Targeting TGFß-activated kinase-1 activation in microglia reduces CAR T immune effector cell-associated neurotoxicity syndrome.

Cancer immunotherapy with chimeric antigen receptor (CAR) T cells can cause immune effector cell-associated neurotoxicity syndrome (ICANS). However, the molecular mechanisms leading to ICANS are not well understood. Here we examined the role of microglia using mouse models and cohorts of individuals with ICANS. CD19-directed CAR (CAR19) T cell transfer in B cell lymphoma-bearing mice caused microglia activation and neurocognitive deficits. The TGFß-activated kinase-1 (TAK1)-NF-κB-p38 MAPK pathway was activated in microglia after CAR19 T cell transfer. Pharmacological TAK1 inhibition or genetic Tak1 deletion in microglia using Cx3cr1CreER:Tak1fl/fl mice resulted in reduced microglia activation and improved neurocognitive activity. TAK1 inhibition allowed for potent CAR19-induced antilymphoma effects. Individuals with ICANS exhibited microglia activation in vivo when studied by translocator protein positron emission tomography, and imaging mass cytometry revealed a shift from resting to activated microglia. In summary, we prove a role for microglia in ICANS pathophysiology, identify the TAK1-NF-κB-p38 MAPK axis as a pathogenic signaling pathway and provide a rationale to test TAK1 inhibition in a clinical trial for ICANS prevention after CAR19 T cell-based cancer immunotherapy.

Myelodysplastic Neoplasms (MDS) with Ring Sideroblasts or SF3B1 Mutations: The Improved Clinical Utility of World Health Organization and International Consensus Classification 2022 Definitions, a Single-Centre Retrospective Chart Review.

Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.

Serologic Response to Vaccine for COVID-19 in Patients with Hematologic Malignancy: A Prospective Cohort Study.

BACKGROUND: Patients with hematological cancers have increased COVID-19 morbidity and mortality, and these patients show attenuated vaccine responses. This study aimed to characterize the longitudinal humoral immune responses to COVID-19 vaccination in patients with hematological malignancies. PATIENTS AND METHODS: We conducted a prospective cohort study, collecting samples from March 2021 to July 2022, from patients seen at a cancer treatment center in London, Ontario, Canada, who met the following eligibility criteria: age ≥18 years, diagnosed with a hematological malignancy, recipient of a COVID-19 vaccine during the study period, and able to provide informed consent. RESULTS: Median anti-S titers (MST) were 0.0, 64.0, and 680.5 U/mL following first (V1), second (V2), and third (V3) vaccine doses, respectively. Patients with lymphoid malignancies' response to vaccination was attenuated compared to myeloid malignancy patients after V2 and V3 (P < .001, P < .01). Active treatment was associated with lower antibody titers (MST 10) compared to treatment 12-24 months (MST 465, P = .04367) and >24 months (MST 1660.5, P = .0025) prior to vaccination. V3 significantly increased antibody titers compared to V2 for patients less than 3 months from treatment. Increasing age was associated with smaller antibody response following V2 (P < .05), but not following V3. Patients receiving anti-CD20 therapy did not demonstrate increased antibody titer levels after V3 (V2 MST 0, V3 MST 0; P > .05). CONCLUSION: We report an attenuated serologic response to COVID-19 vaccination in our study population of patients with hematological malignancy. The immune response to vaccination was affected by patient age, diagnosis, treatment, and timing of treatment exposure.

Clinical Features and Long-Term Outcomes of a Pan-Canadian Cohort of Adolescents and Young Adults with Myeloproliferative Neoplasms: A Canadian MPN Group Study.

Myeloproliferative neoplasms (MPNs) are a group of chronic hematologic malignancies that lead to morbidity and early mortality due to thrombotic complications and progression to acute leukemia. Clinical and mutational risk factors have been demonstrated to predict outcomes in patients with MPNs and are used commonly to guide therapeutic decisions, including allogenic stem cell transplant, in myelofibrosis. Adolescents and young adults (AYA, age ≤45 years) comprise less than 10% of all MPN patients and have unique clinical and therapeutic considerations. The prevalence and clinical impact of somatic mutations implicated in myeloid disease has not been extensively examined in this population. We conducted a retrospective review of patients evaluated at eight Canadian centers for MPN patients diagnosed at ≤45 years of age. In total, 609 patients were included in the study, with median overall survival of 36.8 years. Diagnosis of prefibrotic or overt PMF is associated with the lowest OS and highest risk of AP/BP transformation. Thrombotic complications (24%), including splanchnic circulation thrombosis (9%), were frequent in the cohort. Mutations in addition to those in JAK2/MPL/CALR are uncommon in the initial disease phase in our AYA population (12%); but our data indicate they may be predictive of transformation to post-ET/PV myelofibrosis.

An Approach to the Investigation of Thrombocytosis: Differentiating between Essential Thrombocythemia and Secondary Thrombocytosis.

Background: Thrombocytosis is a common reason for referral to Hematology. Differentiating between secondary causes of thrombocytosis and essential thrombocythemia (ET) is often clinically challenging. A practical diagnostic approach to identify secondary thrombocytosis could reduce overinvestigation such as next generation sequencing (NGS) panel. Methods and Results: All adult patients with thrombocytosis (≥450 × 109/L) who underwent molecular testing at a single tertiary care centre between January 1, 2018 and May 31, 2021 were evaluated. Clinical and laboratory variables were compared between patients with secondary thrombocytosis vs. ET. Clinical variables included smoking, thrombosis, splenectomy, active malignancy, chronic inflammatory disease, and iron deficiency anemia. Laboratory variables included complete blood count (CBC), ferritin, and myeloid mutations detected by NGS. The overall yield of molecular testing was 52.4%; 92.1% of which were mutations in JAK2, CALR, and/or MPL. Clinical factors predictive of ET included history of arterial thrombosis (p < 0.05); active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency were associated with secondary thrombocytosis (p < 0.05). A diagnosis of ET was associated with higher hemoglobin, mean corpuscular volume (MCV), red cell distribution width (RDW), and mean platelet volume (MPV), while secondary thrombocytosis was associated with higher body mass index, white blood cells, and neutrophils (p < 0.01). Conclusion: A practical approach to investigating patients with persistent thrombocytosis based on clinical characteristics such as active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency may assist in accurately identifying patients more likely to have secondary causes of thrombocytosis and reduce overinvestigation, particularly costly molecular testing.

Outpatient consolidation chemotherapy with intermediate dose cytarabine has similar survival and relapses rates in acute myeloid leukemia as compared to high dose cytarabine: A single center analysis.

INTRODUCTION: The last decade has seen advances in delivering outpatient consolidation therapy for acute myeloid leukemia (AML). The standard of care involves high-dose cytarabine or intermediate-dose cytarabine, given twice daily for three alternating days. At the London Regional Cancer Program, we have transitioned the administration of outpatient cytarabine to a once-daily regimen over six consecutive days. The outcomes of a longer duration interval of high-dose cytarabine and intermediate-dose cytarabine is currently unknown. This study aims to assess the feasibility of administering a continuous 6-day protocol of high-dose (HDAC-16) and intermediate-dose cytarabine (IDAC-16) consolidation therapy in the outpatient setting. METHODS: This is a retrospective chart review to analyze AML patients treated with outpatient high-dose or intermediate-dose cytarabine consolidation therapy at the London Regional Cancer Program from January 1, 2019, through November 1, 2022. The primary objective was to determine the outcomes of the 6-day outpatient administration of once daily high-dose cytarabine or intermediate-dose cytarabine. RESULTS: Forty-five patients received 89 cycles of cytarabine as outpatients; males were 55.6% of the total population, with a median age of ~57 years. Our overall 2-year survival of HDAC-16 (57.1%) and IDAC-16 (83.3%) is consistent with the reported literature. There was no difference in delays, relapse rates, and nonrelapse mortality between both HDAC and IDAC groups. The 2-year relapse free survival was 57.1% for HDAC-16 and 66.7% for IDAC-16. CONCLUSION: Outpatient administration of intermediate-dose cytarabine once daily over six consecutive days results in similar overall survival and relapse rates as compared to high dose cytarabine consolidation chemotherapy. Moving to a once daily administration schedule can alleviate logistical and/or accessibility hurdles for outpatient oncology clinics. Prospective randomized trials are needed in this setting to validate our results.

Identification of 58 novel HLA alleles identified in Chinese individuals by next-generation sequencing.

HLA genes are the most polymorphic in the human genome. High resolution HLA typing from 13,870 bone marrow donors in Hong Kong was obtained using Next-generation sequencing (NGS) technology. Among the 67 novel alleles identified, official HLA allele names of 50 novel class I alleles (HLA-A, -B, -C) and 8 novel class II alleles (HLA-DRB1, -DQB1) were assigned by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System.

Novel α7 nicotinic acetylcholine receptor modulators as potential antitussive agents.

A novel series of α7 nicotinic acetylcholine receptor (nAChR) modulators was designed and evaluated for antitussive activity in an in vivo guinea pig model of chemically induced cough. Compound 16 at all tested doses (9.5, 3 and 1 mg/kg) significantly (p < 0.01) reduced the cumulative number of coughs and showed similar results to a positive control (codeine at 30 mg/kg). Among three different administration routes (intraperitoneal, oral and inhalation), compound 16 exerted a significant antitussive effect in guinea pigs at an inhaled dose as low as 0.4 mg/kg (p < 0.05). α7 nAChR modulators may provide a novel, non-narcotic approach to therapy in patients with acute and chronic cough.

A Rational Approach to JAK2 Mutation Testing in Patients with Elevated Hemoglobin: Results from the JAK2 Prediction Cohort (JAKPOT) Study.

BACKGROUND: Erythrocytosis, most often measured as an increase in hemoglobin and/or hematocrit, is a common reason for referral to internal medicine and hematology clinics and a rational approach is required to effectively identify patients with polycythemia vera while avoiding over-investigation. AIM: We aimed to develop and validate a simple rule to predict JAK2 mutation positivity based on complete blood count parameters to aid in the diagnostic approach to patients referred for elevated hemoglobin. SETTING: Internal medicine and hematology clinics at an academic tertiary referral center. PARTICIPANTS: The JAK2 Prediction Cohort (JAKPOT), a large retrospective cohort (n = 901) of patients evaluated by internal medicine and hematology specialists for elevated hemoglobin. DESIGN: JAK2 mutation analysis was performed in all patients and clinical and laboratory variables were collected. Patients were randomly divided into derivation and validation cohorts. A prediction rule was developed using data from the derivation cohort and tested in the validation cohort. KEY RESULTS: The JAKPOT prediction rule included three variables: (i) red blood cell count >6.45×1012/L, (ii) platelets >350×109/L, and (iii) neutrophils >6.2×109/L; absence of all criteria was effective at ruling out JAK2-positivity with sensitivities 94.7% and 100%, and negative predictive values of 98.8% and 100% in the derivation and validation cohorts, respectively, with an overall low false negative rate of 0.4%. The rule was validated for three different methods of JAK2 testing. Applying this rule to our entire cohort would have resulted in over 50% fewer tests. CONCLUSION: In patients with elevated hemoglobin, the use of a simple prediction rule helps to accurately identify patients with a low likelihood of having a JAK2 mutation, potentially limiting costly over-investigation in this common referral population.

Targeting MDM2 enhances antileukemia immunity after allogeneic transplantation via MHC-II and TRAIL-R1/2 upregulation.

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2 inhibition.

Effects of cigarette smoke exposure on a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory autoimmune disease associated with genetic and environmental factors. Cigarette smoking is harmful to health and may be one of the risk factors for MS. However, there have been no systematic investigations under controlled experimental conditions linking cigarette smoke (CS) and MS. The present study is the first inhalation study to correlate the pre-clinical and pathological manifestations affected by different doses of CS exposure in a mouse experimental autoimmune encephalomyelitis (EAE) model. Female C57BL/6 mice were whole-body exposed to either fresh air (sham) or three concentrations of CS from a reference cigarette (3R4F) for 2 weeks before and 4 weeks after EAE induction. The effects of exposure on body weight, clinical symptoms, spinal cord pathology, and serum biochemicals were then assessed. Exposure to low and medium concentrations of CS exacerbated the severity of symptoms and spinal cord pathology, while the high concentration had no effect relative to sham exposure in mice with EAE. Interestingly, the clinical chemistry parameters for metabolic profile as well as liver and renal function (e.g. triglycerides and creatinine levels, alkaline phosphatase activity) were lower in these mice than in naïve controls. Although the mouse EAE model does not fully recapitulate the pathology or symptoms of MS in humans, these findings largely corroborate previous epidemiological findings that exposure to CS can worsen the symptoms and pathology of MS. Furthermore, the study newly highlights the possible correlation of clinical chemistry findings such as metabolism and liver and renal function between MS patients and EAE mice.

Antidepressant efficacy of low-frequency repetitive transcranial magnetic stimulation in antidepressant-nonresponding bipolar depression: a single-blind randomized sham-controlled trial.

BACKGROUND: To examine the antidepressant efficacy and response predictors of R-DLPFC-LF rTMS for antidepressant-nonresponding BD. METHODS: We conducted a single-blind randomized sham-controlled trial for 54 (28 sham, 26 active) patients with antidepressant-nonresponding BD (baseline MADRS ≥ 20). Patients received 15 daily sessions of active or sham neuronavigated rTMS (Figure-of-8 coil, five 1 Hz 60 s 110% RMT trains). Outcome measures included depressive response (≥ 50% MADRS reduction, CGI ≤ 2) and remission (MADRS < 7, CGI = 1) rates, treatment emergent hypo/mania (YMRS), depressive and anxiety symptoms (HAM-A). RESULTS: 48 patients (25 sham, 23 active) completed treatment, with 3 drop-outs each in active and sham groups. Active rTMS did not produce superior response or remission rates at endpoint or 6 or 12 weeks (ps > 0.05). There was no significant group * time interaction (ps > 0.05) in a multivariate ANOVA with MADRS, HAMA and YMRS as dependent variables. Exploratory analysis found MADRS improvement to be moderated by baseline anxiety (p = 0.02) and melancholia (p = 0.03) at week 3, and depressive onset at weeks 6 (p = 0.03) and 12 (p = 0.04). In subjects with below-mean anxiety (HAMA < 20.7, n = 24), MADRS improvement from active rTMS was superior to sham at week 3 (ITT, t = 2.49, p = 0.04, Cohen's d = 1.05). No seizures were observed. Groups did not differ in treatment-emergent hypomania (p = 0.1). LIMITATIONS: Larger sample size might be needed to power subgroup analyses. Moderation analyses were exploratory. Single-blind design. Unblinding before follow-up assessments due to ethical reasons. CONCLUSIONS: 1-Hz 110% RMT (5 × 60 s trains) R-DLPFC-LF rTMS was not effective for antidepressant non-responding BD but may be further investigated at increased dosage and/or in BD patients with low anxiety. Trial registration CCRB Clinical Trials Registry, CUHK, CUHK_CCT00440. Registered 04 December 2014, https://www2.ccrb.cuhk.edu.hk/registry/public/279.

Revisit of Optimal Donor Number Estimation in the Hong Kong Bone Marrow Donor Registry.

High resolution typing of the HLA-DPB1 locus for patient who requested for hematopoietic stem cell transplantation (HSCT) workup has recently become mandatory by the National Marrow Donor Program (NMDP) in order to facilitate matching between donors and recipients for better outcomes. The likelihood of identifying HLA matched donors in Hong Kong, on top of the existing HLA-A, -B, -C, and -DRB1 loci, is revisited in this study. HLA-A, -B, -C, -DRB1 and -DPB1 genotypes of 5,266 volunteer unrelated Chinese donors from the Hong Kong Bone Marrow Donor Registry (HKBMDR), were included in this study. Matching models were employed to determine the matching probabilities for 10/10(DPB1) and 9/10(DPB1) HLA match. The matching probabilities are 20% at 10/10(DPB1) HLA match and 55% at 9/10(DPB1) match, based on the existing 130,000 donors in the HKBMDR. The likelihoods of match become 27% and 65% respectively, by increasing the registry to 250,000. However, if DPB T-cell-epitope (TCE) model is considered in the matching, the probability will increase to 46% at 10/10 DPB1 permissive mismatching. Our findings provide vital information about the future planning on the targeted recruitment size, HLA typing and search strategies of the donor registry and arose the transplant physicians' acceptability to 9/10(DBP1) or 10/10(DBP1) HLA match. Nevertheless, the marrow donor registry has planned for increasing the registry size and bringing down the age of recruited donors which will ultimately enhance patient outcome.

Proteomic Profiling of IgG1 Producing CHO Cells Using LC/LC-SPS-MS3: The Effects of Bioprocessing Conditions on Productivity and Product Quality.

The biopharmaceutical market is dominated by monoclonal antibodies, the majority of which are produced in Chinese hamster ovary (CHO) cell lines. Intense cell engineering, in combination with optimization of various process parameters results in increasing product titers. To enable further improvements in manufacturing processes, detailed information about how certain parameters affect cellular mechanisms in the production cells, and thereby also the expressed drug substance, is required. Therefore, in this study the effects of commonly applied changes in bioprocessing parameters on an anti-IL8 IgG1 producing CHO DP-12 cell line were investigated on the level of host cell proteome expression combined with product quality assessment of the expressed IgG1 monoclonal antibody. Applying shifts in temperature, pH and dissolved oxygen concentration, respectively, resulted in altered productivity and product quality. Furthermore, analysis of the cells using two-dimensional liquid chromatography-mass spectrometry employing tandem mass tag based isotopic quantitation and synchronous precursor selection-MS3 detection revealed substantial changes in the protein expression profiles of CHO cells. Pathway analysis indicated that applied bioprocessing conditions resulted in differential activation of oxidative phosphorylation. Additionally, activation of ERK5 and TNFR1 signaling suggested an affected cell cycle. Moreover, in-depth product characterization by means of charge variant analysis, peptide mapping, as well as structural and functional analysis, revealed posttranslational and structural changes in the expressed drug substance. Taken together, the present study allows the conclusion that, in anti-IL8 IgG1 producing CHO DP-12 cells, an improved energy metabolism achieved by lowering the cell culture pH is favorable when aiming towards high antibody production rates while maintaining product quality.

CD200 expression marks leukemia stem cells in human AML.

The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34- and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34-, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.

Establishing Simultaneous T Cell Receptor Excision Circles (TREC) and K-Deleting Recombination Excision Circles (KREC) Quantification Assays and Laboratory Reference Intervals in Healthy Individuals of Different Age Groups in Hong Kong.

The clinical experience gathered throughout the years has raised awareness of primary immunodeficiency diseases (PIDD). T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) assays for thymic and bone marrow outputs measurement have been widely implemented in newborn screening (NBS) programs for Severe Combined Immunodeficiency. The potential applications of combined TREC and KREC assay in PIDD diagnosis and immune reconstitution monitoring in non-neonatal patients have been suggested. Given that ethnicity, gender, and age can contribute to variations in immunity, defining the reference intervals of TREC and KREC levels in the local population is crucial for setting up cut-offs for PIDD diagnosis. In this retrospective study, 479 healthy Chinese sibling donors (240 males and 239 females; age range: 1 month-74 years) from Hong Kong were tested for TREC and KREC levels using a simultaneous quantitative real-time PCR assay. Age-specific 5th-95th percentile reference intervals of TREC and KREC levels (expressed in copies per µL blood and copies per 106 cells) were established in both pediatric and adult age groups. Significant inverse correlations between age and both TREC and KREC levels were observed in the pediatric age group. A significant higher KREC level was observed in females than males after 9-12 years of age but not for TREC. Low TREC or KREC levels were detected in patients diagnosed with mild or severe PIDD. This assay with the established local reference intervals would allow accurate diagnosis of PIDD, and potentially monitoring immune reconstitution following haematopoietic stem cell transplantation or highly active anti-retroviral therapy in the future.

Evaluation of toxicity of aerosols from flavored e-liquids in Sprague-Dawley rats in a 90-day OECD inhalation study, complemented by transcriptomics analysis.

The use of flavoring substances is an important element in the development of reduced-risk products for adult smokers to increase product acceptance and encourage switching from cigarettes. In a first step towards characterizing the sub-chronic inhalation toxicity of neat flavoring substances, a study was conducted using a mixture of the substances in a base solution of e-liquid, where the standard toxicological endpoints of the nebulized aerosols were supplemented with transcriptomics analysis. The flavor mixture was produced by grouping 178 flavors into 26 distinct chemical groups based on structural similarities and potential metabolic and biological effects. Flavoring substances predicted to show the highest toxicological effect from each group were selected as the flavor group representatives (FGR). Following Organization for Economic Cooperation and Development Testing Guideline 413, rats were exposed to three concentrations of the FGR mixture in an e-liquid composed of nicotine (23 µg/L), propylene glycol (1520 µg/L), and vegetable glycerin (1890 µg/L), while non-flavored and no-nicotine mixtures were included as references to identify potential additive or synergistic effects between nicotine and the flavoring substances. The results indicated that the inhalation of an e-liquid containing the mixture of FGRs caused very minimal local and systemic toxic effects. In particular, there were no remarkable clinical (in-life) observations in flavored e-liquid-exposed rats. The biological effects related to exposure to the mixture of neat FGRs were limited and mainly nicotine-mediated, including changes in hematological and blood chemistry parameters and organ weight. These results indicate no significant additive biological changes following inhalation exposure to the nebulized FGR mixture above the nicotine effects measured in this sub-chronic inhalation study. In a subsequent study, e-liquids with FGR mixtures will be aerosolized by thermal treatment and assessed for toxicity.

Expression of Lmna-R225X nonsense mutation results in dilated cardiomyopathy and conduction disorders (DCM-CD) in mice: Impact of exercise training.

AIMS: To recapitulate progressive human dilated cardiomyopathy (DCM) and heart block in the Lmna R225X mutant mice model and investigate the molecular basis of LMNA mutation induced cardiac conduction disorders (CD); To investigate the potential interventional impact of exercise endurance. METHODS AND RESULTS: A Lmna R225X knock-in mice model in either heterozygous or homozygous genotype was generated. Electrical remodeling was observed with higher occurrence of AV block from neonatal and aged mutant mice as measured by surface electrocardiogram and atrio-ventricular Wenckebach point detection. Histological and molecular profiles revealed an increase in apoptotic cells and activation of caspase-3 activities in heart tissue. Upon aging, extracellular cellular matrix (ECM) remodeling appeared with accumulation of collagen in Lmna R225X mutant hearts as visualized by Masson's trichrome stain. This could be explained by the upregulated ECM gene expression, such as Fibronectin: Fn1, collagen: Col12a1, intergrin: Itgb2 and 3, as detected by microarray gene chip. Also, endurance exercise for 3 month improved the ventricular ejection fraction, attenuated fibrosis and cardiomyocytes apoptosis in the aged mutant mice. CONCLUSIONS: The mechanism of LMNA nonsense mutation induced cardiac conduction defects through AV node fibrosis is due to upregulated ECM gene expression upon activation of cardiac apoptosis. Lmna R225X mutant mice hold the potential for serving as in vivo models to explore the mechanism and therapeutic methods for AV block or myopathy associated with the aging process.