not change [0.01 (0.07, -0.06) Vs. -0.03 (0.09, -0.07), p=0.92]. However, within group analysis show significant decreases in Cav-1 mRNA only during sleep restriction (normal: p=0.09; restricted: p=0.003). Importantly, restricting sleep duration was associated with lowering of insulin (0.6 ± 1.9 Vs. -1.6 ± 1.7 uIU/ml; p=0.003), no change in glucose (-1.5 ± 3.8 Vs. -3.7 ± 4.12 mg/dl, p=0.06) and improvement in HOMA-IR index (0.12 ± 0.44 Vs -0.42 ± 0.43, p=0.002). Conclusion Chronic sleep restriction does not alter the transcription of cell-signaling regulators in abdominal adipose tissue. Moreover, restricting sleep duration without increasing calorie intake did not seem to decrease insulin sensitivity as determined by HOMA-IR. Support NIH grants HL114676, Mayo Clinic CCaTS UL1 TR002377; AHA grant 13POST16420009 to NC

10017 Background: Seviprotimut-L is a vaccine prepared from antigens of 3 human melanoma cell lines, administered with alum. Prior formulations induced T cell and antibody responses and improved survival in a small phase II clinical trial. Part B1 of MAVIS (Melanoma Antigen Vaccine Immunotherapy Study, a three part, Phase III clinical program), was a multicenter, double-blind, placebo-controlled trial to assess efficacy of seviprotimut-L, with the primary endpoint of relapse-free survival (RFS). The goal of Part B1 was to guide design of the pivotal Part B2. Methods: Patients with AJCC v7 stage IIB-III cutaneous melanoma, after surgical resection, age 18-75, ECOG PS 0-1, were randomized 2:1 to seviprotimut-L 40 mcg or placebo, injected subcutaneously every 2 weeks x 5, then monthly x 4, then every 3 months x 9. Patients were stratified by stage (IIB/C, IIIA, IIIB/C). Target enrollment was 325. The study was powered for assessment of RFS, with target hazard ratio (HR) of 0.625, one-sided alpha of 0.10, and power 80%. Final data are presented. Results: 347 patients were randomized. Arms were well-balanced. Treatment-related adverse events (AEs) led to discontinuation in 0.4% and 0%, respectively, for vaccine and placebo arms. There were no treatment-related SAEs. By intent-to-treat (ITT) analysis, RFS was not significantly longer for seviprotimut-L in the full study population but trended toward benefit (HR 0.88). Subgroup analysis based on planned stratification revealed the hazard ratio (HR) for the Stage IIB/IIC subset (randomization stratum, n=111) to be 0.65 (95% CI [0.37, 1.17]), favoring seviprotimut-L. Age can decrease immune competence: RFS was longer with vaccine for patients age <60 overall (N = 191, HR = 0.64 [0.38, 1.08]) and among Stage IIB/C patients (N = 52, HR = 0.32 [0.12, 0.86]). The effect modification interaction p value for age for stage IIB/IIC patients was 0.056. In a multivariable RFS model, for IIB/IIC patients <60 with ulceration (n=38), HR = 0.209 [0.07,0.61]. For overall survival, for patients < 60, HR = 0.41 [0.33,1.14] (n=191, 19 deaths) and for those ≥60, HR = 0.92 [0.39,2.12] (n = 156, 24 deaths). Conclusions: Seviprotimut-L is very well-tolerated. Subgroup efficacy analyses identified populations who may benefit from Seviprotimut-L: those with Stage IIB/IIC melanoma and those under age 60. These data support design of the definitive part B2 of the MAVIS phase III trial to test seviprotimut-L for stage IIB/C patients, with stratification by age and ulceration. Clinical trial information: NCT01546571.

10006 Background: Treatment options are limited for patients with advanced melanoma who have progressed on checkpoint inhibitors and targeted therapies. Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) leverages and enhances the body’s natural defense against cancer and has shown durable responses in heavily pretreated melanoma patients. Methods: C-144-01 is a global Phase 2 open-label, multicenter study of efficacy & safety of lifileucel in patients with unresectable metastatic melanoma who have progressed on checkpoint inhibitors and BRAF/MEK inhibitors, if BRAFv600 mutant. We report on Cohort 2 (N = 66) patients who have received TIL. Tumors were resected at local institutions, processed in central GMP facilities for TIL production, manufactured, cryopreserved & shipped back to sites in a 22-day process. Therapy consisted of one week of lymphodepletion, a single lifileucel infusion, and up to 6 IL-2 doses. ORR was based on RECIST v1.1 by investigator assessment. Data cutoff was Feb 2, 2020. Results: Baseline characteristics: 3.3 mean prior therapies (anti-PD1 100%; anti-CTLA-4 80%; BRAF/MEK inhibitor 23%), high baseline tumor burden (106 mm mean target lesion sum of diameters), 44% liver/brain lesions, 40.9% LDH > ULN. ORR by investigator was 36.4% (2 CR, 22 PR) and DCR was 80.3%. Mean time to response was 1.9 months (range: 1.3-5.6). After a median study follow-up of 17.0 months, median DOR (mDOR) was still not reached. Six responders have progressed, 2 have died and 2 started other anti-cancer therapy without progression. The adverse event profile was consistent with the underlying advanced disease and the lymphodepletion and IL-2 regimens. Additional follow-up data will be available for presentation. Conclusions: Lifileucel treatment results in a 36.4% ORR and mDOR was not reached at 17.0 months of median study follow up in a heavily pretreated metastatic melanoma patients with high baseline disease burden who progressed on multiple prior therapies, including anti-PD1 and BRAF/MEK inhibitors, if BRAFv600 mutant. Clinical trial information: NCT02360579.